Évaluation des entités de recherche

Vague B : campagne d’évaluation 2015 - 2016

Unité de recherche

Dossier d’évaluation

Nom de l’unité : Institut de Génétique et Développement de Acronyme : IGDR

Nom du directeur pour le contrat en cours : Directeur Claude PRIGENT Directeur Adjoint Christian JAULIN

Nom du directeur pour le contrat à venir : Directeur Reynald GILLET Directrice Adjointe Marie-Dominique GALIBERT

Type de demande :

Renouvellement à l’identique  Restructuration □ Création ex nihilo □

Établissements et organismes de rattachement :

Liste des établissements et organismes de l’unité de recherche pour le prochain contrat (tutelles) : - Université de Rennes 1 - CNRS

Choix de l’évaluation interdisciplinaire de l’unité de recherche :

Oui □ Non 

Vague B : campagne d’évaluation 2015 – 2016 Janvier 2015

C o n t e n t s Page Volume I. ASSESSEMENT RECORD

1. Unit Presentation 7 1.1. Scientific Policy 9 1.1.1. Our missions 9 1.1.2. Our scientific objectives 9 1.1.3. Our goals 9 1.1.4. The Institute’s organisation 9 1.1.5. Positioning in Rennes, Bretagne, and internationally 10 1.2. Business profile 10 1.3. Organisation and life in the Institute 11 1.3.1. Staff evolution 11 1.3.2. Evolution of the budget 12 1.3.3. Organisation of life in the Institute 13 1.3.3.1. People with disabilities 14 1.3.3.2. Social cohesion 14 1.3.3.3. Scientific exchanges 15 1.3.3.4. Seminar cycles 16 1.3.3.5. Directors Committee 16 1.3.3.6. Team Leader Council 16 1.3.3.7. Laboratory Council 17 1.3.3.8. General assembly 17 1.3.3.9. Scientific Advisory Board (SAB) 17 1.3.3.10. Pooled resources 18 1.3.3.11. Functional chart 21 1.4. Highlights 21 1.5. Achievements 22 1.5.1. Scientific production 22 1.5.2. Academic influence and attractiveness 41 1.5.3. Social, economic and cultural interactions 43 1.6. Institutional involvement in research training 46 1.7. Global Strategy and Scientific Perspectives 47 1.7.1. Context 47 1.7.2. Local, regional, national and international development 47 1.7.3. Scientific Objectives 49 1.7.4. Scientific guidelines and strategic choices 49 1.7.5. Scientific guidelines and strategic choices 49 1.7.5.1. Industry and technology transfers 50 1.7.5.2. Scientific goals 50 1.7.5.3. Management and running of the institute 51 1.7.6. Renewed partnerships 52 1.7.7. New scientific topics 52 1.7.8. A new communications agenda 52 1.7.9. List of Researchers and Academics for the next contract 54

2. Team Presentation (plan for each team) 2.1. Team presentation 2.2. Assessment from January 1, 2010, to June 30, 2015 2.2.1. Flow chart of the team (permanent people) 2.2.2. List of staff 2.2.2.1. Permanent staff 2.2.2.2. Temporary staff 2.2.2.3. Permanent staff who left the team during the contract 2.2.2.4. Permanent staff who will be joining the team in Jan 1st 2017 2.2.3. Achievements 2.2.3.1. Scientific achievements 2.2.3.2. Scientific dissemination and influence 2.2.3.3. Interaction with the economic, social and cultural environment 2.4. Research training (if applicable) 2.3. Scientific strategies & perspectives 2.4. Collaboration

2.4.1. Within the IGDR 2.4.2. Other collaborations 2.5. SWOT Annexes: list of production 1. Publications (with IF) 2. Patents (with licence) 3. Conferences (International & National) 4. Funding 5. Training Page Team 1 "Canine " 57 Leader: Catherine ANDRE Team 2 "Kidney cancer: molecular basis of tumorigenesis" 69 Leaders: Yannick ARLOT-BONNEMAINS & Cécile VIGNEAU ➥ "Molecular bases of tumorigenesis: VHL disease" Leader: Yannick ARLOT-BONNEMAINS Team 3 "Tubulin and interacting " 83 Leader: Denis CHRETIEN Team 4 "Architecture and evolution of eukaryotic genetic circuits" 91 Leader: Damien COUDREUSE Team 5 "Genetics of pathologies related to development" 99 Leader: Véronique DAVID Team 6 " expression and oncogenesis" 115 Leader: Marie-Dominique GALIBERT Team 7 "Cytoskeleton and cell proliferation" 129 Leader: Régis GIET Team 8 "Translation and folding" 137 Leader: Reynald GILLET ➥ "Ribosome, bacteria and stress" Team 9 "Oocyte dynamics and implantation in mammals" 149 Leader: Guillaume HALET Team 10 "Structure and molecular interactions" 159 Leader: Jean-François HUBERT ➥ "Dystrophin structure and function in myopathies" Team 11 " and cancer" 171 Leader: Christian JAULIN Team 12 "Epithelia dynamics and mechanics - polarity, division and fate acquisition" 179 Leader: Roland LE BORGNE Team 13 "Membrane traffic and polarity in C. elegans" 191 Leader: Grégoire MICHAUX Team 14 "Integrated functional genomics and biomarkers" 199 Leader: Jean MOSSER ➥ "Translational oncogenomics" Team 15 "Gene expression and development" 213 Leader: Luc PAILLARD Team 16 "Reverse engineering cell division" 223 Leader: Jacques PECREAUX Team 17 "Cell cycle" 237 Leader: Claude PRIGENT Team 18 "Spatio-temporal regulation of transcription in eukaryotes" 253 Leader: Gilles SALBERT Team 19 "Quantitative fluorescence microscopy" 265 Leader: Marc TRAMIER

Team 20 "Genome duplication and maintenance" 277 Leader: Pei-Yun Jenny WU Team 21 " ubiquitylation" 285 Leader: Gwénaël RABUT

Volume II. ANNEXES (UNIT & TEAMS)

Annex 1: "Présentation synthétique" & Executive Summary

Annex 2: Staff turnover

Annex 3: PhD program

Annex 4: External seminars

Annex 5: Frontiers in Biology

Annex 6: Scientific Advisory Board reports

Annex 7: Training

Annex 8: Single document of risk assessment (DUER)

Annex 9: List of contracts

Annex 10: Equipment, platforms

Annex 11: Mutualisation

Annex 12: Detailed organization

Annex 13: Meeting

Annex 14: List of achievements and research products

Annex 15: List of staff (present on June 30, 2015 and which will remain until January 1, 2017)

Annex 16: Internal rules of the IGDR

Annex 17: Summary of the last AERES report (2011)

I. ASSESSMENT RECORD

1. Unit Presentation

History of the IGDR

The "Institute of Genetics and Development of Rennes" (IGDR) is a CNRS research laboratory (UMR) associated with the University of Rennes 1 (UR1).

The laboratory is the result of several evolutions: - Francis GALIBERT’s CNRS Genetic Recombination laboratory moved from the Saint-Louis Hospital in to Rennes. The goal was to develop a new genetics research laboratory on the Health campus in Rennes by joining with the Human Genetics laboratory. - The Biology and Genetics of Development department then moved from the Rennes Sciences campus to the Health one to join with and strengthen the Genetic Recombination laboratory. - This association led to the creation of a Laboratory of Genetics and Development that evolved into the Institute of Genetics and Development of Rennes (IGDR). - Since its creation, the IGDR has recruited 7 research teams headed by young group leaders mainly working on cell division, cell biology and development biology. - Today, the IGDR hosts 20 research teams and works at the intersection of research, education and medicine. In , the IGDR is currently the major research institute dedicated to basic research in biology.

IGDR Timeline

1988: Francis GALIBERT creates the “Recombination Genetics” (UPR 41 CNRS) laboratory at the Saint- Louis Hospital in Paris. 1993: F. GALIBERT and his Recombination Genetics laboratory moved onto the Health campus of Rennes and was joined by Jean-Yves LE GALL’s “Human Genetics” laboratory. 1996: Michel PHILIPPE and his “Biology and Genetics of Development” (URA 256 CNRS-UR1) department leave the Rennes Sciences campus to join F. GALIBERT. 2000: The resulting laboratory directed by Beverley H. OSBORNE was renamed the “Laboratory of Genetics and Development” (UMR 6061). 2004: Claude PRIGENT became director of the laboratory and renamed it the “Institute of Genetics and Development of Rennes”. 2007: Recruitment of the first CNRS ATIP team in Rennes (double ATIP in Bio Cell & Bio Development): “Epithelia dynamics and mechanics,” Roland LE BORGNE. Recruitment of the first AVENIR INSERM team in Rennes: “Membrane traffic and polarity in C. elegans,” Grégoire MICHAUX. 2008: Recruitment of the CNRS ATIP team: “Oocyte dynamics and implantation in mammals,” Guillaume HALET. 2010: Recruitment of the CNRS–UR1 interdisciplinary Physics/Biology chair: Sébastien HUET. Recruitment of the CNRS ATIP interdisciplinary Physics/Biology team: “Reverse engineering cell division,” Jacques PÉCREAUX. 2011: Recruitment of the team “R&D in quantitative fluorescence microscopy,” Marc TRAMIER. 2012: Recruitment of the CNRS ATIP team "Architecture and evolution of eukaryotic genetic circuits," Damien COUDREUSE (ERC starting grant recipient in 2013). The IGDR (CNRS–UR1 UMR 6290) welcomed 4 research teams from the laboratory “Molecular and Cellular Interactions” (CNRS–UR1 UMR 6026). 2013: Recruitment of the ATIP CNRS team”Genome duplication and maintenance,” Pei-Yun Jenny WU (the first foreign group leader of the IGDR, recipient of FRM and AICR starting grants).

1

Administration

The IGDR is a combined research unit under the joint supervision of the University of Rennes 1 (UR1) and the Centre national de la recherche scientifique (CNRS). The UR1 is the local supervisory authority, and the IGDR is hosted on its premises. The CNRS is the French public research organization, and acts as the national supervisory authority for the IGDR. The two supervising organizations are under the jurisdiction of the Ministry of National Education, Higher Education and Research. The CNRS and the university: - Employ technicians, engineers, researchers (CNRS) and teacher/researchers (UR1) working in the IGDR - Award the IGDR an annual operating budget. Within the CNRS, the IGDR is administratively affiliated to the Institut National des Sciences Biologiques (INSB), directed by Catherine JESSUS. Laurent KODJABACHIAN is the Scientific Deputy Director of the INSB in charge of the IGDR. The main section within the CNRS in charge of the IGDR is section 22, the secondary one is 21, and we wish to add section 24 as a third. The IGDR is affiliated with the University of Rennes 1 under President Guy CATHELINEAU (with Eric BELLISSANT, Dean of Medicine, and Hubert LERIVRAY, Director of the UFR Life Sciences and Environment). It is also partner of the Vie- Agro-Santé (VAS) Doctoral School directed by Nathalie THERET. The IGDR participates in the "Structure Fédérative de Recherche" Biology Service Unit (Biology Health Innovation & Technology) directed by Thierry GUILLAUDEUX.

Location

The 20 research teams that make up the IGDR are located in 5 buildings on 2 main sites:

Health campus of Villejean (west of Rennes): - Building 4, School of Medicine: ground floor, second and third floors (14 teams) - Building 5, School of Medicine: ground floor (1 team) - Building 8, School of Medicine: first floor (1 team) - Building BMT-HC, Pontchaillou Hospital (1 team)

Scientific and technological campus of Beaulieu (east of Rennes): - Building 13 (3 teams)

These 3 teams must be relocated to the Health campus as soon as possible. The university committed to doing this some time between 2012 and 2016.

2

Research fields

The IGDR currently brings together 20 research teams, 6 have settled with contract ATIP CNRS, AVENIR INSERM. The IGDR teams work on genetics and cell Biology, developmental biology and structural biology. We use interdisciplinary approaches (physics, mathematics, computer science) to address biological problems. We also study human pathologies, such as cancers and genetic and orphan diseases.

Technological facilities

The group is organized around the many facilities it operates. Members of the IGDR also carry out the scientific direction of four IBiSA-labeled technological platforms. Three of these are administrated by the Unité Mixte de Service (UMS) CNRS 3480 / US INSERM 018 BIOSIT: the "Microscopy, Rennes Imaging Center" (MRic) platform, the "DNA chips" platform and the H2P2 histopathology platform. The fourth, the CaniDNA CRB-Anim (PIA1) platform, is run by the IGDR. The CaniDNA platform needs to be administered by BIOSIT in the future

Staff

As of June 30, 2015, the IGDR hosts approximately 212 people. This includes 129 permanent and 83 non-permanent staff; 34 researchers (25 CNRS and 9 INSERM); 38 teacher/researchers (25 university and 13 university/hospital); 53 permanent technical staff (29 CNRS, 3 CHU, 1 CRLCC and 20 university); 12 non-permanent technical staff (8 CNRS, 2 university and 2 SATT); 1 CDI university worker (salary paid by the IGDR); 15 post-doctoral fellows; 29 PhD students; 23 Master’s degree students; and 2-7 others (AHU, ATER, DREM, 3 PH, PUEM).

1.1. Scientific policy

1.1.1. Our missions

Develop research in cell biology, developmental biology and genetics: - to promote excellence in science; - to promote the transfer of our research: • transfer to industry • transfer to hospitals

1.1.2. Our scientific objectives

To explore the regulation, dynamics and robustness of cell division and identity.

1.1.3. Our goals

In a word: multiple! - Multi-scale Going from the molecule to the cell (2D/3D) to the living organism - Multi-model Bacteria, yeast, flies, amphibians, worms, fish, mice, dogs and humans - Multi-disciplinary Involving biology, chemistry, physics, mathematics and computer science

1.1.4. The Institute’s organisation

The beginning of the present contract was marked by the merger of 2 joint CNRS/university institutes: the UMR 6061 (14 teams) and the UMR 6026 (9 teams). In the end, the merger was uneven. Of the 9 original UMR 6026 teams, 3 joined an INSERM unit (IRSET) instead, leaving 6 who wanted to participate in the merger. Unfortunately, two these teams proposed scientific projects, which were not acceptable. Therefore, only four UMR 6026 teams joined the fourteen UMR 6061 teams in the creation of the new IGDR UMR 6290. Two additional ATIP teams then joined the project. The IGDR is currently structured around twenty research teams and one administrative team. To consolidate the merger of the two UMRs and to stimulate scientific exchange, two departments and two

3

programs were created. Each research team was placed under the aegis of one of the two departments, "Cell Biology and Development" or "Biology and expression of the genome." Every team was then invited to participate in one or both of the programs, "Biology of Human Pathologies" and "Biology and Interdisciplinarity." This institutional organisation has functioned well, indicating that the merger has been successful. A new, more flexible structure is being proposed for the next contract.

1.1.5. Positioning in Rennes, Bretagne, France and internationally

The IGDR is the main research institute on the Health campus of the University of Rennes 1 (even if three research teams remain located on the Science campus). Athough dedicated to basic research, we are also very active in medical research, with direct involvement for instance in trying to find the origin of and treatment for cancers and genetic deseases. The IGDR is the only CNRS biology research institute in Rennes in the fields of Cell Biology, Development Biology, Genetics and Structural Biology. It is also currently the largest research institute dedicated to basic biology research in France’s Grand West (Bretagne & Pays de Loire). In France, not only the CNRS but also AVISAN (the alliance that regroups all the French life science organisations) consider the IGDR to be a major player. The IGDR has recruited many young researchers from the CNRS, INSERM and the university. Most if not all of them were external recruitments, proving the attractiveness of the IGDR. Of these recruits, 6 (including 2 during the present contract) were awarded the ATIP/AVENIR, and 1 received an FRM starting grant. At the European level, the IGDR hosts an ERC starting grant (Damien COUDREUSE). Several people in the IGDR are also financed by Europe: Catherine ANDRE (7PCRD-Health-F4 and EUROSTARS OSEO); Damien COUDREUSE (FP7-PEOPLE Marie Curie); Marie-Dominique GALIBERT (ERG and FP7-PEOPLE Marie Curie); Sébastien HUET (FP7-PEOPLE Marie Curie); Claude PRIGENT (InterReg TC2N); Erwan WATRIN (TARGET- CdLS); and Pei-Yun Jenny WU (FP7-PEOPLE Marie Curie). The IGDR is now an internationally-known institute. As suggested by the last AERES committee, the IGDR recruited a foreign group leader, Pei-Yun Jenny WU, a US citizen and ATIP, FRM and AICR starting grants. The IGDR also hosts PhD students and post-docs of different nationalities. Currently, several PhDs under joint supervision work at the IGDR, 1 from Quebec City and 2 from Munich. The working language of the IGDR for conferences and group meetings is now English. A SAB contributes to the dissemination of our reputation in science, and the long list of renowned international scientists who have accepted to lecture here clearly shows that the IGDR, its researchers and their work have international significance.

1.2. Business profile

Academic Interaction with Research Training though Team Total research the environment support research Team 1 42.5% 1% 42.5% 14% 100% C. ANDRE

Team 2 Y. ARLOT- 55.6% 0,4% 22.2% 22.2% 100% BONNEMAINS & C. VIGNEAU

Team 3 50.0% 0% 33.3% 16.7% 100% D. CHRETIEN

Team 4 33.3% 0% 66.7% 0% 100% D. COUDREUSE

Team 5 58% 0% 4% 38% 100% V. DAVID

Team 6 57% 0,2% 14.3% 28.6% 100% M.D. GALIBERT

Team 7 49% 1% 33% 17% 100% R. GIET

Team 8 32.5% 1.5% 32% 34% 100% R. GILLET

4

Academic Interaction with Research Training though Team Total research the environment support research Team 9 75% 0% 25% 0% 100% G. HALET

Team 10 38.8% 0.2% 23% 38% 100% J.F. HUBERT

Team 11 100% 0% 0% 0% 100% C. JAULIN

Team 12 56% 0% 33% 11% 100% R. LE BORGNE

Team 13 64.4% 0.1% 33.3% 0.2% 100% G. MICHAUX

Team 14 26.9% 0.1% 53% 20% 100% J. MOSSER

Team 15 60% 1% 11% 28% 100% L. PAILLARD

Team 16 50% 3% 37% 10% 100% J. PECREAUX

Team 17 66.6% 0.1% 25% 8.3% 100% C. PRIGENT

Team 18 35% 0.1% 49.9% 15% 100% G. SALBERT

Team 19 0% 0% 100% 0% 100% M. TRAMIER

Team 20 50% 0% 40% 10% 100% P.Y.J. WU

1.3. Institutional organisation and life

1.3.1. Staff evolution (Annex 2)

Staff turnover, 2010-2015 CNRS INSERM University of Rennes 1 Researcher/ Technical Researchers Technical staff Researchers Teachers staff Arrivals 7 13 1 4 1 Departures 1 11 0 3 4

Personnel movements during the new contract, 2017-2022 CNRS INSERM University of Rennes 1 Researcher/ Technical Researchers Technical staff Researchers Teachers staff Arrivals 1 0 0 5 4 Departures 1 1 1 7 1

5

The IGDR is a joint venture between the CNRS and the University of Rennes 1. Our expectation was that both administrations would provide an equal number of staff for the institute’s expansion. In reality when looking at the number of staff obtained from each during the period from January 2011 to June 2015, the allocation is seen to be uneven. For the technical staff, 10 positions were obtained from the CNRS and only 1 from the university; for the researchers, 8 positions came from the CNRS and 2 from the university.

1.3.2. Evolution of the budget

Financial support from CNRS and from the university

The numbers presented below do not take into account the salaries for permanent university positions, or building and related charges. The figures are those of the UMR 6061 (2010 and 2011) and the UMR 6290 (2012-2015). Since January 2012 when UMR 6290 was created, there was a: - 25% increase of the CNRS budget - 9.7% decrease of the university budget

Budget Salaries CNRS University CNRS University 2010: € 240 000 134 742 2 400 000 2011: € 200 000 134 742 2 486 416 2012: € 300 000 207 158 4 316 213 2013: € 310 000 191 942 4 094 879 2014: € 348 000 197 347 4 462 828 2015: € 373 000 178 528 4 181 294

As shown below, the budget allocated by the CNRS clearly shows a continuous increase, whereas the university’s budget decreases slightly.

Total budget (again without salaries)

2010: 2 363 416 € 2011: 2 154 344 € 2012: 4 702 973 € 2013: 4 774 990 € 2014: 5 570 112 € 2015: 4 941 304 € (as of June 1, 2015)

6

1.3.3. Organisation of life in the Institute

To consolidate the merger of the two institutes, and to stimulate interaction between staff members, five working groups with specific missions were created, and membership is voluntary.

#1: improving social cohesion Thomas DERRIEN – Gérard CALLIONI – Olivier DELALANDE – Céline CALLENS This group organizes or coordinates social events together with several members of the Institute they participated to the organisation of (see 1.3.3.2). Members of the IGDR now meet regularly for various activities, including regular BBQs, film showings in the new conference room, evenings watching football matches during the World Cup, weekly jogging, sporting events, etc.

#2: improving participation in external and internal seminars Gwénaël RABUT – Pei-Yun Jenny WU – Pascale QUIGNON This group reorganised our conferences, and the workgroup was a real success. Now each Thursday at 12:30 we have a one-hour internal seminar consisting of either two conferences on two different topics presented by two different groups, or a one-hour conference given by a group leader. Every Friday at 11:00 we have an external seminar featuring an invited speaker.

#3: improving the relationships between the IGDR and the university Stéphane DESCHAMPS – Franck CHESNEL – Catherine LE GOFF-GAILLARD – Céline RAGUÉNÈS- NICOL This objective has not been met. We also need to improve the local image of the IGDR, especially with students in order to continue to attract internship and PhD candidates.

#4: setting up of an IGDR PhD program (Annex 3) Jacques PÉCREAUX – Sébastien HUET – Grégoire MICHAUX – Guillaume HALET The PhD program was launched two years ago and it is already a success. The PhD program is intended to participate in a European appeal for PhD fellowships for the IGDR. The group organized specific training sessions, which were highly appreciated by the participating PhD students. When PhD students participate in the Thursday internal seminars, the workgroup gives a debriefing to help improve their presentations. Most of the research teams are pleased with the initiative.

#5: increasing technical staff promotion and successful from grant applications and CNRS/INSERM competitive exams David GILOT – Christian JAULIN – Claude PRIGENT – Hugues HERVÉ This group did a great job. See the following list: 11 CNRS employees promoted since 2012: BILLETTE DE VILLEMEUR Hervé IE1C to IEHC 2012 TRAMIER Marc IR2 to IR1 2013 MOTTIER Stéphanie IE2 to IE1C 2013 BOURDAIS Anne TCN to AI 2013 CREMET Jean-Yves IE1C to IEHC 2014 COUTURIER Anne AI to IE2 2014 RIO Anne-Gaëlle TCN to TCS 2015 CALLIONI Gérard TCS to TCE 2015 HERVE Hugues IE2 to IE1C 2015 GATTET Nadine TCS to AI 2015 TRAMIER Marc IR1 to IRHC 2015 3 university employees promoted: PASCAL Aude TCS to TCE 2012 BECOT Nathalie ATRF2 to ATRF1 2013 BOTHEREL Nadine ATRF P2 to ATRF P1 2014 7 CNRS researchers have been recruited: COUDREUSE Damien CR1 CNRS 01/01/2012 DERRIEN Thomas CR2 CNRS 01/10/2012 PECREAUX Jacques CR1 CNRS 01/10/2012 TROADEC Marie-Bérengère CR1 CNRS 01/10/2012 WU Pei-Yun Jenny CR1 CNRS 01/10/2012 BOUVRAIS Hélène CR2 CNRS 01/01/2015 PINOT Mathieu CR2 CNRS 01/10/2015

7

1 INSERM researcher has been recruited: CORRE Sébastien CR1 INSERM 01/10/2012

1.3.3.1. People with disabilities

The IGDR is committed to the hosting of disabled scientists and to adapting the laboratory spaces to their needs.

The teams of Jean-François HUBERT and Reynald GILLET hosted a PhD/post-doc student, Aurélie NICOLAS, who permanently uses a motorized wheelchair: - a handicapped parking spot has been attributed on both campuses (Villejean and Beaulieu); - a slope has been constructed in Beaulieu to facilitate access to the building.

Catherine ANDRÉ’s team is hosting a PhD student, Morgane BUNEL, who is permanently in a motorized wheelchair: - doors have been changed on the 2nd floor where she works; - a refuge room (also used as a treatment room) has been constructed on the 2nd floor; - a handicapped-accessible washroom has been constructed on the 2nd floor; - the outside door (west) has been changed to automatically open; - a parking spot has been attributed to her on the campus; - she has been authorized to be accompanied by her helper dog.

Jacques PECREAUX’s team recruited a CNRS CR2 researcher, Hélène BOUVRAIS, who is permanently in an wheelchair: - doors will be changed between the parking area and the 3nd floor where she works; - refuge and treatment rooms will be constructed on the 3nd floor, - an on-campus parking spot has been attributed to her; - a handicapped-accessible washroom will be constructed on the 3nd floor.

In close collaboration with the CNRS and the University of Rennes 1, the IGDR will pursue its efforts to allow disabled staff to thrive in our environment.

1.3.3.2. Social Cohesion

To encourage bonding in the IGDR, various non-scientific events are regularly organized: - Every year one or two crews participate in the "National Sail-Research Challenge" sailing event organized by the CNRS CAES (Comité d'Action et d'Entraide Sociales). This competition brings lab teams together for a 4-day regatta on the Atlantic coast, with several races aboard one-design sailboats. - Several times a week, many membres of the IGDR jog 6-11 km together. They then participate in various races (10 km, half marathon and marathon) organized by different towns around Rennes or by charitable organisations such the Ligue contre le cancer. - Fooball matches are regularly held on the Health campus in late afternoons. - In spring and summer, the IGDR organises BBQs that bring the IGDR staff together on a regular basis, an initiative that has been very successful.

8

- Other sports activities such as bowling, laser tag and bubble football are also organized. - Cinema Evenings: the day before a new cinema release of a movie, the IGDR often projects the preceding movie in the series.

To increase job satisfaction, a shower has been installed in building 4 (Health campus) for those who practice sports and for people who cycle to work, and we plan to install at least one more in the near future.

1.3.3.3. Scientific exchanges

The mission of the two-department, two-program system has been to promote scientific exchange. Each team was asked to join one of the departments and to participate in at least one program. The Biology of the Cell and Development department was headed for a year by Jacek KUBIAK then by Gregoire MICHAUX, while the Biology and Expression of Genomes department has been driven from the beginning by Reynald GILLET. Marie-Dominique GALIBERT led the Biology of Human Pathologies program from the beginning, while the Interdisciplinary program was driven for one year by Arnaud BONDON then by Sébastien HUET.

The events organised by both the departments and the programs have been remarkably effective.

- DEPARTMENT - Biology and Expression of Genomes (BEG) The department organised a "Science Café" which takes place every first Tuesday of the month from 12:45 to 1:45. These monthly "no PowerPoint" exchanges are organized around a coffee: no projector, only discussions and "chalk talks." The round tables bring IGDR members together to freely discuss a topic related to our scientific activities. Anyone can suggest a topic for the next session and the choice is by concensus. This is then discussed in a series of three sessions. So far, the topics discussed (three times each) have been: - Computational resources (calculation, visualization and storage) available to biological researchers in Rennes - RNA (different classes of RNA; available methods for studying RNA in the IGDR; good laboratory practices for working with RNA; RNA and the origins of life) - The new generation of high-throughput sequencing methods (for DNA, RNA and proteins) - Publish or perish? This was followed by a specific workshop dedicated to scientific writing held January 15-16, 2015 by Hervé MAISONNEUVE (www.redactionmedicale.fr). The public is usually limited but highly motivated. Student and post-doc participation in these round tables is always appreciated by, as this is an opportunity to very freely discuss science.

"Department symposium": A large biannual symposium is held which assembles members of the department to discuss and present their data in front of the entire institute. We organize flash movie presentations, posters, a buffet, and informal exchanges. The public is large (more than a hundred participants in each symposium), and the meetings have been very productive. - The first was held on February 7, 2012, at the DIAPASON cultural center on the Beaulieu campus - The second was held on June 24, 2014, in collaboration with the "BioPatho" program hosted by Marie-Dominique GALIBERT.

All events are open to all IGDR members.

- DEPARTMENT - Biology of the Cell and Development (BiCD) For most of the 2011-2015 contract, the BiCD department organized bi-weekly internal seminars showcasing presentations by group leaders and permanent researchers. This provided the opportunity to get general overviews of research areas and for technical seminars on subjects ranging from the use of statistics in biology (Yann LE CUNFF) to high-resolution microscopy (Marc TRAMIER). Starting in mid 2014, these seminars were incorporated into the internal IGDR seminar program, originally dedicated just to PhD student and postdoc presentations. The BiCD department also organized several round table discussions dedicated to soft skills (how to find a postdoc, how to attend a meeting) and to technical questions (CRISPR-Cas9 methodologies). We

9

have also started to invite internationally-recognised speakers such as Yves BARRAL (ETH Zurich). These activities have allowed the department to promote internal collaborations, and have helped to identify experts within the IGDR that students and postdocs can consult with technical questions.

- PROGRAM - Biology of human pathologies (Bio-Patho) The Bio-Patho program was started to encourage interteam interaction between basic science and translational research. Researchers, post-docs, students and technicians are all welcome. About forty people gathered on Friday afternoons an average of four times a year to exchange expertise and promote knowledge. The first central question was the choice of the most accurate model for validating the function of a favourite gene. The pros and cons of each model (yeast, drosophila, C. elegans, zebrafish, xenopus, dog, chicken egg, oocyte, eukaryote/KD cells, nude/knockout mice and tumour sample collection) were discussed by the experts of the field present in the Institute. The second theme centred on the type of questions that can be addressed with life imaging, fluorescent imaging, Flim by Fret, NGS, etc., along with discussions on how to successfullly set up such experiments. In 2014, the Bio-Patho program joined the BEG department in organizing a one-day meeting. The aim of this meeting was to link high throughput expression studies to human pathologies, and the meeting was a success.

- PROGRAM - Interdisciplinary The interdisciplinary program was initiated by a one-day institute-wide meeting on May 21, 2013 during which the teams developing interdisciplinary methodologies presented their work. This indicated the need for the organisation of workshops to disseminate our advanced methods throughout the IGDR. Since a majority of the interdisciplinary approaches developed are related to fluorescence imaging, several workshops were organized on this topic in association with the MRic imaging platform (BIOSIT facility). One-week workshops were organized in March 2014 and June 2015 to showcase advanced fluorescence microscopy methods. We also set up a training session in image processing approaches during December 2014. In addition to imaging workshops, a one-day session on "Statistical methods for biology" was organized internally at the IGDR in March 2015. Altogether, these different training sessions contributed to improving the interdisciplinary methodology skills of IGDR members.

1.3.3.4. Seminar cycles

Every Thursday at 12:30, two speakers from different IGDR teams give a 30-minute presentation. Occasionally a group leader introduces his team’s research project in a one-hour lecture. Every Friday, the IGDR organizes external seminars with a personality invited by an IGDR team. Since 2010, 136 speakers have been invited. (See Annex 4) Four times a year the IGDR organizes prestigious conferences for the series "Frontiers in Biology" created by Pei-Yun Jenny WU and Damien COUDREUSE (Annex 5). A lunch with the lecturer and students and post-docs is organized on this occasion, and the day ends with wine and cheese.

1.3.3.5. Directors Committee

The IGDR has adopted a Directors Committee which includes the Director, Deputy Director and General Secretary, the leaders of each department and program, and a representative of the technical staff. This committee meets every other Monday at 9:00 and has been very effective in discussing and preparing certain issues before presenting them to the Team Leader and Laboratory councils.

1.3.3.6. Team Leader Council

The Team Leader Council is consulted on scientific strategy and the group sets the IGDR’s scientific guidelines. Important decisions to be made by the Director are discussed and voted upon. For instance, the Team Leader Council would discuss the creation of a research team, and they approved the creation of Gwenael RABUT’s new team. Upon request the council ranks grant applications according to the scientific priorities of the IGDR.

10

1.3.3.7. Laboratory Council

The Laboratory Council usually meets after the Team Leader Council, about every two months. The group is informed of the decisions taken by the team leaders, and when these decisions affect life within the institute they are subjected to a vote.

1.3.3.8. General Assembly

A general assembly is scheduled once a year to present the IGDR budget and the promotions obtained, to discuss recruitment issues and to answer questions.

1.3.3.9. Scientific Advisory Board (SAB)

The SAB is composed of European scientists who are not working in France. The task of the SAB is to evaluate the research projects of the IGDR teams based on a European level of excellence. The creation of in-house teams is also subject to SAB evaluation.

The SAB is chaired by: - Jonathan PINES (The Gurdon Institute, Cambridge, UK)

Members of the SAB are: - David FITZPATRICK (MRC, Edinburgh, UK) - François NEDELEC (EMBL, Heidelberg, Germany) - Juan VALCARCEL (CGR, Barcelona, Spain) - Radu ARICESCU (MRC, Oxford, UK) - Marcos GONZALEZ GAITAN (University of Geneva, Switzerland)

On February 4-6, 2015, the SAB evaluated the IGDR’s projects and teams (report in Annex 6), and the board did an outstanding job. Its main recommendations for the Institute as a whole were the following: 1) Unify the IGDR on one campus; 2) Increase cohesion in the Institute (through retreats and group leader talks); 3) Introduce mentoring and appraisals; 4) Introduce rewards for success; These suggestions are now our priorities.

Most team leaders appreciated the work of the SAB, who gave each team specific comments which highlighted the strengths and weaknesses of their projects and/or presentations.

It must be noted that:

- A few days before the SAB evaluation was scheduled, the "Kidney cancer: Molecular basis of tumorogenesis" team co-hosted by two team leaders (Yannick ARLOT-BONNEMAINS CNRS DR2 and Cécile VIGNEAU PU-PH) saw the resignation of three of its researchers (3 PU-PH including the leader C. VIGNEAU). The team and project had originally been designed as a researcher/physician partnership to study kidney cancer. The SAB felt that this team had just suffered the equivalent of an earthquake, and that they could therefore not evaluate the quickly thrown-together project created after the resignations. After scientific discussion, Yannick ARLOT-BONNEMAINS then proposed a project around VHL and cancer that continues one of the main projects of the former team. A special evaluation committee with oncology experts was organized June 12, 2015. Their task was to evaluate the project and the team with an eye toward the presentation of the team to the HCERES. The committee included Alain EYCHENE (Institut Curie, Paris), Brigitte BRESSAC PAILLERETS (Institut Gustave Roussy, Paris) and Alex DUVAL (Hopital Saint-Antoine, Paris) (Annex 6, report in French).

- A new team was proposed to the SAB and approved. It stems from Claude PRIGENT’s team and will be led by Gwénaël RABUT.

The SAB is a great tool for improving our research and its presentation. It must continue, perhaps meeting even more often (at mid-term) and to address specific questions. 11

1.3.3.10. Pooled resources

A centralized administrative team The administration of the IGDR is run by a team, which includes the Director (Claude PRIGENT), Deputy Director (Christian JAULIN), General Secretary (Hugues HERVÉ), Executive Secretary (Géraldine LE PROVOST) and four administrative accountants (Isabelle AGNERAY, Erwan ALAIN, Nadine GATTET and Nathalie ROY-LAVERGNE). Geraldine LE PROVOST is in charge of Human Resources and Dorothy PETROFF is in charge of Communications/PR.

Management and accounting The accounting service ensures the management of the budget lines for 20 research teams and for the IGDR as a whole. In 2014 alone, they handled 4700 accounting operations in. According to the CNRS, this should correspond to the workload of five people, although it was done in fact by four overworked ones. The service is located in the main IGDR building (#4) on the Villejean. The service is organized around four people who are exclusively CNRS staff. They carry out current management operations (orders, deliveries, invoices, missions, travel, hotel …). They also assist researchers with various parts of their research contracts: during the conception of the project’s financial package, during the contract itself and then at the end to help with the financial documents. In 2014, to meet the constraint of multiple locations we reorganized the distribution of IGDR order forms, fully implementing the online exchange system set up in early 2012. Beginning in 2015, research teams were trained to use the CNRS Geslab tool to directly enter their operational and equipment needs. This reorganization allowed the teams to use an intuitive tool adapted to their needs, giving 62 trained users increased autonomy. Various budget reports are now available in real time via the web and for pre-order entries. For each of their contracts, the users can see the amount of money available. Geslab’s structure also allows for the full consolidation of the budgets managed by the two supervisory bodies (CNRS and the University of Rennes 1) and via optimization of the CNRS management tool Geslab. For purchases less than 2 000 €, each manager has a CNRS credit card, allowing the teams to benefit from an efficient payment service via their CNRS appropriations. This facilitates travel bookings, payment of meeting fees, online shopping and local purchases such as restaurants. It should also be noted that four team leaders (of 20) expressed the need for this type of payment of which 2 are at Beaulieu. The management service also offers budgetary advances to teams in difficulties, and these lending transactions are listed and traceable.

Communications/PR Communications officer Dorothée PETROFF was recruited in October 2013 to a newly created position, supporting the research teams and the IGDR as a whole. This has freed up time for researchers. She now provides advice and support for the development and promotion of work and research initiatives as well as in the organization of scientific events, diffusion of information and improvement of signages in the IGDR buildings. The website and extranet have also helped in the development and implementation of a communications strategy for the Institute, while also providing a tool adapted to the expressed needs of the staff for internal and external communication. Our relationships with the communication services of the CNRS and the University of Rennes 1 must be strengthened to facilitate the exchange of information and coordination of practices. In addition, we need to increase the IGDR’s presence and importance locally and regionally, building on our own professional network The main long-term PR task is to develop the reputation of the Institute and its scientific, cultural and social interactions, at both local and international levels. It is difficult to quantify the results of different recently-initiated PR initiatives. We must clarify some procedures and standardize them for the daily uses and attitudes of the Institute members.

12

The following SWOT analysis identifies the strengths and weaknesses of the Institute, and the opportunities and threats in the PR context:

STRENGTHS WEAKNESSES  Full-time PR person  Indifference, ignorance or mistrust of PR  Students and young researchers are  Internal fractures make horizontal concerned by the issues of digital communication difficult communication and the attractiveness  Collecting information difficult and vitality of the Institute  Strong culture of independence of researchers,  Research themes are consistent; easy complicating the sense of belonging to the IGDR INSTITUTE promote in terms of PR  Delayed responses of overloaded researchers  Initiatives to promote the results of leads to considerable loss of time in the PR project progress research of IGDR  No long-term strategic vision for  The aspiration to excellence of IGDR communications could involve PR  Scientific research: a profession that arouse curiosity quickly

OPPORTUNITIES THREATS  Unique position of IGDR in Brittany:  The institute is scattered over several buildings strong visibility potential and campuses, discouraging links and exchanges  Rennes: an important center for the  Careers in communication are not considered a dissemination of scientific knowledge (ex: priority in the research sector CONTEXT Espace des Sciences, Sciences Ouest, etc.)  Basic research is difficult to understand for non-  Biology and health are sectors that specialist audiences, which complicates PR efforts generate "social curiosity"  Infrastructure and communication tools  Links with university and CNRS PR available are sometimes not optimized or obsolete services

Human resources

The HR service is run by Géraldine LE PROVOST, who is in charge of the procedures related to the recruitment of people within the IGDR. For example just in 2014 this included 60 contracts and amendments, 87 internship agreements internship 30 gratuities requests. HR manages hospitality issues and logistics (building access, restauration and catering reservations, email addresses, phone numbers, backup server access, etc.) along with the leaves/absences of the entire IGDR staff.

Staff training Nadine GATTET and Gaëlle PALIERNE are in charge of the IGDR training plan. They also ensure the diffusion of training propositions and they handle IGDR staff training requests and help coordinate the training organized by the IGDR. (See the Plan de Formation d'Unité in Annex 7.) Assessment of the training requested and carried out from 2010 to 2015:

2010 2011 2012 2013 2014 2015 Total

Number of courses 22 142 40 52 57 / 313

Number of people receiving training 22 102 34 100 79 / 337

Number of courses requested / 68 111 64 102 80 425

For 2010, the needs assessment had not been made. For 2015 the assessment of the training received has not yet been done.

Safety The health and safety of IGDR staff are provided by a team of 4 employees (“AP” for agents de prévention): Catherine CHAPUIS, Anne COUTURIER, Gaëlle LE DEZ and Aude PASCAL. Anne COUTURIER is also the corresponding agent for radioactivity in the IGDR. Since the institute is dispersed over 5

13

buildings and 3 sites (Health and Science campuses and the CHU), an assistant AP has also been chosen for each building. (See the "document unique d'évaluation des risques" in Annex 8.)

Finances The total budget of the IGDR was 5 570 000€ in 2014 (without salaries). The IGDR gets 45% of its funding from the CNRS and the university to run the institute. 20 000€ is then set aside to either accompany the installation of a team arriving with a label (ATIP/AVENIR for example) or to be used for equipment purchases or in emergencies. The rest of the support base is redistributed to research teams in proportion to its number of statutory personnel, staff members accounting for a share regardless of their status. 5% of the basic support of the IGDR (excluding specific investments) is paid to the SFR BIOSIT as part of the Rennes biology labs resource pooling. The IGDR also retains 20% (not including salary or equipment) of all research contract and grant monies received by its members (see the list of contracts in Annex 9). This money is used for the general expenses of the IGDR. Although this 20% contribution is essential to run the IGDR, the contribution from each team is unequal. The year 2013 is shown here as an example: 5 teams contributed to 75% of the IGDR budget.

The philosophy of the Institute is to consider that each arriving researcher must be able to start work one week after arriving, and the IGDR relies heavily on shared services to achieve this goal.

Logistics The IGDR uses several shared services (Annexes 10 and 11). A plastic shop with an annual budget of about 100 000€ has been a success since its creation. CNRS technician Gérard CALLIONI is in charge of the workshop, and he is efficient and professional. His position is essential to the Institute. Plastic purchases are grouped, which forces us to place larger orders, but allowing for price negotiations and very significant savings. Nathalie BECOT (UR1 - Health Campus) and Solange SALMON (UR1 - Sciences campus) are in charge of the glass laundry. Because of the wide dispersion of its research teams, the IGDR uses 6 laundries. Two of these are located in building 4 and completely managed by the IGDR, however unfortunately there is only one person in charge of their operation and they are on the ground and 3rd floors. Fortunately a second UR1 agent will be recruited in late 2015. The other laundries are shared with other research units. Nathalie BÉCOT is also in charge of the preparation of common solutions and buffers as well as food for Drosophila. The IGDR orders common products such as chemicals, antibodies, enzymes, and these are stocked in a Promega freezer, Qiagen and Macherey Nagel cabinets.

Description of equipment, technology platforms (Annex 10) Several members of the IGDR are the scientific directors of national technological platforms (IBiSAs). The IGDR is organized around various shared equipment and technical facilities (See Annex 3 for a more detailed description):

14

- Animal facilities for xenopus (IGDR), drosophilae (IGDR), C. elegans (IGDR) and mice (BIOSIT). - Cell culture rooms for bacteria, yeast cells and mammalian cells. - Specific equipment includes but is not limited to Storm & Odyssey imagers and a yeast micromanipulator. - Technological platforms include spectroscopy (supervised by BIOSIT), protein purification, computer systems, bioinformatics and DNA sequencing facilities (IGDR). - Four IBiSA platforms have been created by the IGDR with IGDR members as Scientific Directors. These are the CaniDNA platform (IBiSA and PIA1-IGDR) and the Microscopy-Rennes imaging center (MRic), the health genomics platform and the precision histo-pathology platform (all IBiSA, Biogenouset and BIOSIT).

1.3.3.11. Functional chart (Annex 12)

1.4. Highlights

• UMR 6290 began with a culture clash between two units. An advisory firm was consulted successfully to unify the group leaders around the IGDR project. A training session was held in Rennes and then a team leader retreat was organised, establishing the UMR on a solid foundation. 15

• The recruitment of two ATIP teams, Damien COUDREUSE and Pei-Yun Jenny WU, followed by their recruitment as CR1 CNRS.

• ERC Starting F-Grant for Damien COUDREUSE.

• CRB Anim (PIA1: Plan Investissement d’Avenir # 1) for Catherine ANDRÉ’s team.

• FHU CAMIN (Avenir 2014 Investment Plan) co-led by M.D. GALIBERT, with 7 IGDR teams participating.

• Relocation of the three teams led by Christian JAULIN, Damien COUDREUSE and Pei-Yun Jenny WU. They were moved to a separate building on campus to allow for the complete renovation of the ground floor of building 4, and to build a new cafeteria and conference room.

• The inauguration of the IGDR was marked by a keynote lecture given by Professor Jean-Louis MANDEL (IGBMC Strasbourg; Academy of Sciences and College de France), the presence of Laurent KODJABACHIAN (Deputy Scientific Director of INSB CNRS which is in charge of the IGDR) and Guy CATHELINEAU, President of the University of Rennes 1. (See Annex 13 Meetings)

• "Frontiers in Biology" conferences with prestigious guests such as as (Nobel 2001), Frank ULHMANN, David ALLIS, Adrian BIRD, Michael YOUNG, Venkatraman RAMAKRISHNAN (Nobel 2009) Linda PARTRIDGE, Jason CHIN, Michael HALL and others (See Annex 5)

• Scientific journal covers include: Cell Mol Biol (team LE BORGNE), RNA Biol (team GILLET), J Mol Biol (team GILLET), J Cell Sci (team PRIGENT), Dev Cell (team LE BORGNE), Biol of the Cell (Team ARLOT-BONNEMAINS & VIGNEAU), Trends in Biochemical Sciences (Team GILLET), EMBO report (Team PRIGENT), Development (Team MICHAUX) and Biochemistry (Team GILLET).

• The excellence of our work is attested by publications in journals of very high impact

• New teams to be created are first presented to the team leaders followed by a vote, then to the SAB. For instance, Gwénaël RABUT, CR1 INSERM researcher in Claude PRIGENT’s team, asked to be allowed to set up his own team within the IGDR starting in January 2017. This was approved by the team leaders on the condition of obtaining significant funding for several years including a salary for technical assistance. The SAB also expressed a favourable opinion, so this team is being established.

• The death of three technical staff in one year: Jean-Yves CREMET (IE CNRS), Pierre CADIC (T CNRS) and Gérard FENECH (T CNRS). The IGDR was very affected by the death of these colleagues and friends.

1.5. Achievements

1.5.1. Scientific production (Annex 14)

Number of total publications in peer-reviewed journals: 603 Number of collaborative papers: 288 Number of publications with team researchers as first/last authors (with some in th every top journals like Nature): 315

Some examples : 2010 : Nat Struct Mol Biol. 2010 Feb;17(2):216-21 Team PRIGENT/ARLOT-BONNEMAINS

16

2011: Nature, 2011, 478(7367):97-102 Team DAVID Mol. Cell, 2011, 43(3):488-495. Team PRIGENT/ARLOT-BONNEMAINS Nat. Methods., 2011, 9(1) :45-46 Team SALBERT 2012: Nature Genetics, 2012, 44(2) :140-7 Team ANDRÉ Science, 2012, 335(6074):1366-9 Team GILLET Lancet Oncol., 2012, 13(9) :936-945 Team GALIBERT Nature, 2012, 489(7415):313-7 Team PRIGENT & team JAULIN Nature Structural and Molecular Biology, 2012, 19(11) :1124-31 Team PAILLARD 2013: Nature Genetics, 2013, 45(5) :531-536 Team ARLOT-BONNEMAINS/VIGNEAU Journal of the American Medical Association, 2013 ;309 :232 Team GALIBERT Developmental Cell, 2013, 24(3):242-55 Team LE BORGNE Nature Nanotechnology, 2013, 8(3):199-205 Team LE BORGNE Nature Communications, 2013, 4:1641 Team PRIGENT Nature, 2013, 501(7468) :564-568 Team SALBERT 2014: Nature, 2014, 511 :184-190 Team GALIBERT N Engl J Med., 2014, 371(9):1771-1780 Team GILLET Nature, 2014, 516(7531):410-413 Team PRIGENT Nature Chemical Biology, 2014, 10(5):350-7 Team TRAMIER Nature, 2014, 513(7518):375-381 Team ANDRÉ & team SALBERT Molecular Cell, 2014, 53(4) :655-62 Team WU 2015: Nature Structural and Molecular Biology, 2015 [In press] Team GILLET Nat Commun. 2015 May 28;6:720.5 Team HUBERT

Presentation of activities and research results. Highlight of the IGDR member achievements.

Team Catherine ANDRÉ 1 - MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers. Proc. Natl. Acad. Sci. U. S. A., 2010, 107(33) : 14697-14702 Myopathy in Labrador Retrievers as a model for human myopathy. Mutations in the MTM1 gene encoding myotubularin cause X-linked myotubular myopathy (XLMTM), a well-defined subtype of human centronuclear myopathy. Seven male Labrador Retrievers, age 14-26 wk, were clinically evaluated for generalized weakness and muscle atrophy. Muscle biopsies showed variability in fiber size, centrally placed nuclei resembling fetal myotubes, and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions. Ultrastructural studies confirmed the centrally located nuclei, abnormal perinuclear structure, and mitochondrial accumulations. Wild-type triads were infrequent, with most exhibiting an abnormal orientation of T tubules. MTM1 gene sequencing revealed a unique exon 7 variant in all seven affected males, causing a nonconservative missense change, p.N155K, which haplotype data suggest derives from a recent founder 17

in the local population. Analysis of a worldwide panel of 237 unaffected Labrador Retrievers and 59 additional control dogs from 25 other breeds failed to identify this variant, supporting it as the pathogenic mutation. Myotubularin protein levels and localization were abnormal in muscles from affected dogs, and expression of GFP-MTM1 p.N155K in COS-1 cells showed that the mutant protein was sequestered in proteasomes, where it was presumably misfolded and prematurely degraded. These data demonstrate that XLMTM in Labrador Retrievers is a faithful genetic model of the human condition. 2 - Identification of genomic regions associated with phenotypic variation between dog breeds using selection mapping. PloS Genet., 2011, 7(10) :e1002316 Genomic region at the origin of phenotypic diversity in dog. The extraordinary phenotypic diversity of dog breeds has been sculpted by a unique population history accompanied by selection for novel and desirable traits. Here we perform a comprehensive analysis using multiple test statistics to identify regions under selection in 509 dogs from 46 diverse breeds using a newly developed high-density genotyping array consisting of >170,000 evenly spaced SNPs. We first identify 44 genomic regions exhibiting extreme differentiation across multiple breeds. Genetic variation in these regions correlates with variation in several phenotypic traits that vary between breeds, and we identify novel associations with both morphological and behavioral traits. We next scan the genome for signatures of selective sweeps in single breeds, characterized by long regions of reduced heterozygosity and fixation of extended haplotypes. These scans identify hundreds of regions, including 22 blocks of homozygosity longer than one megabase in certain breeds. Candidate selection loci are strongly enriched for developmental . We chose one highly differentiated region, associated with body size and ear morphology, and characterized it using high-throughput sequencing to provide a list of variants that may directly affect these traits. This study provides a catalogue of genomic regions showing extreme reduction in genetic variation or population differentiation in dogs, including many linked to phenotypic variation. The many blocks of reduced haplotype diversity observed across the genome in dog breeds are the result of both selection and genetic drift, but extended blocks of homozygosity on a megabase scale appear to be best explained by selection. Further elucidation of the variants under selection will help to uncover the genetic basis of complex traits and disease. 3 - Novel origins of copy number variation in the dog genome. Genome Biology, 2012, 13(8) :R73 CNV candidates for generating breed-specific phenotypes. Background: Copy number variants (CNVs) account for substantial variation between genomes and are a major source of normal and pathogenic phenotypic differences. The dog is an ideal model to investigate mutational mechanisms that generate CNVs as its genome lacks a functional ortholog of the PRDM9 gene implicated in recombination and CNV formation in humans. Here we comprehensively assay CNVs using high-density array comparative genomic hybridization in 50 dogs from 17 dog breeds and 3 gray wolves. Results: We use a stringent new method to identify a total of 430 high-confidence CNV loci, which range in size from 9 kb to 1.6 Mb and span 26.4 Mb, or 1.08%, of the assayed dog genome, overlapping 413 annotated genes. Of CNVs observed in each breed, 98% are also observed in multiple breeds. CNVs predicted to disrupt gene function are significantly less common than expected by chance. We identify a significant overrepresentation of peaks of GC content, previously shown to be enriched in dog recombination hotspots, in the vicinity of CNV breakpoints. Conclusions: A number of the CNVs identified by this study are candidates for generating breed-specific phenotypes. Purifying selection seems to be a major factor shaping structural variation in the dog genome, suggesting that many CNVs are deleterious. Localized peaks of GC content appear to be novel sites of CNV formation in the dog genome by non-allelic homologous recombination, potentially activated by the loss of PRDM9. These sequence features may have driven genome instability and chromosomal rearrangements throughout canid evolution. 4 - PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans. Nat Genet. 2012 Jan 15;44(2) Demonstration that dog genetics can be used to find genes responsible for monogenic diseases shared by dogs and humans. Ichthyoses comprise a heterogeneous group of genodermatoses characterized by abnormal desquamation over the whole body, for which the genetic causes of several human forms remain unknown. We used a spontaneous dog model in the golden retriever breed, which is affected by a lamellar ichthyosis resembling human autosomal recessive congenital ichthyoses (ARCI), to carry out a genome-wide association study. We identified a homozygous insertion-deletion (indel) mutation in PNPLA1 that leads to a premature stop codon in all affected golden retriever dogs. We subsequently found one missense and one nonsense mutation in the catalytic domain of human PNPLA1 in six individuals with ARCI from two families. Further experiments highlighted the importance of PNPLA1 in the formation of the epidermal lipid barrier. This study identifies a new gene involved in human ichthyoses and provides insights into the localization and function of this yet uncharacterized member of the PNPLA protein family.

18

5 – Plan Investissement d’Avenir (PIA1): CRB Anim 2013 : CaniDNA The acknowledgement of dog genetics by a French institution after a long-term investment. The CaniDNA bio-bank has been created and is maintained at the l'Institut Génétique et Développement de Rennes (CNRS/Université Rennes1) in Rennes by Dr. Catherine ANDRÉ in collaboration with ANTAGENE laboratory, in (Dr. Anne Thomas) and the Laboratory of Molecular and Cellular Genetics of the National Veterinary School of Maisons Alfort (Dr. Laurent TIRET) and the National Veterinary School of (Dr Lionel MARTIGNAT). CaniDNA contains dog DNA from any breed affected or not with genetic diseases, and includes their genealogical and clinical data. More than 10,000 DNA from blood samples have been collected by numerous French and European veterinary practices, the French web site for hereditary eye diseases and French veterinary schools. CaniDNA also contains resources freely available such as a table of ongoing research projects, a table of genetic diseases, a table of the available genetic tests, links and contacts. The aim of CaniDNA is to collect DNA samples for veterinary and medical genetics projects using DNA samples, accessible upon request. Genomic and genetic research has made the identification of genes involved in genetic diseases possible, thus aiding in: - the development of genetic tests for diagnosis and prediction in veterinary research. - the identification of novel genes and their functions in human homologous diseases. 6 – A mutation in the FAM83G gene in dogs with hereditary footpad hyperkeratosis (HFH). PloS Genetics, 2014, 10(5) :e1004370 Identification of a mutation associated with hyperkeratosis in dogs. Hereditary footpad hyperkeratosis (HFH) represents a palmoplantar hyperkeratosis, which is inherited as a monogenic autosomal recessive trait in several dog breeds, such as e.g. Kromfohrländer and Irish Terriers. We performed genome-wide association studies (GWAS) in both breeds. In Kromfohrländer we obtained a single strong association signal on 5 (p(raw) = 1.0×10(-13)) using 13 HFH cases and 29 controls. The association signal replicated in an independent cohort of Irish Terriers with 10 cases and 21 controls (p(raw) = 6.9×10(-10)). The analysis of shared haplotypes among the combined Kromfohrländer and Irish Terrier cases defined a critical interval of 611 kb with 13 predicted genes. We re-sequenced the genome of one affected Kromfohrländer at 23.5× coverage. The comparison of the sequence data with 46 genomes of non-affected dogs from other breeds revealed a single private non-synonymous variant in the critical interval with respect to the reference genome assembly. The variant is a missense variant (c.155G>C) in the FAM83G gene encoding a protein with largely unknown function. It is predicted to change an evolutionary conserved arginine into a proline residue (p.R52P). We genotyped this variant in a larger cohort of dogs and found perfect association with the HFH phenotype. We further studied the clinical and histopathological alterations in the epidermis in vivo. Affected dogs show a moderate to severe orthokeratotic hyperplasia of the palmoplantar epidermis. Thus, our data provide the first evidence that FAM83G has an essential role for maintaining the integrity of the palmoplantar epidermis. 7 – A canine Arylsulfatase G (ARSG) mutation leading to a sulfatase deficiency is associated with neuronal ceroid lipofuscinosis. Proc. Natl. Acad. Sci. U. S. A., 2010, 107(33):14775-14780 Identification of a mutation associated with neuronal ceroid lipofuscinosis in dogs. Neuronal ceroid lipofuscinoses (NCLs) represent the most common group of inherited progressive encephalopathies in children. They are characterized by progressive loss of vision, mental and motor deterioration, epileptic seizures, and premature death. Rare adult forms of NCL with late onset are known as Kufs' disease. Loci underlying these adult forms remain unknown due to the small number of patients and genetic heterogeneity. Here we confirm that a late-onset form of NCL recessively segregates in US and French pedigrees of American Terrier (AST) dogs. Through combined association, linkage, and haplotype analyses, we mapped the disease locus to a single region of canine chromosome 9. We eventually identified a worldwide breed-specific variant in exon 2 of the Arylsulfatase G (ARSG) gene, which causes a p.R99H substitution in the vicinity of the catalytic domain of the enzyme. In transfected cells or leukocytes from affected dogs, the missense change leads to a 75% decrease in sulfatase activity, providing a functional confirmation that the variant might be the NCL- causing mutation. Our results uncover a protein involved in neuronal homeostasis, identify a family of candidate genes to be screened in patients with Kufs' disease, and suggest that a deficiency in sulfatase is part of the NCL pathogenesis. 8 - The genomic substrate for adaptive radiation in African cichlid fish. Nature. 2014 Sep 18;513(7518):375-81 A collaborative work on fish evolution. Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African

19

lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.

Team Yannick ARLOT-BONNEMAINS & Cécile VIGNEAU 1 - Confinement induces actin flow in a meiotic cytoplasm. Proc. Natl. Acad. Sci. U. S. A., 2012, 109(29) :11705-11710 Collaborative work on oocyte maturation with F. CHESNEL. In vivo, F-actin flows are observed at different cell life stages and participate in various developmental processes during asymmetric divisions in vertebrate oocytes, cell migration, or wound healing. Here, we show that confinement has a dramatic effect on F-actin spatiotemporal organization. We reconstitute in vitro the spontaneous generation of F-actin flow using Xenopus meiotic extracts artificially confined within a geometry mimicking the cell boundary. Perturbations of actin polymerization kinetics or F-actin nucleation sites strongly modify the network flow dynamics. A combination of quantitative image analysis and biochemical perturbations shows that both spatial localization of F-actin nucleators and actin turnover play a decisive role in generating flow. Interestingly, our in vitro assay recapitulates several symmetry-breaking processes observed in oocytes and early embryonic cells. 2 - Recessive mutations in DGKE cause atypical hemolytic-uremic syndrome. Nature Genetics, 2013, 45(5) :531-536 Nathalie RIOUX-LECLERC participated in the identification of a mutation causing Hemolytic- Uremic syndrome. Pathologic thrombosis is a major cause of mortality. Hemolytic-Uremic syndrome (HUS) features episodes of small-vessel thrombosis resulting in microangiopathic hemolytic anemia, thrombocytopenia and renal failure. Atypical HUS (aHUS) can result from genetic or autoimmune factors that lead to pathologic complement cascade activation. Using , we identified recessive mutations in DGKE (encoding diacylglycerol kinase ɛ) that co-segregated with aHUS in nine unrelated kindreds, defining a distinctive Mendelian disease. Affected individuals present with aHUS before age 1 year, have persistent hypertension, hematuria and proteinuria (sometimes in the nephrotic range), and develop chronic kidney disease with age. DGKE is found in endothelium, platelets and podocytes. Arachidonic acid-containing diacylglycerols (DAG) activate protein kinase C (PKC), which promotes thrombosis, and DGKE normally inactivates DAG signaling. We infer that loss of DGKE function results in a prothrombotic state. These findings identify a new mechanism of pathologic thrombosis and kidney failure and have immediate implications for treating individuals with aHUS.

3 - Insulin-like Growth Factor Messenger RNA-binding Protein 3 Expression Helps Prognostication in Patients with Upper Tract Urothelial Carcinoma. European Urology, 2014, 66(2) :379-85 Nathalie RIOUX-LECLERC participated in a study identifying IMP3 as a biomarker in upper-tract urothelial carcinoma. Background: Upper tract urothelial carcinoma (UTUC) is a clinically heterogeneous disease that lacks high-quality trials that provide definitive prognostic markers. Insulin-like growth factor messenger RNA binding protein 3 (IMP3) has been associated with outcomes in urothelial carcinoma of the bladder but was not yet studied in UTUC. Objective: To evaluate the association of the oncofetal protein IMP3 with oncologic outcomes in patients with UTUC treated with radical nephroureterectomy (RNU). Design, setting, and participants: We investigated the expression of IMP3 and its association with clinical outcomes using tissue microarrays constructed from 622 patients treated with RNU at seven international institutions between 1991 and 2008. Intervention: All patients were diagnosed with UTUC and underwent RNU. Outcome measurement and statistical analysis: Uni- and multivariable Cox regression analyses evaluated the association of IMP3 protein expression with disease recurrence, cancer-specific mortality, and all-cause mortality. Results and limitations: IMP3 was expressed in 12.2% of patients with UTUC (n=76). The expression was tumor specific and correlated with higher stages/grades. Within a median follow-up of 27 mo (interquartile range [IQR]: 12-53), 191 patients (25.4%) experienced disease recurrence, and 165 (21.9%) died of the disease. Patients with IMP3 demonstrated significantly worse recurrence-free survival (27.4% vs 75.1%; p<0.01), cancer-specific

20

survival (34.5% vs 78.9%; p<0.01), and overall survival (15.6% vs 64.8%; p<0.01) at 5 yr compared with those without IMP3. In multivariable Cox regression analyses, which adjusted for the effects of standard clinicopathologic features, IMP3 expression was independently associated with disease recurrence (hazard ratio [HR]: 1.87; p<0.01), cancer-specific mortality (HR: 2.15; p<0.01), and all-cause mortality (HR: 2.07; p<0.01). Major limitations include the retrospective design and relatively short follow-up time. Conclusions: IMP3 expression is independently associated with disease recurrence, cancer-specific mortality, and all-cause mortality in UTUC. IMP3 may help improve risk stratification and prognostication of UTUC patients treated with RNU.

Team Denis CHRETIEN 1 - Transport of fibroblast growth factor 2 in the pericellular matrix is controlled by the spatial distribution of its binding sites in heparan sulfate. PLoS Biology, 2012, 10(7):e1001361 Laurence DUSCHESNE collaborative work for a Plos Biology paper. The heparan sulfate (HS) chains of proteoglycans are a key regulatory component of the extracellular matrices of animal cells, including the pericellular matrix around the plasma membrane. In these matrices they regulate transport, gradient formation, and effector functions of over 400 proteins central to cell communication. HS from different matrices differs in its selectivity for its protein partners. However, there has been no direct test of how HS in the matrix regulates the transport of its partner proteins. We address this issue by single molecule imaging and tracking in fibroblast pericellular matrix of fibroblast growth factor 2 (FGF2), stoichiometrically labelled with small gold nanoparticles. Transmission electron microscopy and photothermal heterodyne imaging (PHI) show that the spatial distribution of the HS-binding sites for FGF2 in the pericellular matrix is heterogeneous over length scales ranging from 22 nm to several µm. Tracking of individual FGF2 by PHI in the pericellular matrix of living cells demonstrates that they undergo five distinct types of motion. They spend much of their time in confined motion (∼110 nm diameter), but they are not trapped and can escape by simple diffusion, which may be slow, fast, or directed. These substantial translocations (µm) cover distances far greater than the length of a single HS chain. Similar molecular motion persists in fixed cells, where the movement of membrane PGs is impeded. We conclude that FGF2 moves within the pericellular matrix by translocating from one HS-binding site to another. The binding sites on HS chains form non-random, heterogeneous networks. These promote FGF2 confinement or substantial translocation depending on their spatial organisation. We propose that this spatial organisation, coupled to the relative selectivity and the availability of HS-binding sites, determines the transport of FGF2 in matrices. Similar mechanisms are likely to underpin the movement of many other HS-binding effectors. 2 - The formation of ordered nano-clusters controls cadherin anchoring to actin and cell-cell contact fluidity. J Cell Biol. 2015 Jul 20;210(2):333-46 Laurence DUSCHESNE collaborated on a J Cell Biol paper. The heparan sulfate (HS) chains of proteoglycans are a key regulatory component of the extracellular matrices of animal cells, including the pericellular matrix around the plasma membrane. In these matrices they regulate transport, gradient formation, and effector functions of over 400 proteins central to cell communication. HS from different matrices differs in its selectivity for its protein partners. However, there has been no direct test of how HS in the matrix regulates the transport of its partner proteins. We address this issue by single molecule imaging and tracking in fibroblast pericellular matrix of fibroblast growth factor 2 (FGF2), stoichiometrically labelled with small gold nanoparticles. Transmission electron microscopy and photothermal heterodyne imaging (PHI) show that the spatial distribution of the HS-binding sites for FGF2 in the pericellular matrix is heterogeneous over length scales ranging from 22 nm to several µm. Tracking of individual FGF2 by PHI in the pericellular matrix of living cells demonstrates that they undergo five distinct types of motion. They spend much of their time in confined motion (∼110 nm diameter), but they are not trapped and can escape by simple diffusion, which may be slow, fast, or directed. These substantial translocations (µm) cover distances far greater than the length of a single HS chain. Similar molecular motion persists in fixed cells, where the movement of membrane PGs is impeded. We conclude that FGF2 moves within the pericellular matrix by translocating from one HS-binding site to another. The binding sites on HS chains form non-random, heterogeneous networks. These promote FGF2 confinement or substantial translocation depending on their spatial organisation. We propose that this spatial organisation, coupled to the relative selectivity and the availability of HS-binding sites, determines the transport of FGF2 in matrices. Similar mechanisms are likely to underpin the movement of many other HS-binding effectors.

Team Damien COUDREUSE Architecture and logic of the eukaryotic cell cycle, ATIP 2012 then ERC 2013 The introduction of synthetic biology to the IGDR. Control of cell proliferation is at the basis of cellular life, and the mechanisms underlying this

21

process are conserved throughout eukaryotes. However, the complexity of the regulatory network driving the cell division cycle has made it difficult to understand how this essential function originated and how it is fundamentally controlled. We are applying a synthetic approach using fission yeast to decipher the core architecture and evolution of the cell cycle system. Our projects build on a model of minimal cells that efficiently progress through the entire cycle with a single oscillating module harboring cyclin-dependent kinase (CDK) activity. On one hand, we are focusing on revealing the importance of the way cell cycle control is organized for its functioning and robustness. From imposing complex patterns in the dynamics of CDK activity to rewiring the control network molecularly and spatially, we are uncovering the basic rules governing cell proliferation at both the population and single-cell levels. In addition, our synthetic approach allows us to assess why the natural regulation has been selected through evolution over alternative circuits that appear compatible with life. On the other hand, we use the broad range of "synthetic" cells that we have generated as theoretical and experimental models to understand how basic genetic circuits driving essential processes evolve when challenged. This is achieved through experimental evolution of minimal yeast cells that are grown under various constraints for hundreds of generations. Taking advantage of our successful efforts to develop powerful tools, from microfluidic devices to automated culture systems for laboratory evolution, and of a fruitful collaboration to apply mathematical modeling to our synthetic circuits, our research proposes a unique approach to understanding cell cycle logic and evolution

Team Véronique DAVID 1 - Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2 locus. Nature, 2011, 478(7367):97-102 A collaborative work showing our involvement in the international arrayCGH network. Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.

2 - six collaborative papers in Am J Hum Genet attest to our expertise in clinical genetics: - Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias. American Journal of Human Genetics, 2011, 15(89):7-14 - Identification of mutations in TMEM5 and ISPD as a cause of severe cobblestone lissencephaly. American Journal of Human Genetics, 2012, 91(6):1135-1143 - PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy. American Journal of Human Genetics, 2013, 93(1):141-149 - MFAPS loss-of-function mutations underscore the involvement of matrix alteration in the pathogenesis of familial thoracic aortic aneurysms and dissections American Journal of Human Genetics, 2014, 95(6):736-743 - Mutations in SLC13A5 cause autosomal-recessive epileptic encephalopathy with seizure onset in the first days of life. American Journal of Human Genetics, 2014 Jul 3;95(1):113-20 - Submicroscopic deletions at 13q32.1 cause congenital microcoria. American Journal of Human Genetics, 2015 Apr 2;96(4):631-9

22

Team Marie-Dominique GALIBERT 1- USF-1 is critical for maintaining genome integrity in response to UV-induced DNA photolesions. PLoS Genet. 2012 Jan;8(1):e1002470 Deepening the understanding of the abnormal clearance of UV-induced mutations. An important function of all organisms is to ensure that their genetic material remains intact and unaltered through generations. This is an extremely challenging task since the cell's DNA is constantly under assault by endogenous and environmental agents. To protect against this, cells have evolved effective mechanisms to recognize DNA damage, signal its presence, and mediate its repair. While these responses are expected to be highly regulated because they are critical to avoid human diseases, very little is known about the regulation of the expression of genes involved in mediating their effects. The Nucleotide Excision Repair (NER) is the major DNA-repair process involved in the recognition and removal of UV-mediated DNA damage. Here we use a combination of in vitro and in vivo assays with an intermittent UV-irradiation protocol to investigate the regulation of key players in the DNA-damage recognition step of NER sub-pathways (TCR and GGR). We show an up-regulation in gene expression of CSA and HR23A, which are involved in TCR and GGR, respectively. Importantly, we show that this occurs through a p53 independent mechanism and that it is coordinated by the stress-responsive transcription factor USF-1. Furthermore, using a mouse model we show that the loss of USF-1 compromises DNA repair, which suggests that USF-1 plays an important role in maintaining genomic stability. 2- Imatinib after induction for treatment of children and adolescents with Philadelphia- chromosome-positive acute lymphoblastic leukaemia (EsPhALL): a randomised, open-label, intergroup study. Lancet Oncol., 2012, 13(9) :936-945 Collaborative work by Virgine GANDEMER (Pediatric Haematology/Oncology department at the hospital). Background: Trials of imatinib have provided evidence of activity in adults with Philadelphia- chromosome-positive acute lymphoblastic leukaemia (ALL), but the drug's role when given with multidrug chemotherapy to children is unknown. This study assesses the safety and efficacy of oral imatinib in association with a Berlin–Frankfurt–Munster intensive chemotherapy regimen and allogeneic stem-cell transplantation for paediatric patients with Philadelphia-chromosome-positive ALL. Methods: Patients aged 1–18 years recruited to national trials of front-line treatment for ALL were eligible if they had t(9;22)(q34;q11). Patients with abnormal renal or hepatic function, or an active systemic infection, were ineligible. Patients were enrolled by ten study groups between 2004 and 2009, and were classified as good risk or poor risk according to early response to induction treatment. Good-risk patients were randomly assigned by a web-based system with permuted blocks (size four) to receive post-induction imatinib with chemotherapy or chemotherapy only in a 1:1 ratio, while all poor-risk patients received post-induction imatinib with chemotherapy. Patients were stratified by study group. The chemotherapy regimen was modelled on a Berlin–Frankfurt–Munster high-risk backbone; all received four post-induction blocks of chemotherapy after which they became eligible for stem-cell transplantation. The primary endpoints were disease-free survival at 4 years in the good-risk group and event-free survival at 4 years in the poor-risk group, analysed by intention to treat and a secondary analysis of patients as treated. The trial is registered with EudraCT (2004-001647-30) and ClinicalTrials.gov, number NCT00287105. Findings: Between Jan 1, 2004, and Dec 31, 2009, we screened 229 patients and enrolled 178: 108 were good risk and 70 poor risk. 46 good-risk patients were assigned to receive imatinib and 44 to receive no imatinib. Median follow-up was 3·1 years (IQR 2·0–4·6). 4-year disease-free survival was 72·9% (95% CI 56·1–84·1) in the good-risk, imatinib group versus 61·7% (45·0–74·7) in the good-risk, no imatinib group (p=0·24). The hazard ratio (HR) for failure, adjusted for minimal residual disease, was 0·63 (0·28–1·41; p=0·26). The as-treated analysis showed 4-year disease-free survival was 75·2% (61·0–84·9) for good-risk patients receiving imatinib and 55·9% (36·1–71·7) for those who did not receive imatinib (p=0·06). 4-year event-free survival for poor-risk patients was 53·5% (40·4–65·0). Serious adverse events were much the same in the good-risk groups, with infections caused by myelosuppression the most common. 16 patients in the good-risk imatinib group versus ten in the good-risk, no imatinib group (p=0·64), and 24 in the poor-risk group, had a serious adverse event. Interpretation: Our results suggests that imatinib in conjunction with intensive chemotherapy is well tolerated and might be beneficial for treatment of children with Philadelphia-chromosome-positive ALL. Funding: Projet Hospitalier de Recherche Clinique- Cancer (France), Fondazione Tettamanti-De Marchi and Associazione Italiana per la Ricerca sul Cancro (Italy), Novartis Germany, Cancer Research UK, Leukaemia Lymphoma Research, and Central Manchester University Hospitals Foundation Trust. 3 - SPECT/CT for sentinel lymph node detection in patients with melanoma. Journal of the American Medical Association, 2013 ;309 :232 A letter to the Edotor of JAMA by Alain DUPUY. To the Editor: Dr Stoffels and colleagues1 evaluated disease-free survival in a series of consecutive patients with melanoma seen in a single center who underwent sentinel lymph node (SLN)

23

excision after preoperative single-photon emission computed tomography/computed tomography (SPECT/CT) detection. A comparison was made to historical controls from the same center who had SLN detection without preoperative SPECT/CT. The SPECT/CT cohort presented with significantly more SLNs detected per patient and a higher number of positive SLNs per patient. Being in the standard cohort rather than in the SPECT/CT cohort was strongly associated with worse disease-free survival (hazard ratio, 4.11; 95% CI, 1.25-13.51) in a multivariate Cox model including SLN status and other important prognostic factors. 4 - p53 requires the stress sensor USF1 to direct appropriate cell fate decision. PLoS Genet. 2014. May 15;10(5):e1004309 New non-canonical function for the transcription factor USF1. Genomic instability is a major hallmark of cancer. To maintain genomic integrity, cells are equipped with dedicated sensors to monitor DNA repair or to force damaged cells into death programs. The tumor suppressor p53 is central in this process. Here, we report that the ubiquitous transcription factor Upstream Stimulatory factor 1 (USF1) coordinates p53 function in making proper cell fate decisions. USF1 stabilizes the p53 protein and promotes a transient cell cycle arrest, in the presence of DNA damage. Thus, cell proliferation is maintained inappropriately in Usf1 KO mice and in USF1- deficient melanoma cells challenged by genotoxic stress. We further demonstrate that the loss of USF1 compromises p53 stability by enhancing p53-MDM2 complex formation and MDM2-mediated degradation of p53. In USF1-deficient cells, the level of p53 can be restored by the re-expression of full-length USF1 protein similarly to what is observed using Nutlin-3, a specific inhibitor that prevents p53-MDM2 interaction. Consistent with a new function for USF1, a USF1 truncated protein lacking its DNA-binding and transactivation domains can also restore the induction and activity of p53. These findings establish that p53 function requires the ubiquitous stress sensor USF1 for appropriate cell fate decisions in response to DNA-damage. They underscore the new role of USF1 and give new clues of how p53 loss of function can occur in any cell type. Finally, these findings are of clinical relevance because they provide new therapeutic prospects in stabilizing and reactivating the p53 pathway. 5 - CD9, a key actor in the dissemination of lymphoblastic leukemia, modulating CXCR4-mediated migration via RAC1 signaling. Blood. 2015 in press Deciphering the central role of CD9 in lymphoblastic leukemia: clinical implications. CD9, a member of the tetraspanin family, has been involved in hematopoietic and leukemic stem cell homing. We investigated the role of CD9 in the dissemination of B acute lymphoblastic leukemia (B- ALL) cells be stably downregulating CD9 in REH and NALM6 cells. CD9 expression was associated with higher levels of REH cells adhesion to fibronectin and CXCR4-mediated migration. Death occurred later in NOD/SCID mice receiving for transplantation REH cells depleted of CD9 than in mice receiving control cells. After CXCL12 stimulation, CD9 promoted the formation of long cytoplasmic actin-rich protusions. We demonstrated that CD9 enhanced RAC1 activation in both REH cells and blasts from patients. Conversely, the overexpression of a competing CD9 C-terminal tail peptide in REH cytoplasm decreased RAC1 activation and cytoplasmic extensions formation in response to CXCL12. Finally, the inhibition of RAC1 activation decreased migration in vitro and the depletion of RAC1 protein from transplanted REH cells increased mice survival. Furthermore, a testis conditioned medium induced the migration of REH and NALM6 cells, and this migration was impeded by an anti-CD9 antibody. The level of CD9 expression also influenced the homing of these cells in mice testes. These findings demonstrate, for the first time, that CD9 plays a key role in the CXCR4-mediated migration and engraftment of B-ALL cells in the bone marrow or testis, through RAC1 activation. 6 - Plan Investissement d’Avenir : FHU CAMin Marie-Dominique GALIBERT and Thierry LAMY, co-directors of the FHU, to coordinate cancer research in Rennes. The University Hospital Federation "Cancer Microenvironment and Innovation" (FHU Camin) brings together the main actors, clinicians, researchers, teachers of the University Hospital of Rennes, Universities of Rennes 1 and 2 and the Centre for the Fight Against Cancer Eugène Marquis invested daily in the fight against cancer. It aims to optimize oncology care sector within the two institutions (CHU and CLCC) while integrating basic research components, translational and clinical site Rennes heavily involved in the theme of cancer. The ambition of the CAMin project is to strengthen an oncology structuring in clinical axes, promote translational research and strengthen the links between research teams and between researchers and other clinicians. At national level this clinico-biological program is part of the objectives of the INCa cancer Plan 3. 7 - Aryl hydrocarbon receptor control of a disease tolerance defence pathway. Nature. 2014 Jul 10;511(7508):184-90 David GILOT participated in a paper for Nature. Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other

24

inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long- term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness. 8 - Impact of early molecular response in children with chronic myeloid leukemia treated in the French Glivec phase 4 study. Blood, 2014, 124(15) :2408-2410 Virgine GANDEMER participated in a paper for Blood. Studies in adults have shown that an early molecular response to imatinib predicts clinical outcome in chronic myeloid leukemia (CML). We investigated the impact of the BCR-ABL1 transcript level measured 3 months after starting imatinib in a cohort of 40 children with CML. Children with a BCR-ABL1/ABL ratio higher than 10% at 3 months after the start of imatinib had a larger spleen size and a higher white blood cell count compared with those with BCR-ABL1/ABL ≤10%. Children with BCR- ABL1/ABL ≤10% 3 months after starting imatinib had higher rates of complete cytogenetic response and major molecular response at 12 months compared with those with BCR-ABL1/ABL >10%. With a median follow-up of 71 months (range, 22-96 months), BCR-ABL1/ABL ≤10% correlated with better progression- free survival. Thus, early molecular response at 3 months predicts outcome in children treated with imatinib for CML. This trial was registered at www.clinicaltrials.gov as #NCT00845221.

Team Regis GIET Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells. J Cell Biol. 2014 Mar 31;204(7):1111-21 Three different IGDR teams describe a new player in spindle assembly. The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

Team Reynald GILLET 1 - Elucidation of the self-assembly pathway of Lanreotide octapeptide into beta-sheet nanotubes: role of two stable intermediates. J. Am. Chem. Soc., 2010, 132(12):4230-4241 This work was a result of the long-standing local collaboration of our team with the Institute of Physics of Rennes (IPR). Nanofabrication by molecular self-assembly involves the design of molecules and self-assembly strategies so that shape and chemical complementarities drive the units to organize spontaneously into the desired structures. The power of self-assembly makes it the ubiquitous strategy of living organized matter and provides a powerful tool to chemists. However, a challenging issue in the self-assembly of complex supramolecular structures is to understand how kinetically efficient pathways emerge from the multitude of possible transition states and routes. Unfortunately, very few systems provide an intelligible structure and formation mechanism on which new models can be developed. Here, we elucidate the molecular and supramolecular self-assembly mechanism of synthetic octapeptide into nanotubes in equilibrium conditions. Their complex hierarchical self-assembly has recently been described at the mesoscopic level, and we show now that this system uniquely exhibits three assembly stages and three intermediates: (i) a peptide dimer is evidenced by both analytical centrifugation and NMR translational diffusion experiments; (ii) an open ribbon and (iii) an unstable helical ribbon are both visualized by transmission electron microscopy and characterized by small angle X-ray scattering. Interestingly, the structural features of two stable intermediates are related to the final nanotube organization as they set, respectively, the nanotube wall thickness and the final wall curvature radius.

25

We propose that a specific self-assembly pathway is selected by the existence of such preorganized and stable intermediates so that a unique final molecular organization is kinetically favored. Our findings suggests that the rational design of oligopeptides can encode both molecular- and macro-scale morphological characteristics of their higher-order assemblies, thus opening the way to ultrahigh resolution peptide scaffold engineering. 2 - Control of peptide nanotube diameter by chemical modifications of an aromatic residue involved in a single close contact. Proc. Natl. Acad. Sci. U. S. A., 2011, 108:7679-7684 This work was a result of the long-standing local collaboration of our team with the Institute of Physics of Rennes (IPR) Supramolecular self-assembly is an attractive pathway for bottom-up synthesis of novel nanomaterials. In particular, this approach allows the spontaneous formation of structures of well- defined shapes and monodisperse characteristic sizes. Because nanotechnology mainly relies on size- dependent physical phenomena, the control of monodispersity is required, but the possibility of tuning the size is also essential. For self-assembling systems, shape, size, and monodispersity are mainly settled by the chemical structure of the building block. Attempts to change the size notably by chemical modification usually end up with the loss of self-assembly. Here, we generated a library of 17 peptides forming nanotubes of monodisperse diameter ranging from 10 to 36 nm. A structural model taking into account close contacts explains how a modification of a few Angström of a single aromatic residue induces a fourfold increase in nanotube diameter. The application of such a strategy is demonstrated by the formation of silica nanotubes of various diameters. 3 - Decoding in the absence of a codon by tmRNA and SmpB in the ribosome. Science. 2012 Mar 16;335(6074):1366-9 This is the first X-ray structure of a ribosome ongoing trans-translation. This work is the result of the long-standing collaboration of our team with Dr V. RAMAKRISHNAN (2009 Nobel prize in chemistry for his outstanding work on ribosome structures). In bacteria, ribosomes stalled at the end of truncated messages are rescued by tmRNA, a bifunctional molecule that acts as both a tRNA and mRNA, and SmpB, a small protein that works in concert with tmRNA. Here we present the crystal structure at 3.2 Å resolution of a tmRNA fragment, SmpB and elongation factor Tu bound to the ribosome. The structure shows how SmpB plays the role of both the anticodon loop of tRNA and portions of mRNA to facilitate decoding in the absence of an mRNA codon in the A site of the ribosome, and explains why the tmRNA-SmpB system does not interfere with normal translation.

4 – tmRNA-SmpB: a journey to the center of the bacterial ribosome EMBO J, 29:3810-3818, 2010 (IF2010: 10.12) This article has been highlighted by: -"Have you seen...?" article in EMBO J (Let's see how tmRNA rescues a stuck ribosome. Klaholz BP. EMBO J. 2010 Nov 17;29(22):3747-9); the Faculty of 1000 rated it an 8 (must read); and the Institut des Sciences biologiques of the CNRS spotlighted it (http://www.cnrs.fr/insb/recherche/parutions/articles2010/r-gillet.htm). Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalysing peptide bond formation between amino acids. When bacterial ribosomes stall on incomplete messages, the trans-translation quality control mechanism is activated by the transfer-messenger RNA bound to small protein B (tmRNA–SmpB ribonucleoprotein complex). Trans-translation liberates the stalled ribosomes and triggers degradation of the incomplete proteins. Here, we present the cryo-electron microscopy structures of tmRNA–SmpB accommodated or translocated into stalled ribosomes. Two atomic models for each state are proposed. This study reveals how tmRNA–SmpB crosses the ribosome and how, as the problematic mRNA is ejected, the tmRNA resume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag-encoding sequence. This provides a structural basis for the transit of the large tmRNA–SmpB complex through the ribosome and for the means by which the tmRNA internal frame is set for translation to resume. 5 - 2012 Junior member IUF (Institut Universitaire de France) Reynald GILLET was the first IUF junior member in the IGDR. This prestigious and honorific distinction is awarded to very few (<2%) professors, and aims to recognize, encourage, and promote high-quality and interdisciplinary research done by university professors at the international level. Disciplinary sector: Biology Medicine Health - Specialty: Biochemistry - Molecular Biology - Themes: - protein synthesis and folding - Ribosome - Cryo-electron microscopy.

26

6 - Preparing the 60S ribosomal subunit for translation: structural basis of eIF6 release, Nature Structural and Molecular Biology, 2015 [In press] This is the first high-resolution cryo-EM structure of the large robosomal subunit of SBDS, paving the way to a better understanding of the control of ribosome maturation and its link with cancer. Article embargoed.

Team Guillaume HALET Novel role for p110β PI 3-kinase in male fertility through regulation of androgen receptor activity in Sertoli cells. PloS Genetics 2015 Jul 1 ;11(7) :e1005304 Guillaume HALET participated in a Plos Genet collaborative paper. The organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110β and AR have previously been reported to functionally interact.

Team Jean-François HUBERT 1 - Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45. Ann. Neurol., 2015, 77:668-674 Aurélie NICOLAS & Elisabeth LE RUMEUR collaborated on a study of the link between phenotypes and dystrophin exon 45. Objective: Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion-exon 45 (Δ45)-may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients. Methods: Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions. Results: As expected, deletions of either exons 45 to 47 (Δ45-47) or exons 45 to 48 (Δ45-48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45-46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44-45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype. Interpretation: The observation that Δ45-46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46-48) are better potential therapies than skipping of exon 46 alone. 2 - Abnormal splicing switch of DMD’s penultimate exon compromises muscle fiber maintenance in Myotonic Dystrophy. Nat Commun. 2015 May 28;6:7205 The demonstration that abnormal splicing of dystrophin exon 78 compromises muscle fibres in Myotonic Dystrophy type 1. Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear- retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs 27

the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1.

Team Roland LE BORGNE 1 - AP-1 controls the trafficking of Notch and Sanpodo toward E-cadherin junctions in sensory organ precursors. Curr. Biol., 2011, 21(1):87-95 By controlling the endocytic recycling of the Notch cofactor Sanpodo, AP-1 is shown to negatively regulate of Notch signaling during asymmetric cell division. In Drosophila melanogaster, external sensory organs develop from a single sensory organ precursor (SOP). The SOP divides asymmetrically to generate daughter cells, whose fates are governed by differential Notch activation. Here we show that the clathrin adaptor AP-1 complex, localized at the trans Golgi network and in recycling endosomes, acts as a negative regulator of Notch signaling. Inactivation of AP-1 causes ligand-dependent activation of Notch, leading to a fate transformation within sensory organs. Loss of AP-1 affects neither cell polarity nor the unequal segregation of the cell fate determinants Numb and Neuralized. Instead, it causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-cadherin is localized. Endocytosis-recycling assays reveal that AP-1 acts in recycling endosomes to prevent internalized Spdo from recycling toward adherens junctions. Because AP-1 does not prevent endocytosis and recycling of the Notch ligand Delta, our data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling. 2 - Septins regulate the contractility of the actomyosin ring to enable adherens junction remodeling during cytokinesis of epithelial cells. Dev Cell. 2013 Feb 11;24(3):242-55. + front page Article from LE BORGNE's lab published back-to-back with those of the LECUIT and BELLAÏCHE labs present complementary findings on the regulation of adhesion and its impact on cell division. How adhesive contacts with neighbors may affect epithelial cell cytokinesis is unknown. We report that in Drosophila, septins are specifically required for planar (but not orthogonal) cytokinesis. During planar division, cytokinetic furrowing initiates basally, resulting in a contractile ring displaced toward the adherens junction (AJ). The formation of new AJ between daughter cells requires the disengagement of E-Cadherin complexes between mitotic and neighboring cells at the cleavage furrow, followed by the assembly of E-Cadherin complexes on the daughter-daughter interface. The strength of adhesion with neighbors directly impacts both the kinetics of AJ disengagement and the length of the new AJ. Loss of septins causes a reduction in the contractility of the actomyosin ring and prevents local disengagement of AJ in the cleavage furrow. By modulating the strength of tension induced by neighbors, we uncover a mechanical function for septins to overcome the extrinsic tension induced by neighboring interphasic cells. 3 - Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles. Nat Nanotechnol. 2013 Mar;8(3):199-205 An interdisciplinary collaborative work. Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment. 4 - Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor. Curr Biol. 2013 Apr 8;23(7):581-7 A link between AP-1 and Numb. In metazoans, unequal partitioning of the cell-fate determinant Numb underlies the generation of distinct cell fates following asymmetric cell division. In Drosophila, during asymmetric division of the sensory organ precursor (SOP) cell, Numb is unequally inherited by the pIIb daughter cell, where it antagonizes Notch. Numb inhibits Notch partly through inhibiting the plasma membrane localization of Sanpodo (Spdo), a transmembrane protein required for Notch signaling during asymmetric cell division.

28

Numb, by binding to Spdo and α-Adaptin, was proposed to mediate Spdo endocytosis alone or bound to Notch in the pIIb cell, thereby preventing Notch activation. However, in addition to endocytosis, Numb also controls the postendocytic trafficking and degradation of Notch in mammals and negatively regulates basolateral recycling in C. elegans. Thus, whether Numb promotes the endocytosis of Spdo is a question that requires experimental demonstration and is therefore investigated in this article. Based on internalization assays, we show that Spdo endocytosis is restricted to cells in interphase and requires AP- 2 activity. Surprisingly, the bulk endocytosis of Spdo occurs properly in numb mutant SOP, indicating that Numb does not regulate the steady-state localization of Spdo via Spdo internalization. We report that Numb genetically and physically interacts with AP-1, a complex regulating the basolateral recycling of Spdo. In numb mutant organs, Spdo is efficiently internalized and recycled back to the plasma membrane. We propose that Numb acts in concert with AP-1 to control the endocytic recycling of Spdo to regulate binary-fate decisions. 5 – inDroso Spin-off of the IGDR The first business unit emanating from the IGDR. Spin-off of the Institute of Genetics and Development of Rennes (R. LE BORGNE’s lab), inDroso offers a unique all-inclusive service: the production of custom-engineered CRISPR flies, Drosophila melanogaster, to the fly research community. To generate the CRISPR flies, inDroso implements the latest CRISPR/Cas9 genome editing technology. Disrupting a gene, tagging an endogenous protein, modeling a disease or functionalizing a fly line, InDroso at locus genome editing service provides optimal tools, custom engineered CRISPR flies, to support fly lab’s research projets. inDroso won the 2015 i-LAB award organized by the French Ministry of Higher Education and Research and BPIiFrance for the creation of innovative technological companies. 6 - Drosophila E-Cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth. PNAS 2015 accepted for publication A link between tissue growth and cell-cell contact maintenance. This work addresses the interplay between membrane trafficking, cell adhesion and tissue integrity maintenance in the Drosophila female germline. The Clathrin adaptor complex AP-1 is shown to regulate the trafficking of E-Cadherin to ring canals (RCs), a structure resulting from incomplete cytokinesis and allowing intercellular communication. E-Cad assembles adhesive clusters surrounding RCs, which as revealed by electron microscopy analyses organize a dense microvilli meshwork wrapping around RCs. While dispensable for RCs biogenesis and maturation, AP-1 and E-Cadherin are required for the maintenance of RCs anchoring to plasma membrane at the onset of vitellogenesis, when cells experience exponential growth and increased mechanical stress. Our study unravels a novel unanticipated function for E-Cadherin in the maintenance of RC anchoring to plasma membrane.

Team Grégoire MICHAUX PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division. J Cell Biol, accepted. A new pathway regulates spindle positioning during asymmetric division During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway which is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In C. elegans one-cell embryos, myosin localizes at the anterior cortex while the mitotic spindle localizes towards the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin- independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow towards the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically-localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis.

Team Jean MOSSER 1 - A 4-gene signature associated with clinical outcome in high-grade gliomas. Clin Cancer Res. 2011 Jan 15;17(2):317-27 A 4-gene signature for scoring the outcome of high-grade gliomas. Purpose: Gene expression studies provide molecular insights improving the classification of patients with high-grade gliomas. We have developed a risk estimation strategy based on a combined analysis of gene expression data to search for robust biomarkers associated with outcome in these tumors. Experimental design: We performed a meta-analysis using 3 publicly available malignant gliomas microarray data sets (267 patients) to define the genes related to both glioma malignancy and

29

patient outcome. These biomarkers were used to construct a risk-score equation based on a Cox proportional hazards model on a subset of 144 patients. External validations were performed on microarray data (59 patients) and on RT-qPCR data (194 patients). The risk-score model performances (discrimination and calibration) were evaluated and compared with that of clinical risk factors, MGMT methylation status, and IDH1 mutational status. Results: This interstudy cross-validation approach allowed the identification of a 4-gene signature highly correlated to survival (CHAF1B, PDLIM4, EDNRB, and HJURP), from which an optimal survival model was built (P < 0.001 in training and validation sets). Multivariate analysis showed that the 4-gene risk score was strongly and independently associated with survival (hazard ratio = 0.46; 95% CI, 0.26-0.81; P = 0.007). Performance estimations indicated that this score added beyond standard clinical parameters and beyond both the MGMT methylation status and the IDH1 mutational status in terms of discrimination (C statistics, 0.827 versus 0.835; P < 0.001). Conclusion: The 4-gene signature provides an independent risk score strongly associated with outcome of patients with high-grade gliomas. 2 - Genome-wide association study identifies TF as a significant modifier gene of iron metabolism in HFE hemochromatosis. Journal of Hepatology 2015 Mar;62(3):664-72 A new polymorphism in the tranferrin gene is associated with HFE-associated hereditary hemochromatosis. Background & aims: Hereditary hemochromatosis (HH) is the most common form of genetic iron loading disease. It is mainly related to the homozygous C282Y/C282Y mutation in the HFE gene that is, however, a necessary but not a sufficient condition to develop clinical and even biochemical HH. This suggests that modifier genes are likely involved in the expressivity of the disease. Our aim was to identify such modifier genes. Methods: We performed a genome-wide association study (GWAS) using DNA collected from 474 unrelated C282Y homozygotes. Associations were examined for both quantitative iron burden indices and clinical outcomes with 534,213 single nucleotide polymorphisms (SNP) genotypes, with replication analyses in an independent sample of 748 C282Y homozygotes from four different European centres. Results: One SNP met genome-wide statistical significance for association with transferrin concentration (rs3811647, GWAS p value of 7×10(-9) and replication p value of 5×10(- 13)). This SNP, located within intron 11 of the TF gene, had a pleiotropic effect on serum iron (GWAS p value of 4.9×10(-6) and replication p value of 3.2×10(-6)). Both serum transferrin and iron levels were associated with serum ferritin levels, amount of iron removed and global clinical stage (p<0.01). Serum iron levels were also associated with fibrosis stage (p<0.0001). Conclusions: This GWAS, the largest one performed so far in unselected HFE-associated HH (HFE-HH) patients, identified the rs3811647 polymorphism in the TF gene as the only SNP significantly associated with iron metabolism through serum transferrin and iron levels. Because these two outcomes were clearly associated with the biochemical and clinical expression of the disease, an indirect link between the rs3811647 polymorphism and the phenotypic presentation of HFE-HH is likely.

Team Luc PAILLARD CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex. Nat Struct Mol Biol. 2012 Nov;19(11):1124-31 A collaborative work highlighting IGDR expertise in methodology. The exon junction complex (EJC) is a central effector of the fate of mRNAs, linking nuclear processing to mRNA transport, translation and surveillance. However, little is known about its transcriptome-wide targets. We used cross-linking and immunoprecipitation methods coupled to high- throughput sequencing (CLIP-seq) in human cells to identify the binding sites of the DEAD-box helicase eIF4AIII, an EJC core component. CLIP reads form peaks that are located mainly in spliced mRNAs. Most expressed exons harbor peaks either in the canonical EJC region, located ~24 nucleotides upstream of exonic junctions, or in other noncanonical regions. Notably, both of these types of peaks are preferentially associated with unstructured and purine-rich sequences containing the motif GAAGA, which is a potential binding site for EJC-associated factors. Therefore, EJC positions vary spatially and quantitatively between exons. This transcriptome-wide mapping of human eIF4AIII reveals unanticipated aspects of the EJC and broadens its potential impact on post-transcriptional regulation.

Team Jacques PECREAUX 1 - Reverse engineering cell division (CeDRE), ATIP CNRS 2010 A true multidisciplinary team in the IGDR, combining biology, physics, mathematics and computer simulation. How biological systems can perform tasks with such an extraordinary fidelity and robustness despite their complexity and variability? Few studies address this question in mechanical biological systems such as cell division (mitosis), during which not only but also cell contents are dispatched in a strongly controlled way. Cell division’s complex and stereotypic choreography is driven

30

by interactions of molecular motors and cytoskeletal filaments that constantly grow or shrink. Cell division’s complex choreography is driven by the interactions of cytoskeletal motors and filaments. This proposal asserts that physics is essential to understand not only the molecular players’ details, but also their signaling and regulation. Asymmetric division, where daughter cell size and contents differ, is dynamic, thus facilitating reverse engineering of mechanisms. Until now, molecular biology and in vitro biophysics have characterized the players, while simulations only accounted for cell-level phenotypes. I envision an experimental systems biophysics strategy, quantitatively linking these micro- and macroscopic approaches. This will be relevant to basic and applied cell division, and microfilaments research, particularly infection. Starting with the nematode embryo familiar to me, I will pursue fruit- fly, and later mammalian cells, more relevant to medicine. In the C. elegans embryo, the spindle is centered until anaphase onset, then elongated by internal forces and likely by cortical motors pulling on astral microtubules. The spindle also moves towards the posterior and oscillates transversely. I hypothesize that cell division diversity and stereotypic choreography can be modeled only including spindle-internal, centering, and cortical forces, derived from microscopic characteristics and interactions. I will focus on three key assertions: (1) microtubule dynamics, cortical forces, and kinesins determine the length of the spindle; (2) the spindle is centered by astral microtubules pushing on the cortex; (3) pulling motors are targeted to the cortex along astral microtubules instead of diffusing. Using my multidisciplinary training, I will lead a team performing systems biophysics modeling to account for collective behaviors, validation by in vivo experiments, and quantification by advanced image processing. UMR 6061 will benefit from this approach, while fostering the study through its expertise in the proposed model organisms and in cell division. 2 - Evolutionary comparisons reveal a positional switch for spindle pole oscillations in Caenorhabditis embryos. Journal of Cell Biology, 2013, 201(5):653-62 Jacques PECREAUX collaborated on a J Cell Biol Paper. During the first embryonic division in Caenorhabditis elegans, the mitotic spindle is pulled toward the posterior pole of the cell and undergoes vigorous transverse oscillations. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of its close relative Caenorhabditis briggsae. Compared with C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by interspecies changes in the regulation of the cortical Gα–GPR–LIN-5 complex. However, we found that in both species (1) a conserved positional switch controls the onset of spindle oscillations, (2) GPR posterior localization may set this positional switch, and (3) the maximum amplitude of spindle oscillations is determined by the time spent in the oscillating phase. By investigating microevolution of a subcellular process, we identify new mechanisms that are instrumental to decipher spindle positioning.

Team Claude PRIGENT 1 - The TFIIH subunit Tfb3 regulates cullin neddylation. Mol Cell. 2011 Aug 5;43(3):488-95 Gwénaël RABUT finished a work on neddylation that had begun during his post-doc. Cullin proteins are scaffolds for the assembly of multisubunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING domain of Hrt1 nor required the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 and the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING domain protein Tfb3 controls activation of a subset of cullins. 2 - Nucleophosmin/B23 activates Aurora A at the centrosome through phosphorylation of serine 89. J Cell Biol. 2012 Apr 2;197(1):19-26 A new interphase activator of Aurora-A kinase. Aurora A (AurA) is a major mitotic protein kinase involved in centrosome maturation and spindle assembly. Nucleophosmin/B23 (NPM) is a pleiotropic nucleolar protein involved in a variety of cellular processes including centrosome maturation. In the present study, we report that NPM is a strong activator of AurA kinase activity. NPM and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. In contrast with previously characterized AurA activators, NPM does not trigger autophosphorylation of AurA on threonine 288. NPM induces phosphorylation of AurA on serine 89, and this phosphorylation is necessary for activation of AurA. These data were confirmed in vivo, as depletion of NPM by ribonucleic acid interference eliminated phosphorylation of CDC25B on S353

31

at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA. 3 - HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle. Nature. 2012 Sep 13;489(7415):313-7 Erwan WATRIN and his colleagues found a new mutated gene present in Cornelia de Lange syndrome. Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S- phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the ‘used’ cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations. 4 - CSN- and CAND1-dependent remodelling of the budding yeast SCF complex. Nature Communications, 2013, 4:1641. Gwénaël RABUT collaborated on a Nat. Comm. paper. Cullin-RING ligases (CRLs) are ubiquitin E3 enzymes with variable substrate-adaptor and - receptor subunits. All CRLs are activated by modification of the cullin subunit with the ubiquitin-like protein Nedd8 (neddylation). The protein CAND1 (Cullin-associated-Nedd8-dissociated-1) also promotes CRL activity, even though it only interacts with inactive ligase complexes. The molecular mechanism underlying this behaviour remains largely unclear. Here, we find that yeast SCF (Skp1-Cdc53-F-box) Cullin-RING complexes are remodelled in a CAND1-dependent manner, when cells are switched from growth in fermentable to non-fermentable carbon sources. Mechanistically, CAND1 promotes substrate adaptor release following SCF deneddylation by the COP9 signalosome (CSN). CSN- or CAND1-mutant cells fail to release substrate adaptors. This delays the formation of new complexes during SCF reactivation and results in substrate degradation defects. Our results shed light on how CAND1 regulates CRL activity and demonstrate that the cullin neddylation-deneddylation cycle is not only required to activate CRLs, but also to regulate substrate specificity through dynamic substrate adaptor exchange. 5 - Aurora A is involved in central spindle assembly through phosphorylation of Ser 19 in P150Glued. J Cell Biol. 2013 Apr 1;201(1):65-79. + Biobytes The design of an ATP analog-sensitive isoform of Aurora-A. Knowledge of Aurora A kinase functions is limited to premetaphase events, particularly centrosome maturation, G2/M transition, and mitotic spindle assembly. The involvement of Aurora A in events after metaphase has only been suggested because appropriate experiments are technically difficult. We report here the design of the first human Aurora A kinase (as-AurA) engineered by chemical genetics techniques. This kinase is fully functional biochemically and in cells, and is rapidly and specifically inhibited by the ATP analogue 1-Naphthyl-PP1 (1-Na-PP1). By treating cells exclusively expressing the as-AurA with 1-Na-PP1, we discovered that Aurora A is required for central spindle assembly in anaphase through phosphorylation of Ser 19 of P150Glued. This paper thus describes a new Aurora A function that takes place after the metaphase-to-anaphase transition and a new powerful tool to search for and study new Aurora A functions 6 - Sororin pre-mRNA splicing is required for proper sister chromatid cohesion in human cells. EMBO Rep. 2014 Sep;15(9):948-55. + front cover 4 publications on the same topic: 2 back-to-back in EMBO Rep, and 2 back-to-back in EMBO J. Sister chromatid cohesion, which depends on cohesin, is essential for the faithful segregation of replicated chromosomes. Here, we report that splicing complex Prp19 is essential for cohesion in both G2 and mitosis, and consequently for the proper progression of the cell through mitosis. Inactivation of splicing factors SF3a120 and U2AF65 induces similar cohesion defects to Prp19 complex inactivation. Our data indicate that these splicing factors are all required for the accumulation of cohesion factor Sororin, by facilitating the proper splicing of its pre-mRNA. Finally, we show that ectopic expression of Sororin corrects defective cohesion caused by Prp19 complex inactivation. We propose that the Prp19 complex and the splicing machinery contribute to the establishment of cohesion by promoting Sororin accumulation during S phase, and are, therefore, essential to the maintenance of genome stability.

32

7 - Protein quality control at the inner nuclear membrane. Nature. 2014 Dec 18;516(7531):410-3 Gwénaël RABUT & his collaborators found a new nucear protein degradation process. The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. 8 - Annexin A2 is required for the early steps of cytokinesis. EMBO Rep. 2015 Apr;16(4):481-9 Christelle BENAUD found a new function for Annexin2. Cytokinesis requires the formation of an actomyosin contractile ring between the two sets of sister chromatids. Annexin A2 is a calcium- and phospholipid-binding protein implicated in cortical actin remodeling. We report that annexin A2 accumulates at the equatorial cortex at the onset of cytokinesis and depletion of annexin A2 results in cytokinetic failure, due to a defective cleavage furrow assembly. In the absence of annexin A2, the small GTPase RhoA-which regulates cortical cytoskeletal rearrangement-fails to form a compact ring at the equatorial plane. Furthermore, annexin A2 is required for cortical localization of the RhoGEF Ect2 and to maintain the association between the equatorial cortex and the central spindle. Our results demonstrate that annexin A2 is necessary in the early phase of cytokinesis. We propose that annexin A2 participates in central spindle-equatorial plasma membrane communication.

Team Gilles SALBERT 1 - Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers. Genome Res., 2011, 21(4) :555-565 A link between DNA methylation levels and FOXA1 binding. Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis- regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation. 2 - Sebastien HUET recruited MCU UR1 on a Chair CNRS/UR1 in 2010 joined Salbert’s team in 2012 First true interdisciplinarity recruitment to the IGDR. Recruited to teach in the Biology department, Sebastien HEUT was the first interdisciplinary physics/biology staff member. He arrived from Jan ELLENBERG’s Heidelberg laboratory, bringing an expertise in photonic microscopy (optics) and in DNA chromatin remodelling after microscopy-induced DNA damage.

33

3 - Wapl is an essential regulator of chromatin structure and chromosome segregation. Nature. 2013 Sep 26;501(7468):564-8 Sébastien HUET participated in a Nature paper. Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher- order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

Team Marc TRAMIER Golgi sorting regulates organization and activity of GPI proteins at apical membranes. Nat Chem Biol. 2014 May;10(5):350-7 A collaborative work with Marc TRAMIER’s former lab. Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi- derived homoclusters are required for their subsequent plasma membrane organization into cholesterol- dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface.

Team Pei-Yun Jenny WU 1 - Genome duplication and maintenance, AICR/FRM/ATIP starting grants (2012/2013) The first international group leader in the IGDR. Eukaryotic DNA synthesis initiates at origins of replication distributed throughout the genome. The program of DNA replication is defined by the timing of origin firing during S phase and by the efficiency of origin usage, which represents the probability that an origin is used in a population of cells. The maintenance of genome integrity requires the accurate duplication and transmission of genetic information. To achieve this, cells employ multiple layers of regulation, including the coordinated assembly of complexes for DNA synthesis and specific checkpoint systems that verify completion of replication prior to the onset of mitosis. These mechanisms are conserved throughout eukaryotes and are critical for proper cell growth and proliferation. For example, changes in origin usage have been associated with developmental stages and differentiation, and defects in DNA synthesis as well as alterations in checkpoint signaling have been linked to many human diseases and shown to promote cancer progression. However, it remains surprisingly unclear how alterations in the pattern of replication and defects in checkpoint function contribute to changes in both cell physiology and genome maintenance. Our work uses the fission yeast Schizosaccharomyces pombe as a model to investigate the controls that that regulate origin selection and ensure genome fidelity. 2 - Replication origin selection regulates the distribution of meiotic recombination. Mol Cell. 2014 Feb 20;53(4):655-62 First demonstration of the functional importance of the replication program for cellular physiology. The program of DNA replication, defined by the temporal and spatial pattern of origin activation, is altered during development and in cancers. However, whether changes in origin usage play a role in regulating specific biological processes remains unknown. We investigated the consequences of modifying origin selection on meiosis in fission yeast. Genome-wide changes in the replication program of premeiotic S phase do not affect meiotic progression, indicating that meiosis neither activates nor requires a particular origin pattern. In contrast, local changes in origin efficiencies between different replication programs lead to changes in Rad51 recombination factor binding and recombination

34

frequencies in these domains. We observed similar results for Rad51 when changes in efficiencies were generated by directly targeting expression of the Cdc45 replication factor. We conclude that origin selection is a key determinant for organizing meiotic recombination, providing evidence that genome- wide modifications in replication program can modulate cellular physiology.

1.5.2. Academic influence and attractiveness

Catherine ANDRE - Member of the CNRS National Committee Section 21 "Organisation, Expression, Evolution des génomes. Bioinformatique et Biologie des systems." - Member of the US national canine cancer biobank CCOGC (Canine Comparative Oncology and Genomic Consortium). - Created a canine genetics group of 30 veterinarians from the French Association of Companion Animal Veterinarians (AFVAC). - Member of the International Conference on Advances in Canine and Feline Genomics and Inherited Diseases.

Yannick ARLOT-BONNEMAINS - Member of the council of the French group of peptides and proteins and scientific editorial board member involved in the organization of the 15th and 19th congresses of the GFPP (member since 2007). - Member of the French Society of Theoretical Biology and involved in the organization of the 31st congress as a scientific editorial board member (2011-2013).

Denis CHRETIEN - Member of the Administration Council of the French Society for Microscopies (SFµ, 2011-14); involved in the organization of the 13th and 14th congresses as a Scientific Advisory Board member. - From 2010 to 2014, he was member of the editorial board of Biology of the Cell, and as such was the co-editor of a special issue on microtubules.

Véronique DAVID - Member of the National Council of Universities (CNU) in Biochemistry (section 44-01) from 2013 onward.

Valérie DUPE - Active member of the Société Française de Biologie du Développement (SFBD) (2007-2013).

Francis GALIBERT - Full member of the National Academy of Medicine Pharmaceutical Sciences section since 2009.

Marie-Dominique GALIBERT - Is currently supervisor of the IGDR "Biology and human pathologies" department and will be nominated as the Deputy Director of the Institute for the next (2017-2022) contract. - Member of the board of the Rennes CHU hospital’s scientific committee (CRBSP). - Co-director of the University/Hospital Alliance for Cancer-Microenvironment-Innovation (FHU CAMIn). - Scientific director of the H2P2 (histopathology) core facility since 2010. - Member of the Ligue Nationale Contre le Cancer’s scientific committee section #1 (2010-2014). - Member of the board of the European Society of Pigment Cell Report since 2010.

Reynald GILLET - Current supervisor of the IGDR’s "Biology and Expression of Genomes" department comprised of 9 teams and more than 120 people. Will be nominated as Director of the Institute for the next contract (2017- 2022). - Created a "Sciences Café" within the Institute in 2012. - Elected member of the Scientific Board of the University of Rennes 1 in 2012; member of its steering committee (comité de pilotage) since 2015. - Elected member of the board of the French Society of Biochemistry and Molecular Biology (SFBBM) 2010-2014. Board member of the SFBBM commitee dedicated to "Teaching Biochemistry." - Co-organizer of the national meeting of the "Institut Universitaire de France" in Rennes, France in June 2016. - Participated in the "Sciences and Arts" exhibition commemorating the international year of crystallography that took place from April through June 2014 on the Beaulieu Sciences campus, University of Rennes 1.

35

Emmanuel GIUDICE - Has been an expert for Go Capital (https://www.gocapital.fr/). - Member of the CNRS National Committee, Section 20 " Biologie moléculaire et structurale, biochimie"

Daniel GUERRIER - Member of the INSERM CSS2 Specialized Scientific Committee 2 "Genetics, Epigenetics, Cancerology".

Guillaume HALET - F1000 Biology Associate Faculty Member since 2010. - Honored in 2013 as one of the “Top 10% Best Reviewers” by the editor of Molecular Human Reproduction.

Christophe HITTE - Member of the Scientific Review Committee for the Future Investments structure of France Genomics around the theme of "Environmental and Agricultural Genomics " (2012 to present). - Member of the Scientific council of the AERES (Agence de l’Evaluation de le Recherche et de l’Enseignement Superieure) - Member of the Evaluation Commission of the National Research Agency (ANR) for the theme “Genomics, genetics, bioinformatics and systems biology” (SVSE6) from 2011 to 2013. - Associate Editor of BMC Veterinary Research since 2011. - Recording secretary for the expert evaluation of project FP7 COFUND program (2013-2018). - Member of the Days program committee on “Open Biology, Informatics and Mathematics” (JOBIM) for the conference which took place in Rennes during July 2012.

Sébatien HUET - Member of the Executive Committee of the GDR3070 "CellTiss” national research network.

Christian JAULIN - Member of the ANR SVSE2 Committee. - Member of the Ligue Nationale Contre le Cancer’s Scientific Committee section #1. - Member of CNRS section 26 (2008-2012).

Roland LE BORGNE - Member of the Ligue Nationale Contre le Cancer’s Scientific Committee section #1.

Stéphanie LE BRAS - Member of the CNRS National Committee Section 22 "Biologie cellulaire, développement, évolution- développement, reproduction."

Agnès MEREAU - Board member of the French Society for Biochemistry (SFBBM).

Grégoire MICHAUX - Board member of the French Society for Cell Biology (SBCF) since 2008 and editor of the SBCF newsletter and website. - Board member of the IGDR PhD program, and coordinator of the IGDR “Cell and Developmental Biology” department.

Jean MOSSER - Member of the INCa NGS working group. - Member of the INCa bioinformatics network in NGS transfer on cancer molecular diagnostic. - Member of the National Evaluation Committee for the Translational Cancer Research training program (AVIESAN – INCa), 2015. - Member of the Scientific Evaluation Committee of the "Groupement Interrégional de Recherche Clinique et d’Innovation Grand Ouest."

Luc PAILLARD - Member of the CNU 65 (Conseil National des Universités), 2011-2015.

Claude PRIGENT - Elected member of the Board of Directors at UR1, 2008-2012. - Elected member of CNRS section 26 (2004-2007 and 2008-2012). - Active member of the SBCF until 2012.

36

- Permanent member of the UICC (Union for International Cancer Control) since 2002. - President of the ANR SVSE2 committee from 2011 to 2013. - Member of the LNCC National Scientific Council section #1 from 2011 to 2015. - Representative of the President of UR1 for the “comité de gestion” of the Biology Station at (2008-2012). - Representative of Alain FUCHS President and the CNRS DG within the Scientific Council and for the Board of Directors of the Cancéropôle Grand Ouest since 2013. - Board of Directors representative of the President of UR1 at the Biology Station at Roscoff since 2008. - Scientific Director of the IBISA Microscopy Rennes Imaging Center (MRic) core facility since 2009. - Associate Professor at the University of Laval, Quebec, from April 2014 to April 2017.

Gwénaël RABUT - Member of the Scientific Council of the University of Rennes 1.

Daniel THOMAS - Designated in 2011 as an honorary member of the French Society of Microscopies (SFµ).

Marc TRAMIER - Member of the Scientific Committee and Coordinator of the Bio-Imaging axis of GIS BioGenOuest, a network of technological platforms in western France. - Member of the scientific committee of the CNRS-supported "Microscopie et Imagerie du Vivant" GDR. - Member of the Steering Committee of the CNRS-supported RTmfm Technological Network (Multidimensional Fluorescence Microscopy). - Member of the National User and National Advisory Committees for France Bio-Imaging. - Member of the Organizing Committee of the CNRS MiFoBio (Functional Biology Microscopy) school.

Erwan WATRIN - Member of the Medical Council of the French Cornelia de Lange syndrome association (AFSCDL) since 2013.

Organisation of meetings (Annex 13)

Denis CHRETIEN & Marc TRAMIER Scientific Committee members for the 13ème symposium of the French Microscopy Society, held in Nantes in July 2013.

Claude PRIGENT Organisation of the international 34th SBCF "Cell Cycle Cancer & Development" meeting, May 2011 in Saint Malo.

Erwan WATRIN Organisation of a symposium on cohesinopathies during the annual conference of the German, Austrian and Switzerland Society of Human Genetic in Dresden at the end of 2013.

IGDR PhD students and post-doc Organisation of a Young Life Sciences Symposium on "DNA damage response in physiology and disease" in Rennes in October 2014.

1.5.3. Social, economic and cultural interactions

Franck CHESNEL - Participed in "Des élèves invitent des chercheurs" (organised by the Rectorat of Ille & Vilaine), a presentation of careers in research in biology - CNRS : University program courses objectives, staff: 7 h in 3 high schools in March-April of 2010. - Participed in the half-day "Carrefour des métiers en collèges" at the La Binquenais secondary school in Rennes in 2011, 2012, 2014 and 2015.

Guillaume HALET - Interviewed in 2015 by Sciences Ouest magazine (in association with the Science Center in Rennes) for a special issue dedicated to current research developments in reproductive biology, destined for a lay audience. - Participated in the "Science café" that followed. (A podcast is available at http://www.espace-sciences.org.)

37

Christophe HITTE - Member of the Scientific Committee for a temporary exhibition at the Cité des Sciences et de l'Industrie in Paris called "Chiens & Chats l'expo, Vous en ressortirez moins bête" (April 2015 to February 2016). - Interviewed during Marie RICHEUX’s French culture radio program "Pas la peine de crier : L’aventure génétique du canidé" on February 17, 2015 (4-5PM) at the Maison de la Radio in Paris. (A podcast is available at http://www.franceculture.fr/emission-pas-la-peine-de-crier-les-chiens-15-l’aventure- genetique-du-canide-2014-02-17).

Yann LE CUNFF - Participated in the IGR-IAE Rennes management school’s intradisciplinary seminar "L’open innovation, clé de renouveau économique pour la Bretagne" in January 2014, presenting the talk "Illustrations de pratiques d'innovation ouverte." - Participated in a technical seminar organized by the Rennes Chamber of Commerce and Industry called "Et si l’open innovation devenait une pratique ordinaire de nos organisations pour stimuler l'innovation?" in March 2014. Presentation was entitled "Prérequis pour aider à mettre en œuvre une démarche d’open innovation." - Presented "Le rôle de l’approche pluridisciplinaire dans l’innovation" during the conference "360 possibles" organised by Bretagne Développement Innovation, October 2014 (http://www.360 possibles.fr). - Provided methodological support for an IGR-IAE seminar on "L’économie collaborative, nouveau pilier de développement pour la Bretagne?", February 2015. - Published "La ville, écosystème d’innovations" in the magazine Place Publique (Rennes).

Agnès MEREAU - Scientific photograph exhibition during the "Festival des sciences," a science fair for the general public.

Agnès MEREAU & Stéphane DESCHAMPS - Presentation to high school students of research jobs during the "Forum des lycées" and "Forum des métiers."

Sylvie ODENT - Sponsor of the Oscars for entrepreneurs in the Ille et Vilaine department, 2014. - Took part in an emission on TV Rennes, February 2014. - Appeared in the newspaper Ouest France in February 2014. - Took part in a general public conference for the “les Mardi Santé” series in the Rennes CHU teaching hospital, presenting "et si c’était une maladie génétique" in December 2014. - Contributor of text and videos on genetics for the website of the French Biomedical Agency.

Luc PAILLARD & Serge HARDY - Presented talks about genetics and genomics for high school students as part of the series "Amphithéâtres des lycéens" (L. PAILLARD in 2013-2014, S. HARDY in 2015).

Claude PRIGENT - Member of the Scientific Council of the Rennes "Espace des Sciences" since 2007.

Gilles SALBERT - Members of the team are regularly involved in meetings with high school students to explain the many facets of biological research and to introduce the different associated professions.

Industrial contracts

Yannick ARLOT-BONNEMAINS - Service contract with the Biotrial Contract Research Organization.

Jean MOSSER - Industrial contract with Roche Laboratories, an ANTiCIPe project entitled "Advancing monitoring of NSCLC Treated with EGFR TKI: Molecular diagnosis is circulating tumor DNA."

38

- Partnership with the Biotrial Contract Research Organization for developing xenograft models with primary glioblastoma cell lines.

Jacques PÉCREAUX & Marc TRAMIER - Shared research and development program with the company Photonlines.

Pei-Yun Jenny WU & Damien COUDREUSE - Collaborators with the Rennes-based start-up Cherry Biotech for the development of microfluidic devices used in live-cell imaging. Both teams have been awarded collaborative research grants from the regional government of Brittany for this project.

Technology transfers and econnomic developments

Marie-Dominique GALIBERT - SATT Ouest Valorisation technological development program: New therapeutic opportunities in melanoma based on the identification of a microRNA sponge that predicts overall patient survival.

Daniel GUERRIER - SATT Ouest Valorisation technological development program VARITI-5: development and testing of a diagnostic test showing normal and pathophysiological states of the human uterus.

Roland LE BORGNE - Rennes Métropole/SATT Ouest Valorisation "inDroso functional genomics" project based on the development of genome-editing using CRISPR/Cas9 methodology.

Marc TRAMIER & Jacques PÉCREAUX - SATT Ouest Valorisation technological development program of “POMM” optimal control of multidimensional microscopy (Pilotage Optimal pour la Microscopie multidimensionnelle.

Patents and licensing

Catherine ANDRE - International patent awarded to C. ANDRE et al. November 17, 2009 for “Methods for diagnosing skin diseases,” patent # 03116-01 (Ref: VD/ACV BET 09P1294). European patent accepted June 2014, US patent process ongoing. - International licence received for the ANTAGENE animal DNA test lab in Lyon, France, since April 2010.

Team ARLOT-BONNEMAINS - Patent awarded June 2014 for “Antibodies against pVHL172 in the development or prevention of renal tumors,” Patent # 14305925.1 -1402 reference: BEP: 130403EPDP. - French patent awarded in April 2015 to Y. ARLOT-BONNEMAINS and F. JOUAN for “Technology for organotypic culture for human kidney cancer tissues,” patent # FR-1553755.

Christian DELAMARCHE In process with Ouest Valorisation: patent on the “Metamyl” A METa-Predictor for AMYLoid Proteins” software by C. DELAMARCHE, M. EMILY (AgroCampus) and A. TALVAS (IRISA).

Team GALIBERT - Patent awarded March 18, 2012 for “New compounds as key modulators of the pigmentation,” patent # WO2013156738 with US CN JP EP extensions. - Patent awarded November 6, 2013 for “Identification of a natural miRNA sponge and its use in managing human melanoma aggressiveness,” patent # EP13306524 with PCT/EP2014/073961 extensions.

Daniel GUERRIER Patent awarded to D. GUERRIER and K. MORCEL for "ITIH5 as a diagnostic marker of uterine development and functional defects," extended to Europe and the USA. European patent # 13709136.9-1405; North American patent # USP201261610590.

Claude PRIGENT - Patents awarded to C. PRIGENT and Anne MARTIN for “Anticorps monoclonal anti-aurora-a, son procede d'obtention, et ses utilisations dans le diagnostic et le traitement des cancers,” patent numbers CA2489214A1, CA2489214C, DE60325725D1, EP1511771A1, EP1511771B1, US7514231 and US20070117163. Licensed to SEROTEC, ABCAM, EMD BIOSCIENCE, HYCULT BIOTECHNOLOGY, SIGMA, ZYMED and CELL SIGNALING TECHNOLOGY. 39

- Patent awarded to C. PRIGENT, David REBOUTIER, Marie-Bérengère TROADEC and Patrick SALAUN FOR “Protéine Aurora A mutée sensible à un inhibiteur,” patent # 13 52932, publication # 3 003 871.

Julien ROUL, Jacques PECREAUX, Marc TRAMIER - Patent filed in 2014 for "Procédé de pilotage multi-modules fonctionnels incluant un dispositif d’imagerie multi-longueur d’onde, et système de pilotage correspondant," INPI # 14/52766. - Copyrighted "FLIMager" software which was submitted in 2012 to the French Agence pour la Protection des Programmes, registration # IDDN.FR.001.140028.000.S.P.2012.000. 31235.

1.6. Institutional involvement in research training

- The IGDR’s PhD program (Annex 3) was set up in 2013 and is based on training-through-research. It aims to open perspectives by exposing students and postdoctoral fellows to all of the IGDR’s research topics. We introduce a global vision of the various fields in biology, and provide them with practical training in soft skills. Since its inception, 26 PhD students and post-docs have been trained.

- Pedagogical responsibilities at all levels in the University of Rennes 1: The IGDR counts 39 professors and associate and assistant professors as full members. Therefore, more than 7500 hours of university courses are devised and given each year by members of the IGDR, mainly in Biology and Health Sciences. Numerous members are deeply committed to the academic life and to the training of future scholars. Among others, notable responsibilities are as follows: - Xavier LE GOFF and Franck CHESNEL are members of the board of the VAS (Life Sciences, Agronomy and Health) Doctoral School. Several members of the IGDR contribute to the three main committees of VAS. X. LE GOFF is the President of the Research Committee of the VAS, while F. CHESNEL is the Vice-president of the "Career development and international mobility" Committee there. - Véronique DAVID is responsible for the exam after the first year of medical studies and forewoman of the jury. - Christian DELAMARCHE is in charge of a Master’s degree in Bioinformatics and Genomics (60 students) and in charge of the Continuing Education Center. - Marie-Dominique GALIBERT is in charge of the Master 2 Cancer program. - Carole GAUTIER-COURTEILLE is in charge of the Master 1 Biology and Health program. - Reynald GILLET has been in charge of the Master’s degree in Biochemistry since 2012. - Emmanuel GIUDICE is in charge of the Bioinformatics educational team within the Biology department. He represents the Biology department at the university’s Informatics Committee. - Serge HARDY has been in charge of the Bachelor’s degree in Life Sciences (> 1,400 students) since 2012. - Jean-François HUBERT is in charge of the Master’s degree in Agronomy-Biology-Health (550 students). - Stéphanie LE BRAS manages internship offers in public and private academic research in France and abroad (https://etudes.univ-rennes1.fr/master-scmv/themes/Stages/Stages_proposes). She also developed a professional alumni network using LinkedIn. - Yann LE CUNFF is strongly committed to developing innovative "learning through research" programs for undergraduate and graduate biology students. - Hubert LERIVRAY has been the director of the UFR SVE (Health, Life, Science) (> 2,500 students) since 2010. - Luc PAILLARD has been in charge of a Master’s degree in molecular and cell biology (120 students) since 2008. - Céline RAGUENES-NICOL heads the educational team in Cellular Biology. - Several members of the IGDR are in charge of numerous teaching modules, from Bachelor’s to Master’s degrees, mainly in biology and health studies (Medicine and Pharmacy).

- Responsibilities in other Universities - Claude PRIGENT is an Associate Professor at the Université Laval in Quebec City, Canada, for the period 2014-2016.

40

- Reynald GILLET and Yannick ARLOT-BONNEMAINS are in charge of the courses in molecular biology and biology of the cell for the International Master’s degree in Molecular and Medicinal Chemistry in Hanoi, Viet-Nam.

- Training - Since 2012, Denis CHRETIEN and his team organize an annual one-week course on cryo-electron tomography under the auspices of the French Society for Microscopies. It is directed towards PhD students, technical staff and research scientists, and is open to both academic institutes and private companies. - Marc TRAMIER and several members of the IGDR organize annual hands-on courses. These have included: light microscopy applied to biology with imaging facilities (2011, 2012, 2014); image analysis with imaging facilities (2011, 2013, 2014); protein interactions by FRET/FLIM and 2 color FCS for experts with RTmfm (2014); and advanced techniques in light microscopy to study the dynamics and molecular interactions, using FRAP / FRET / FLIM / FCS / Photomanipulation with imaging facilities (2015). - The IGDR’s organization of "Frontiers in Biology" seminars has been presented several times in the local press and was recently validated as a PhD student module in the VAS doctoral school. - In January 2015, PhD students underwent two days of training in how to write scientific articles by Hervé MAISONNEUVE, Associate professor in Health Sciences.

1.7. Global Strategy and Scientific Perspectives

1.7.1.Context The precedent contract began in January 2012 with the fusion of two institutes: the UMR 6026 Molecular and Cellular Interactions and the UMR 6061 Institute of Genetics and Development of Rennes. Although the combination corresponded to the creation of a new CNRS UMR 6290, the name IGDR was kept for the new group. Unfortunately the union was unbalanced, with 4 teams from UMR 6026 and 14 from UMR 6061. Among the 4 teams coming from UMR 6026, 3 were structural biologists, while the teams from the UMR 6061 were cell and developmental biologists and geneticists. This important operation had to be a success since the UMR 6290 had become the only CNRS laboratory working on cell biology, developmental biology, genetics and structural biology in Rennes. The first and main objective therefore for the director was the construction of an institute around research teams often working on very different projects using very different approaches. The hope was that the teams would learn from each other and gain in excellence. And we can be proud: it worked (for instance, see the list of publications with high impact factor). The second objective was to create an institute which people would be proud to work in, to create a sense of belonging for IGDR members. We are on our way to succeeding in this goal. The mission of the IGDR as a CNRS institute is to develop outstanding research in biology and this has been accomplished in the past contract. This mission will remain unchanged in the future, always prioritizing scientific quality. However as the research landscape has changed in France and Europe particularly in regards to funding, we have reinforced our projects that involve data transfer to industry and hospitals. Efforts will also be made to increase the visibility of our scientific projects, with a stronger emphasis on issues appealing to the general public. Building on our expertise, an overall scientific project has emerged: to explore the dynamics and the robustness of cell division and cell identity.

1.7.2. Local, regional, national and international development

Local (Rennes) Each Rennes research institute must have a truly unique identity. The IGDR is the only CNRS institute dedicated to basic science in the fields of genetics, and cell, developmental and structural biology. Thanks to close collaborations with the neighboring teaching hospital, we are working in the cancer and genetic disease research fields.

Regional (West of France) In a collaboration between Rennes and Nantes Universities, a new university proiect has been

41

proposed: the "COMUE UBL" (Community of Universities and Establishments in the Bretagne and Loire). Collaborations with our colleagues from Nantes have been ongoing for a long time, and Nantes and Rennes have truly independent research identities. The biology research in Nantes is mainly oriented towards the hospital, with extensive transfers like the Institut de Recherche en Santé which hosts several INSERM laboratories. In Rennes there are two main research intitutes in biology: the IRSET, which is public health-oriented, and the IGDR, which is basic research-oriented. Both local institutes complement one another well, and should go well with Nantes, and the local and regional compatabilities will be cultivated and maintained. The IGDR also collaborates with laboratories in Brest, Roscoff and Angers. The facilities directed by IGDR members participate in the IBISA GIS and also in the Western France life sciences and environment core facility network Biogenouest GIS. The IGDR also participates in the "Canceropole Grand Ouest." This partnership will be further developped as the IGDR is the main research institute taking part in the FHU CAMIn (Programme Investissement d’Avenir) dedicated to cancer.

National (France) The IGDR is now one of the main French reseach institutes for basic research. Team members collaborate with most of the major institutes in France. Members of the IGDR are involved in many committees that manage research in France, including the IUF, CNRS, scientific societies, charity organisations (ARC, LNCC), the French National Research Agency (ANR), etc. We will increase participation in the administration of research by encouraging people to apply to such committees.

International (Europe/World) The working langage for all IGDR conferences is English. We are motivated to recruit foreign PhD and post-doctoral students. Members of the IGDR will be encouraged to apply for all European grants, especially for the European Research Council. Collaboration with European laboratories is already ongoing, such as for PhDs under joint supervision. Further cooperation with foreign laboratories is encouraged. CNRS International Projects for Scientific Cooperation (PICS) applications are also promoted as the first step toward creating an International Associated Laboratory.

IGDR SWOT STRENGTHS - The excellence of our research. - We have great expertise in genetics, cell biology, developmental biology and structural biology. - We have great expertise in microscopy. - Our attractivity of the IGDR. - We have many of young dynamic group leaders. - Team projects are evaluated by a SAB. WEAKNESSES - Too little local recognition of our excellence. - The IGDR is located on two sites leaving 3 isolated teams. The unification of all the teams of the IGDR on the Health Sciences campus was promised by the university in 2010 but it has not happened. - There is a shortage of space. This led us to reject several applications from group leaders who wanted to set up research groups in the IGDR (ATIP and even ERC applicants). This also makes the IGDR the research institute with the highest density of researchers in Rennes. - Certain research groups continue to working as self-contained units, with little investment in the IGDR community. - International recruiting and grant applications are limited (although there are some individual initiatives). OPPORTUNITIES - Unifying the group on one campus will definitely strengthen the IGDR. Moving the anatomy laboratory that currently occupies the first floor of the main IGDR building would help. - Moving the electron microscopy facility from the Sciences campus to the Health one will increase the usage of the technology. - The acquisition of a new state-of-the-art TEM machine and camera will greatly improve our work and expertise.

42

- In Rennes, We are paving the way for the creation of a Research Center of Excellence for Biology that has no equivalent in the West of France. THREATS - Like many labs in France, funding difficulties are a serious threat to the institute’s continuing success. - Inequality in team funding raises questions about the IGDR’s model wherein 20% of each grant received is retained to run the IGDR. Some of the teams have very low financial resources and are thus not supporting the IGDR financially. - Space is a huge issue. It has become urgent that all the teams of the IGDR are regrouped together. We also urgently need more space to host new research teams. - The lack of technical support is a major issue. Research groups are working without technical support, the IGDR administration is working on a just-in-time basis (an additional technician is needed), the EM facility on the Sciences campus has no engineer, the Xenopus facility has no technician, one of the washing facilities has no technician, etc.

1.7.3. Scientific Objectives

TO EXPLORE THE REGULATION, DYNAMICS AND ROBUSTNESS OF THE DIVISION AND IDENTITY OF THE CELL.

1.7.4. Scientific guidelines and strategic choices

- Encourage technological development around microscopy (photonics and EM) Move research teams and facilities to the Health campus Buy a new TEM with camera

- Continue to support interdisciplinary approaches New recruited team members must prove interdisciplinary interests

- Develop organoids and gene editing approaches New recruited team members will have to use such approaches

- Develop a bio-informatics facility (will need to recruit more people) Set up a facility and ensure that team engineers share their knowledge and expertise

- Recruit an senior team member (through an external call for applications) Obtain lab space that is attractive enough to attract a senior group leader

- Encourage international initiatives (PhD programs, PICS, Erasmus)

- Recruit an Institute-wide lab manager for global technical assistance

1.7.5. Scientific guidelines and strategic choices

The IGDR is deeply committed to the conception of innovative multidisciplinary approaches. Our research will combine structural aspects, physics, mathematics and digital technology to work towards a quantitative and dynamic understanding of life.

Three major research programs will be developed:

1 - From molecule to cell (structure versus dynamics) Discover how does structure impact the function of a molecule, or a complex of several molecules? How does the plastic network of actors/proteins in dynamic interactions reflect the robustness of an organelle or the cell?

2 - From cell to organism (an identity case) Understand how a single cell gives rise to a whole organism, adapted to external constraints and formed by the interplay of hundreds of different cell types?

3 - From genes to cancer and genetic diseases Find genetic predispositions and alterations of causality; understand their individual and collaborative impacts on the genesis and development of genetic diseases. How does a cell become 43

cancerous, how can we detect it and how can we eliminate it?

Some diseases and conditions that will be studied: Acute lymphoblastic leukemia B (V. GANDEMER); melanoma (M.-D. GALIBERT); glioma (J. MOSSER); VHL diseases (Y. ARLOT- BONNEMAINS); genetic diseases (C. ANDRÉ); myopathies (J.-F. HUBERT); holoprosencephaly (V. DAVID); infertility (G. HALET); cohesinopathies (E. WATRIN); ciliopathies (C. PRIGENT); and skin stability defects (L. PAILLARD).

Tools and approaches: the biology of tomorrow at the crossroads of knowledge. Interdisciplinarity will remain a priority for the IGDR. We will promote the development of translational research; high throughput sequencing and data analysis; structural biology; quantitative biology; mechano-transduction; microfluidics; and simulations.

1.7.5.1. Industry and technology transfers

Transfer to Health Remaining in fundamental sciences but in the context of the development of personalized medicine and bed-to-bench and back approaches, organoids appear to be an important direction to go in. They could be transferred in personalized drug testing or else to hospitals and putatively to private companies (re-inforced by D1). Aspect of robustness and dynamics also appear to have a transfer potential. Studies on cancer or primary cells in their environment need strong contacts with hospitals: this is both an opportunity and a strength of the IGDR. For instance, here are some solid recognized experts at the Rennes CHU university hospital: - Melanoma (M.-D. GALIBERT); - Child acute leukemia (V. GANDEMER); - Glioblastoma (J. MOSSER); - Holoprosencephaly (V. DAVID).

Transfer to Industry Transfer of expertises/tools to industry and academia should be supported and encouraged. Some areas that are currently doing so include: - microfluidics with Cherry-Biotech (D. COUDREUSE); - microscopy with Photonlines (M. TRAMIER and J. PECREAUX); - Drosophila genetics with InDroso (R. LE BORGNE); - dog genetics with the PIA CRB ANIM (C. ANDRE). We can probably do a better job at this. It might be relevant to have a skilled person (“IP Officer” for instance) who is experienced with science and intellectual property. This person could raise awareness of our initiatives, and should have a good address book and an ease with company interaction. The IP Officer should make experience out of existing stuffs in the IGDR and aid the researchers/labs that want to transfer or valorize their research. The person must be chosen with special care, as previous attempts by CNRS to fill such a post resulted in failure.

Academic transfers We must encourage sharing and transfers within the IGDR at a minimum, and more broadly if possible. Indeed, high-level methodological research even when not readily transferable to industry is still of great interest for the Institute or for academic transfer.

1.7.5.2. Scientific goals

To think about the special features/strengths of the IGDR in order to set our goals, we can use various concepts: - Multi-scale approaches (from structure to organoids to organisms, through molecular assemblies and at the cellular level). This also includes space (diversity) and time (dynamics). - Multi-models (organisms). Indeed, it is clear for us that 2D-cultured cells are no longer the future of cancer biology. Instead we must use various model organisms, 3D cultured cells, organoids prepared from animals but also in a long term from patients to reflect the diversity of the cancers.

44

- Multi-disciplinary: think different! Not only as a biologist. Try to tackle a biological problem through multiple angles: • physics: measure physical aspects of a biological events (speed, force, time, level, etc.); • mathematics: use statistics, modelisation of biological events; • informatics: create virtual representations of biological events.

1.7.5.3. Management and running of the institute

Continue to recruit young leaders Reinforce the Bio-Cell/Bio-Dev aspect of the IGDR, particularly for the study of cancer in its environment (using stem cells, organoids, etc.) by recruiting a young leader in the field. Reinforce R&D in biology. The gap between fundamental science and project ready to be submitted for maturation funding (SATT / ANR /…) must be filled. Reinforce the electron microscope facility (BIOSIT). First by moving the Beaulieu teams to Villejean, which should stimulate several IGDR teams to think structural. The Institute will apply for several grants in order to get better equipment rapidly (for instance in-house access to a microscope equipped with a field emission gun and a direct electron detector). After getting such equipment, recruitment of a new team in the field should be easy.

Recruit an external senior team leader Consider the recruitment of an external senior team leader, with appropriate package, who will work to increase the IGDR’s visibility.

From basic science to technological transfer: improve the links Technological transfers and industry links are prominent in ANR, H2020 and other roadmaps. It is therefore important to ensure that the "basic research → proof of concept → development → transfer" continuum flourishes as a result of institutional support (including management directives, finances, space and recruiting practices). We insist again on the difficulty of making suitable “proofs of concept” to enable the leveraging of development funds.

Develop cooperation and team spirit among the different labs Thanks to the scientific programs that will be set up within the IGDR, meetings will join several labs around a question of interest (e.g. organoids, microscopy, cancer, genetic diseases, RNA, image processing, C. elegans models, cytokinesis, tissue mechanics). Departments could propose topics and initiate meetings. These get-togethers will re-enforce scientific sharing and cohesion in the institute without impinging on team freedom. Communal expenses and investment should be re-evaluated. All of the teams should be well- supported. We should also recognize anyone who contributes to the IGDR as a whole through a reputation for excellence, sharing of science or approaches/methods, supporting or developing of facilities, larger money contributions, scientific demonstrations, and so on. The Institute should also help teams that face temporary monetary difficulties. Encourage inter-team sharing of tools (microscopy, super-resolution, etc.) and approaches. Reinforce the PhD program and motivate all IGDR teams to participate according to their skills.

Attractiveness Make our institute attractive for high-level international PhD students and postdocs. This likely requires the establishment of a strong PhD program, social activities, and better welcoming of foreign post-docs to increase the added value of the time spent here. This goal is also related to the vitality of social activities and English-speaking habits.

Foreign researchers and post-doctoral students Better accompany the teams in welcoming foreign researchers and students. Help them through the maze of visas and other administrative complexities. Being able to offer guesthouse-like housing for one or more months is also very important.

45

1.7.6. Renewed partnerships The IGDR is a joint research unit under contracts with the CNRS and the University of Rennes 1. With the CNRS we are affiliated to sections 22 (cell biology, development, evolution- development, reproduction) and 21 (organisation, expression, evolution of the genomes; bioinformatics, systems biology). We would like to add section 24 (physiology, ageing, tumorigenesis). The partenership with the University of Rennes 1 will be reinforced. This will be done through the development of Master’s degree and PhD projects, the Erasmus program, student hosting and a close association to further develop the medical campus. The collaboration with the CHU teaching hospital will be boosted through the CAMIn project and stronger interactions with clinical departments.

1.7.7. New scientific topics In the near future, the IGDR wishes to promote three main research themes: - Cell division of stem cells; - Cancer research on organoids; - Structural biology. We wish to spread the word and recruit new teams to work on these topics. Invitations will depend entirely on the amount of lab space available from the university.

1.7.8. A new communications agenda Careers in research crystallized "the desire for knowledge, curiosity, imagination" It is important to support these values internally but also to insure a restitution of all the flavour through external communication. IGDR staffs have proposed some ideas and perspectives for improving the working environment and the radiation of the institute where PR could help. - Organize scientific cultural events The form: Public lectures involving scientists from the IGDR presenting on research topics or on more epistemological issues; educational exhibitions; events promoting links between the Arts and Sciences. Locally: The Espace des Sciences and the University Hospital of Rennes offer an annual program of lectures which could include IGDR research presentations; University of Rennes 1’s Diapason center offers a year-round program of scientific culture. - Promote the culture of sharing and dissemination Although it does not seem rooted in the culture of science in France, the dissemination of knowledge (to the students but also the general public) is now included in the missions of most research organizations, including the CNRS. Examples: Establish personal blogs; test innovative forms of knowledge sharing; propose participatory workshops. - Encourage exchanges with other structures and with professional sectors Exchange with private partners (partnerships with the socio-economic and cultural). Forms: Organize meetings or workshops. - Get involved in training It seems important to support students and young researchers in popularization and presentation giving, especially for oral communications. Examples: Organize workshops for students/young researchers; view and analyze oral conferences (such as TEDTalks). - Instill a sense of belonging to the institute; limit internal divisions Technicians and administrative personnel feel very "disconnected" from the work of researchers, even if they help them daily, which increases their sense of exclusion. Why do we work? Feeling involved in a broader mission stimulates motivation. The use of English isolates some workers. The form: Organize popular science activities internally; organize a retreat for all of the staff members (as advised by the SAB); set up one day a week where all personnel speak English in order to promote collective learning. - Assist in the writing of funding applications The search works today in the mode of "project" asks researchers to know to explain and convince that their work is worth funding. However, the different evaluation committees are not always

46

composed of experts in the application submittor’s field. It is therefore important for the researcher to get help, and the PR person could also help out. - Develop stock materials for the PR department Examples: The purchase of a digital photo camera and a digital video camera. - Content production and resource enhancement Examples: Capture audio and/or audiovisual for major conferences; regular creation of articles, video interviews, reports; these could also take a more illustrative form. - Establish a collaborative watch Examples: For contract offers in particular, applications, or legal issues... → Develop a differentiating and creative graphic identity Examples: through scientific event posters.

Conclusion:

IGDR is a major research institute dedicated to basic research in France. The main objectives for improving the dynamic of the Institute during the next contract will be:

At the scientific level: To carry on the current scientific projects mainly dedicated to cell and developmental biology and genetics in order to explore the regulation, dynamics and robustness of the division and identity of the cell.

Towards this aim, the IGDR will be structured around 19 research teams, 1 R&D team, 1 translational team and 1 administrative team.

The project will be supported through 3 equal interactive departments, run by 3 scientific supervisors: 1- From molecule to cell (structure versus dynamics) 2- From cell to organism (an identity case) 3- From genes to cancer and genetic diseases

Transdisciplinarity will remain a priority for the IGDR. Tasks will be performed in biology but approached through multiple other angles such as physics, mathematics, bioinformatics, etc.

47

At the organisational level: To reinforce the stability and attractivity of the Institute:

1) Stability: • Bring together all the IGDR teams on one campus as soon as possible. Indeed, three teams are still located on the east campus of Rennes, far from the core of the Institute. We expect to recover 700 square meters within the main IGDR building (#4), after the anatomy department moves in 2017. This will allow a unified building dedicated to IGDR, with the space for at least one new external team. • Accompany this movement by a restructuration of the microscopy facilities. For the first time, the transfer electron microscopes will be all located within the same MRIC platform, next to the IGDR building. • Improve the communications of the board with the Institute through regular meetings with all of the IGDR members. Organise retreats and group leader talks. Transparency and discussion at all levels is thus ensured. • Set up a shared bio-informatics facility within the IGDR. • Recruit a lab manager for global technical assistance. • Help and encourage collaborations between the teams within the Institute. • Convene the Scientific Advisory Board during the middle term and not so close to the end of the contract.

2) Attractiveness • Support the scientific strategy of the Institute by continuing external recruiting through requests for proposals. We will particularly reinforce the development of structural biology and cell biology, particularly cancer (stem cells/organoids), and will continue to emphasize multidisciplinary approaches. • Increase the visibility of IGDR on the campus through better signage and eventually with an upgraded building facing. • Increase the connections of the IGDR with the hospital (translational research; FHU CAMIn), the University of Rennes 1, and with the other research institutes in and out of Rennes. • Encourage international initiatives (PhD program, PICS, Erasmus, etc.) and the organisation of scientific events (Frontiers in Biology, etc.). • Encourage technological developments within the IGDR and transfers outside it. Reinforce the existing shared facilities. • Continue communication efforts, especially through the new website; improve social cohesion of the staff; continue to use English at scientific events.

1.7.9. List of Researchers and Academics for the next contract (Annex 15)

Name First name Position Employer Team BLANCO Carlos PR1 U RENNES 1 E8 BOUJARD Daniel PR1 U RENNES 1 E8 DAVID Véronique PUPH1 U RENNES 1 - CHU E5 DELIGNIERES-GANDEMER Virginie PUPH2 U RENNES 1 - CHU E6 DUPUY Alain PUPH2 U RENNES 1 - CHU E6 ERMEL Gwennola PR2 U RENNES 1 E8 GALIBERT-ANNE Marie-Dominique PUPH2 U RENNES 1 - CHU E6 GILLET Reynald PR2 U RENNES 1 E8 HARDY Serge PR2 U RENNES 1 E15 HUBERT Jean-François PR2 U RENNES 1 E10 LIEVRE Astrid PUPH2 U RENNES 1 - CHU E14 MOSSER Jean PUPH2 U RENNES 1 - CHU E14 ODENT Sylvie PUPH1 U RENNES 1 - CHU E5 PAILLARD Luc PR2 U RENNES 1 E15 SALBERT Gilles PR1 U RENNES 1 E18

48

BOUSSEMART Lise MCUPH2 U RENNES 1 - CHU E6 CHARTRAIN Isabelle MCF U RENNES 1 E17 DE TAYRAC Marie MCUPH2 U RENNES 1 - CHU E5 DELALANDE Olivier MCF U RENNES 1 E10 DESCHAMPS Stéphane MCFHC U RENNES 1 E15 DUBOURG Christèle MCUPH2 U RENNES 1 - CHU E5 DUCHESNE Laurence MCF U RENNES 1 E3 GAUTIER-COURTEILLE Carole MCF U RENNES 1 E15 GILOT David MCF U RENNES 1 E6 GIUDICE Emmanuel MCF U RENNES 1 E8 GOUDE Ronan MCF U RENNES 1 E8 HUET Sébastien MCF U RENNES 1 E18 LE BRAS Stéphanie MCF U RENNES 1 E12 LE CUNFF Yann MCF U RENNES 1 E16 LE GOFF-GAILLARD Catherine MCF U RENNES 1 E2 LE PERON Christine MCF U RENNES 1 E18 LERIVRAY Hubert MCFHC U RENNES 1 E15 MONNIER Annabelle MCF U RENNES 1 E14 OMILLI Francis MCFHC U RENNES 1 E17 QUIGNON Pascale MCF U RENNES 1 E1 RICHARD-PARPAILLON Laurent MCF U RENNES 1 E7 TRAUTWETTER Annie MCF U RENNES 1 E8 ARLOT BONNEMAINS Yannick DR2 CNRS E2 CHRETIEN Denis DR2 CNRS E3 GIET Régis DR2 CNRS E7 KUBIAK Jacek DR2 CNRS E17 LE BORGNE Roland DR1 CNRS E12 PRIGENT Claude DR1 CNRS E17 ANDRE Catherine CR1 CNRS E1 AUDIC Yann CR1 CNRS E15 BOUVRAIS Hélène CR2 CNRS E16 CHESNEL Franck CR1 CNRS E2 COUDREUSE Damien CR1 CNRS E4 DERRIEN Thomas CR2 CNRS E1 EOT-HOULLIER Grégory CR2 CNRS E11 HALET Guillaume CR1 CNRS E9 LE GOFF Xavier CR1 CNRS E2 MAGNAGHI-JAULIN Laura CR1 CNRS E11 MEREAU Agnès CR1 CNRS E15 METIVIER Raphaël CR1 CNRS E18 MICHAUX Grégoire CR2 CNRS E13 PECREAUX Jacques CR1 CNRS E16 PINOT Mathieu CR2 CNRS E12 TASSAN Jean-Pierre CR1 CNRS E17 TROADEC Marie-Bérengère CR1 CNRS E6 WATRIN Erwan CR2 CNRS E17 WU Pei-Yun CR1 CNRS E20 JAULIN Christian DR2 INSERM E11 BENAUD Christelle CR1 INSERM E17 CORRE Sébastien CR1 INSERM E6 DUPÉ Valérie CR1 INSERM E5 GUERRIER Daniel CR1 INSERM E9 MONIER-TSCHOPE Solange CR1 INSERM E11 PACQUELET Anne CR2 INSERM E13 RABUT Gwénaël CR1 INSERM E21

49

Team 1 "Canine genetics" Leader: Catherine ANDRE

51

2.1. Team presentation

The "Canine genetics" team led by Catherine ANDRE is composed of 15 persons (geneticists, veterinarians, bioinformaticians, technicians, Post-doc, students...). The team works hard since 20 years on human/dog homologous genetic diseases and on the analysis of the canine genome (mapping, sequencing, genome annotation, CNV catalogue...) and on setting a National canine sample biobank. The objective being to precisely characterize natural models of human genetic diseases in dogs, to propose homologous models of the diseases and gain knowledge on genetics and therapies benefitting both biomedical and veterinary medicine. The team is highly enriched by a unique expertise on the canine genome and development of NGS analyses. The team also created and developed a National Canine sample biobank (Cani-DNA). The biobank is part of a National funding and structure PIA1 (2008-2020) "Programme Investissement d’Avenir" (projet CRB-Anim), with an international visibility. Owing to the development of these two specific expertises, the team has developed numerous projects, from monogenic rare diseases in dermatology, neurology, ophthalmology... to complex diseases, such as cancers. The past 5 years were dedicated to the characterization of the canine disease models and the team made major findings in dermatology conditions (with the identification of the same altered genes in humans), and in cancers (demonstrating identical and novel genetic alterations in melanomas, sarcomas). Such results, based on an efficient and dynamic team, an AERES 2010 A+ evaluation, secured fundings for 2012-2017 and a strong support of IGDR and CNRS, now constitutes a solid basis with several relevants canine models and dedicated local, national and international collaborations in the medical and veterinary fields. The team objectives for the next years are to decipher the genetic features of the studied diseases (predisposing genes, somatic alterations, gene/environment interactions) and to propose therapeutic targets, and/or pronostic markers and genetic tests to the medical and veterinarian communities.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

ANDRE Catherine CR1 CNRS BOTHEREL Nadine ATRFP1 UR1 DERRIEN Thomas CR2 CNRS HEDAN Benoît IR2 CNRS GALIBERT Francis PUEM UR1 HITTE Christophe IRHC CNRS QUIGNON Pascale MCU UR1 52

2.2.2.2. Temporary staff

CADIEU Edouard Post-doc BUNEL Morgane PhD student (2012-2015) PRIMOT Aline Post-doc CORREARD Solenne PhD student (2015-2018) WUCHER Valentin Post-doc LE BEGUEC Céline PhD student (2015-2018) DE BRITO Clotilde Vétérinaire RAUL Mélanie PhD student (2013-2016) GUILLORY Anne-Sophie Vétérinaire ULVE Ronan PhD student (2013-2016) LAGOUTTE Laetitia IE

2.2.3. Achievements

2.2.3.1. Scientific achievements

Since 2010, the canine model grew in interest in the scientific and medical communites, as original alternative natural models of human genetic diseases, for comparative pathology, genetic and more recently therapeutic objectives.

We already characterized several natural dog breeds models for dermatological conditions: ichthyosis for which we identified a new gene and a new function in human ARCI ichthyosis (Grall et al., 2012) and keratoderma for which we identified the same mutated gene in dogs and human FNEPPK keraoderma (Plassais et al., 2014). The identification of a novel gene and function for ichthyosis, published in Nature Genetics 2012, gave us the opportunity to be highlighted in French medical journals, in the National Press (les Echos, Le Monde…) and by a comment in Science, in addition to selected talks in national and international meetings.

We also characterized several dog breeds models for oncology, such as histiocytic sarcoma, melanomas, lymphomas or gliomas, with several published articles, the 5 past years, mainly dedicated to the definition of homologies of these cancers between dogs and humans and their genetic characteristics: Histiocytic sarcoma: Abadie, Hedan et al., 2009; Hedan et al., 2011, Shearin, Hedan et al., 2012; Melanoma: Gillard et al., 2014; Hitte et al., in prep; de Brito et al., in prep. For the “Melanoma paper” in PCMR, we designed the cover page.

For this research to be efficient and independant, we setteled a solid expertise in the canine genome bioinformatics and developped a canine sample resource, the Cani-DNA Biobank (involving over 5000 French vets, the 4 National Vet Schools and the French Antagene Company). We created many working groups between human and veterinary cliniciens at the national and international levels, with many collaborations. We also secured fundings with US (NIH 2005-2010), European (7eme PCRD 2008-2012) and more recently French funding (INCa 2012-2015; PIA1 2012-2020). In addition, we had the great chance to recruit 3 persons years 2011-2012: Nadine BOTHEREL (research Assistant UR1, a team change), Benoît HÉDAN (a veterinarian, research engineer CNRS) and Thomas DERRIEN (researcher CNRS). This attractivity and trust from CNRS allowed the team to work with a better organization and efficiency.

All genetic diseases research projects are based on the same pipeline (below): sample and clinical data collection, through the Cani-DNA biobank; characterization of the canine disease (clinical, epidemiological, histopathological data) and comparative analyses to precisely define the homologies with the human disease, type and subtype; genetic analyses, through our unique expertise in canine genetics and genomics (genetic linkage analyses, whole genome association studies, NGS sequencing methods – whole genome, exome, locus re-sequencing...); once the genetic alteration(s) is/are found, we try to decipher the function of the gene if not already known and we search for its implication in the human homologous disease.

Flowchart: Analysis of canine genetic diseases:

53

In parallele of the research projects led on canine genetics, Francis GALIBERT (PUEM UR1), leads several projects on functional genomics and phylogenetics of Olfactory Receptors in fishes and mammals. These projects are funded by dedicated grants of Francis GALIBERT.

2.2.3.2. Scientific dissemination and influence

The team deals with numerous international and national collaborations between human genetics and veterinary medicine for each project: - For rare diseases: for dermatology with T. LEEB (Bern, switzerland), J. FISCHER and I. HAUSER (Germany), E. BOURRAT (CHU Paris) and E. GUAGUERE (Vet dermatologist) and GEDAC (Groupe Etude en Dermatology); for neuropathies with J.-M. VALLAT (CHU Limoge); E. LE GUERN (Pitié salpétirière ICM), V. DELAGUE and N. LEVY (CHU/INSERM Marseille) and C. ESCRIOU, J.-L. THIBAULT (Vet neurologists) and GEN (Groupe étude en Neurologie); for epilepsies with H. LOHI (Finland) and Pr. E. LE GUERN (Pitié salpétirière ICM); for retinopathies with C. HAMEL (CHU ) and G. CHAUDIEU (Vet Ophtalmologist and SFEROV and GEMO (Groupes etude en ophtalmologie). - For cancers: on histiocytic sarcoma with Dr. E. OSTRANDER (NIH, US); J. DONADIEU (CHU Paris), J.-Y. BLAY (CHU CRLCC Lyon), J.-F. EMILE (CHU Paris) and J. ABADIE (ENV Nantes) and P. DEVAUCHELLE (MICEN Vet) and GEO (Groupe etude en Oncologie); on Melanomas with M.-D. GALIBERT and T. LESIMPLE (CHU Rennes), F. DEMENAIS and E. MAUBEC (INSERM/CHU Paris) B. VERGER (CHU ), K. LINDBLAD-TOH (Broad Institute, US); on lymphomas with C. THIEBLEMONT and R. FARHEUS (CHU/INSERM Paris), K. TARTE (INSERM Rennes); on gliomas with J. MOSSER (CHU/INSERM Rennes, P. MENEI (CHU Angers) with Vets and GEO.

We, the team leader and team members, are regularely invited for working groups, symposiums, conferences on canine genetics and its interest in human genetic diseases. More recently, we gave 6 talks (out of 40 selected talks) at the “8th International Conference on Advances in Canine and Feline Genomics and Inherited Diseases”, Cambridge, UK.

2.2.3.3. Interaction with the economic, social and cultural environment

From our research results, we already produced 5 genetic tests for the veterinary side, in collaboration with the French animal genetics company, Antagene (Lyon), (all are being commercialized and one obtained an European patent in june 2014 / American patent still pending) and we are developing a genetic risk test for histiocytic sarcoma, the first genetic risk test available in veterinary medicine.

C. ANDRE is a member of the US national canine cancer biobank CCOGC (Canine Comparative Oncology and Genomic Consortium) and has created a Canine Genetics group, composed of 30 veterinarians at the French Association of Companion Animal Veterinarians, AFVAC, France; C. ANDRE is a member of the International Conference on Advances in Canine and Feline genomics and inherited

54

diseases, is an expert for AERES, genetic journals, grant applications, PhD committees... C. HITTE is an expert for ANR, AERES, France génomique and numerous genetic journals and PhD committees.

We are also sollicitated for large exposure conferences, such as: C. ANDRE: Canceropole Grand Ouest, Canceropole Ile de France, Ligue contre le Cancer Ille et Vilaine, Universite de tous les savoirs, Nantes Fevrier 2015; F. GALIBERT: Espace des Sciences de Rennes, 2010, C. ANDRE, 2016. C. HITTE, for the La Vilette Museum exhibition on cats and dogs. We also make numerous lay presentations in national and regional dog shows.

2.3. Projects, scientific strategies & perspectives (5 years)

The main research projects on cancers, and "rare" genetic diseases, will be pursued, organised in 2 branches with bioinformatics/statistics developments and the Cani-DNA Biobank. This organisation brings to the team a highly appreciable independence for the statistical analyses of our results and a continuous development and expertise in genomics/genetics/NGS, as well as an attractivity for the biobank, unique in France, with high quality samples collected, extracted (nucleic acids) and distributed, in the frame of our institutional collaborations.

The expertise and development of bioinformatics and statistics for canine genomic analyses consists in the detailed annotation of the canine genome (CNV catalogue, artificial selection genes, annotation through RNAseq …), sequencing analyses (NGS, exome, RNAseq, Whole genomes, locus re- sequencing), is led by Christophe HITTE recently joined by Thomas DERRIEN, with 1 post-doc and 1 or 2 students. They develop their own research subjects and actively and efficiently participate to the genetic projects of the team, bringing continuous cross expertises and positive critical discussions.

The management and development of the Cani-DNA biobank, is led by Laetitia LAGOUTTE. The last 3 years were devoted to settle collaboration contracts with their scientific and financial sheets with the Antagene company and the 4 Vet Schools and organize the sample collection network. This was a heavy administrative task but now, guarantee Cani-DNA, a National scale and international visibility and attractivity. In addition, we will upgraded our database to a novel “human CRB” type database (LIMS), and we will re-organized Cani-DNA following a process management model to target the dedicated CRB quality certification NF 96900 in 2016/2017. To this aim, we recruit Florent ROLLIN, expert in CRB, to manage the quality processes and database migration.

With such expertises, resources and organisation, we achieved major findings in oncology and monogenic diseases the last 5 years and plan to continue these projects, along with others highly promising models:

1. Rare diseases (P. QUIGNON, A.-S. GUILLORY, M. BUNEL, S. CORREARD, who obtained a PHD funding for 2015/2016): We identified the genetic bases of genodermatoses, rare in humans and highly frequent in dog breeds: an ichthyosis form in the Golden retriever (Grall et al., 2012) and a keratoderma form in the Dogue de Bordeaux breed (Plassais et al. 2014). We will focus on other ichthyoses and keratoderma types in several canine breeds, on neuropathies, epilepsies, and other neurosensoriel defects… for which we have already collected samples and clinical data. The past success stories with genodermatoses showed how the collaboration between different disciplines (clinicicians, histopathologists, bioinformatics), can be efficient and how the dog model is powerful to identify new genes, new functions and transfer to the human homologous conditions. We are aware and ready to adapt genetic stratégies with NGS methods and already launched projects by Whole Genome sequencing of trios. We also launched a new project on a peripheral neuropathy in hunting dog breeds, homologous to Human Hereditary Sensory Autonomous Neuropathies (HSAN), we’ve collected case and control dogs, identified a locus with an extremely high statistical significance, sequenced the locus and… more later !! (Plassais et al., in prep). Other diseases, such as several epilepsy forms in numerous dog breds, retinopathies, are being analysed and are led by Pascale QUIGNON.

2. Cancers: based on the cancer dog models for which we already characterized homologies between dogs and humans, we now plan to decipher their genetic characteristics and they therapeutic potential.

The main objectives are to pursue on:

- Melanomas (C. ANDRE, E. CADIEU, C. DE BRITO and recently A. PRIMOT and C. LE BEGUEC, PhD

55

student who obtained a PHD funding for 2015/2016, directed by C. HITTE on NGS aspects) Melanomas are frequent in dogs, spontaneously occurring and appear at the same localizations as in humans, i.e. skin, mucosal sites, nail matrix and eyes. In dogs, tumors at oral localizations are more frequent and aggressive than at other anatomical sites. Interestingly, dog melanomas are associated with strong breed predispositions and overrepresentation of black-coated dogs. We collected samples and clinical data from melanoma cases and controls and launched several genetic analyses (Exome sequencing, RNAseq and CGH). These analyses represent a unique opportunity to decipher the non-UV linked pathways of human melanomas and specifically the interactions between the genetic causes (exomes and RNAseq experiments are ongoing) and environmental aspects (microbiome of dog mouth, analyses of toxic substances in dog hairs are planed).

- Histiocytic sarcoma and lymphomas (B. HEDAN, and 2 PhD students: M. RAULT and R. ULVÉ) Histiocytic sarcoma, a rare cancer in humans for which no genetic data are known, but highly frequent in the Bernese Mountain Dogs breed with 20% of affected dogs dying from this cancer at the age of 6. We collected a hudge amount of samples (2000 DNAs, 500 cases with tumour/healthy samples in RNA later and formalin), characterized the disease on the clinical histological and epidemiological aspects (Abadie, Hedan et al 2010 ; Hedan et al., 2011). Due to our efforts, the clinical community is now getting organised through a medical network to collect human cases (J. DONADIEU, J.-F. EMILE and J.-Y. BLAY) and complete the poor knowledge on these dramatic multi- metastatic sarcomas. We identified predisposing genomic regions in dogs that have been re-sequenced, results are pending; these findings already allowed us to develop a genetic risk test recently available for breeders (SH pretest). Most recently, through RNAseq experiments, we identified highly relevant somatic driver mutations in targetable pathways, not yet discovered in such human sarcoma; the objective is to search these mutations in human well diagnosed histiocytic sarcoma. These results highlight the power of the dog model to identify the genetic bases of cancers and to propose relevant terapeutic targets. They will soon be submitted. For both projects, we will functionally validate the genetic alterations identified, through siRNA or shRNA of the mutated genes and proliferation tests, owing to the collaboration with M.-D. GALIBERT team, to demonstrate which pathway is involved in tumorigenesis in dogs and humans. When possible, we will use cell lines developped from canine tumours (4 cell lines of canine Histiocytic sarcoma set up by B. HEDAN) to test a first set of drugs prior to test them in dogs for clinical trials.

- Gliomas and lymphomas: We seriously plan to develop the just began and promising models of Gliomas (collaboration with J. MOSSER, IGDR, CHU Rennes) and lymphomas (collaboration with Biosit, team of K. TARTE INSERM, CHU Rennes, and the Biotrial company of Rennes and C. THIEBLEMONT, CHU Paris). Preliminary genetic results are very promising: the identification of gene fusions in 3 canine tumours (glioma, lymphoma and sarcoma) which particularity is to involve the same partner than in the human homologous cancer. This should rapidely be published to strengthen the power and relevance of the dog model in comparative oncology…. (Hedan et al.,in prep).

These projects are in perfect adequation with the local initiatives around the 2014 Labcom ANR funding, just settled by Biosit (T. GUILLAUDEUX) and the CAMIn FHU, joining CHU and research teams (led M.-D. GALIBERT and T. LAMY) on the medical campus of Rennes.

We plan to be part with INCa (French National Institute of Cancer) and the French medical and Vet community, clinical trial with relevant molecules identified through our comparative oncology results and genetic analyses. The objective of such projects are to identify altered genes and pathways in dog cancers and transfer to the human homologous cancers to plan clinical trials to screen new therapies, owing to the targets identified by our genetic analyses in tripartite collaborations with human and Vet clinicians, with “first in dogs” therapeutic trials and a mutual benefit for human and veterinary medicine.

3. Plans to improve our knowledges in canine genetic diseases and genomics and international visisbility:

- In the frame of the PIA1 funding 2012-2020, for Cani-DNA biobank, we plan to provide the community a high quality database and samples in order to increase its use and distribution in coordianation with the sample collection that will be performed during clinical trials for the biomedical community, in conjonction with INCa. The next objective is to find a possibility to gave a permanent position to Laetitia LAGOUTTE, presently contract engineer in the team, who is a great manager for Cani-DNA.

- We also plan to develop a national register on canine genetic diseases and anomalies, implemented

56

by veterinarians to increase the scope of the Cani-DNA biobank and the ability to dispose of canine equivalents of human conditions for many medical fields. In progress with a Web site dedicated to canine genetic diseases (clinical entries and human homologous diseases (initiative of Dr. C. EsCRIOU, DVM, Vet-Agro Sup and AFVAC, C. ANDRE being the genetic expert).

- Improve the canine genome annotation through the expertise of C. HITTE and T. DERRIEN, the discovery of novel functional and regulatory elements owing to the many RNAseq data analyses ongoing and to the strength of relevant and high quality canine samples from Cani-DNA (different tissues, breeds, age, pathologies, …).

- Continue to develop genetic tests for the canine community (Veterinarian, breeders, Kennel clubs...) for diagnosis and prédiction, in collaboration with animal genetics companies.

- Plan to welcome 1 or 2 new researchers with CNRS/INSERM positions, in the team and /or welcome a novel team to share all the expertise, tools, biobank and increasing national and international collaborations.

- Plan to organize in the period 2015-2020, a French/European "Canine model meeting".

2.4. Collaboration

2.4.1. Within the IGDR

With teams working on cancers: melanoma (M.-D. GALIBERT team: with a common INCa INSERM funding 2012-2015); glioblastoma (J. MOSSER team: with a CGO funding -2012-2013 and a pending INCa funding).

2.4.2. Other collaborations

Local collaborations: on lymphomas (K. TARTE team INSERM/CHU) and with the Biotrial company (CIFRE PhD program 2013-2016) ; for the Cani-DNA CRB with the CHU "human CRB" (common IBiSA funding in 2010).

Use of the Biosit platforms: H2P2, Impacell, Plateforme de cytometrie en flux et trie cellulaire sequencing Platform (J. MOSSER).

National and International Collaborations (see 232 chapter): Lots of collaborations since, for a given human genetic disease, I organize working groups composed of the human genetic disease experts, the veterinarians and geneticists, epidemiologists, histopathologists…

2.5. SWOT Analysis STRENGTHS The team: highly motivated, strong, positive and sensible collegues, in a friendly and inventive environement! The dog model : we provide unique opportunities because of an expertise with this model system. Strong potential of a unique just recognized model, novel alternative for experimental models The Cani-DNA resource (CRB): a national resource, internationally recognized and source of many collaborations Coupling in the same team the biological resources (Cani-DNA) and bioinformatics skills with strong expertise on the dog genome and continuous training in NGS analyses WEAKNESSES Dispersion, too many projects (too many diseases models ..), heavy charge of work for the whole team Lots of results, not enough papers ….

57

OPPORTUNITIES International visibility of the team and solid expertise with the Dog model, brings lots of collaboration from the scientific field, the medical and veterinarian fields Funding visibility and opportunities are good (FUI obtained, INCa grant pending, canine associations ...) THREATS No known threat expected since we collaborate and exchange samples with all known groups in Europe and US, working on canine genetics

58

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Abitbol M, Thibaud JL, Olby NJ, Hitte C, Puech JP, Maurer M, Pilot Storck F, Hédan B, Dréano S, Brahimi S, Delattre D, André C, Gray F, Delisle F, Caillaud C, Bernex F, Panthier JJ, Aubin- Houzelstein G, Blot S & Tiret L. A canine ARSG mutation leading to a sulfatase deficiency is associated with neuronal ceroid lipofuscinosis, PNAS, 2010, Aug 17;107(33):14775-80. (IF: 9,81) Beggs AH, Böhm J, Snead E, Kozlowski M, Maurer M, Minor K, Childers MK, Taylor SM, Hitte C, Mickelson JR, Guo LT, Mizisin AP, Buj-Bello A, Tiret L, Laporte J, Shelton GD. MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers. Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14697-702. doi: 10.1073/pnas.1003677107. Epub 2010 Aug 3. (IF : 9,81) Guyon R, Senger F, Rakotomanga M, Sadequi N, Volckaert FA, Hitte C, Galibert F. A radiation hybrid map of the European sea bass (Dicentrarchus labrax) based on 1581 markers: Synteny analysis with model fish genomes. Genomics. 2010 Oct;96(4):228-38. doi: 10.1016/j.ygeno.2010.07.007. Epub 2010 Jul 24. (IF :2,28)

2011 Benbernou N, Robin S, Tacher S, Rimbault M, Rakotomanga M, Galibert F. cAMP and IP3 signaling pathways in HEK293 cells transfected with canine olfactory receptor genes. J Hered. 2011 Sep-Oct;102 Suppl 1:S47-61. doi: 10.1093/jhered/esr033. (IF :2,09) Derrien T, Vaysse A, André C, Hitte C. Annotation of the domestic dog genome sequence: finding the missing genes. Mamm Genome. 2012 Feb;23(1-2):124-31. doi: 10.1007/s00335-011-9372-0. Epub 2011 Nov 11. (IF : 2,89) Galibert F, Quignon P, Hitte C, André C. Toward understanding dog evolutionary and domestication history. C R Biol. 2011 Mar;334(3):190-6. doi: 10.1016/j.crvi.2010.12.011. Epub 2011 Feb 1. (IF : 1,68) Hédan B, Thomas R, Motsinger-Reif A, Abadie J, André C, Cullen J and Breen M. Molecular cytogenetic characterization of canine histiocytic sarcoma: A spontaneous model for human histiocytic cancer identifies deletion of tumor suppressor genes and highlights influence of genetic background on tumor behavior. BMC Cancer. 2011, May, 11. 201. (IF: 3,36) Lequarré AS, Andersson L, André C, Fredholm M, Hitte C, Leeb T, Lohi H, Lindblad-Toh K, Georges M. LUPA: a European initiative taking advantage of the canine genome architecture for unravelling complex disorders in both human and dogs. Vet J. 2011 Aug;189(2):155-9. (IF: 2,17) Vaysse A, Ratnakumar A, Derrien T, Axelsson E, Rosengren Pielberg G, Sigurdsson S, Fall T, Seppälä EH, Hansen MS, Lawley CT, Karlsson EK; The LUPA Consortium, Bannasch D, Vilà C, Lohi H, Galibert F, Fredholm M, Häggström J, Hedhammar A, André C, Lindblad-Toh K, Hitte C, Webster MT. Identification of Genomic Regions Associated with Phenotypic Variation between Dog Breeds using Selection Mapping. PLoS Genet. 2011 Oct;7(10):e1002316. (IF: 8,17)

2012 Berglund J, Nevalainen EM, Molin AM, Perloski M; LUPA Consortium, André C, Zody MC, Sharpe T, Hitte C, Lindblad- Toh K, Lohi H, Webster MT. Novel origins of copy number variation in the dog genome. Genome Biol. 2012 Aug 23;13(8) (IF : 10,8) Grall A, Guaguère E, Planchais S, Grond S, Bourrat E, Hausser I, Hitte C, Le Gallo M, Derbois C, Kim GJ, Lagoutte L, Degorce-Rubiales F, Radner FPW, Thomas A, Küry S, Bensignor E, Fontaine J, Pin D, Zimmermann R, Zechner R, Lathrop M, Galibert F, André C & Fischer J. A PNPLA1mutations cause autosomal recessive congenital ichthyosis in Golden retriever dogs and humans. Nat Genet. 2012 Jan 15;44(2):140-7. (IF: 29,65) Guyon R, Rakotomanga M, Azzouzi N, Coutanceau JP, Bonillo C, D'Cotta H, Pepey E, Soler L, Rodier-Goud M, D'Hont A, Conte MA, van Bers NE, Penman DJ, Hitte C, Crooijmans RP, Kocher TD, Ozouf-Costaz C, Baroiller JF, Galibert F. A high-resolution map of the Nile tilapia genome: a resource for studying cichlids and other percomorphs. BMC Genomics. 2012 Jun 6;13:222. doi: 10.1186/1471-2164-13-222. (IF :3,99) Maurer M, Mary J, Guillaud L, Fender M, Pelé M, Bilzer T, Olby N, Penderis J, Shelton GD, Panthier JJ, Thibaud JL, Barthélémy I, Aubin-Houzelstein G, Blot S, Hitte C, Tiret L. Centronuclear myopathy in Labrador retrievers: a recent founder mutation in the PTPLA gene has rapidly disseminated worldwide. PLoS One. 2012;7(10): Oct 5. (IF : 3,53) Quignon P, Rimbault M, Robin S, Galibert F. Genetics of canine olfaction and receptor diversity. Mamm Genome. 2012 Feb;23(1-2):132-43. (IF: 2,89) Shearin AL*, Hedan B*, Cadieu E, Erich SA, Schmidt EV, Faden DL, Cullen J, Abadie J, Kwon EM, Gröne A, Devauchelle P, Rimbault M, Karyadi DM, Lynch M, Galibert F, Breen M, Rutteman GR, André C, Parker HG, Ostrander EA. The MTAP-CDKN2A Locus Confers Susceptibility to a Naturally Occurring Canine Cancer. Cancer Epidemiol Biomarkers Prev. 2012 Jul;21(7):1019-27. (IF : 4,32)

59

2013 Benbernou N, Esnault S, Galibert F. Activation of SRE and AP1 by olfactory receptors via the MAPK and Rho dependent pathways. Cell Signal. 2013 Jun;25(6):1486-97. doi: 10.1016/j.cellsig.2013.02.019. Epub 2013 Mar 22. (IF :4,32) Ollivier M, Tresset A, Hitte C, Petit C, Hughes S, Gillet B, Duffraisse M, Pionnier-Capitan M, Lagoutte L, Arbogast RM, Balasescu A, Boroneant A, Mashkour M, Vigne JD, Hänni C. Evidence of coat color variation sheds new light on ancient canids. PLoS One. 2013 Oct 2;8(10). (IF : 3,53) Steijger T, J.F. Abril, P.G. Engström, F. Kokocinski, J.F. Abril, M. Akerman, T. Alioto, G. Ambrosini, S.E. Antonarakis, J. Behr, P. Bertone, R. Bohnert, P. Bucher, N. Cloonan, T. Derrien, S. Djebali, J. Du, S. Dudoit, P.G. Engström, M. Gerstein, T.R. Gingeras, D. Gonzalez, S.M. Grimmond, R. Guigo, L. Habegger, J. Harrow, T.J. Hubbard, C. Iseli, G. Jean, A. Kahles, F. Kokocinski, J. Lagarde, J. Leng, G. Lefebvre, S. Lewis, A. Mortazavi, P. Niermann, G. Rätsch, A. Reymond, P. Ribeca, H. Richard, J. Rougemont, J. Rozowsky, M. Sammeth, A. Sboner, M.H. Schulz, S.M.J. Searle, N.D. Solorzano, V. Solovyev, M. Stanke, T. Steijger, B.J. Stevenson, H. Stockinger, A. Valsesia, D. Weese, S. White, B.J. Wold, J. Wu, T.D. Wu, G. Zeller, D. Zerbino, M.Q. Zhang, T.J. Hubbard, R. Guigo, J. Harrow, P. Bertone, Assessment of transcript reconstruction methods for RNA-seq. Nat Methods 2013 Dec;10(12):1177-84.

2014 Azzouzi N, Barloy-Hubler F, Galibert F. Inventory of the cichlid olfactory receptor gene repertoires: identification of olfactory genes with more than one coding exon. BMC Genomics. 2014 Jul 11;15:586. doi: 10.1186/1471-2164-15-586. (IF :3,99) Brawand D, Wagner CE, Li YI, Malinsky M, Keller I, Fan S, Simakov O, Ng AY, Lim ZW, Bezault E, Turner-Maier J, Johnson J, Alcazar R, Noh HJ, Russell P, Aken B, Alföldi J, Amemiya C, Azzouzi N, Baroiller JF, Barloy-Hubler F, Berlin A, Bloomquist R, Carleton KL, Conte MA, D'Cotta H, Eshel O, Gaffney L, Galibert F, Gante HF, Gnerre S, Greuter L, Guyon R, Haddad NS, Haerty W, Harris RM, Hofmann HA, Hourlier T, Hulata G, Jaffe DB, Lara M, Lee AP, MacCallum I, Mwaiko S, Nikaido M, Nishihara H, Ozouf-Costaz C, Penman DJ, Przybylski D, Rakotomanga M, Renn SC, Ribeiro FJ, Ron M, Salzburger W, Sanchez-Pulido L, Santos ME, Searle S, Sharpe T, Swofford R, Tan FJ, Williams L, Young S, Yin S, Okada N, Kocher TD, Miska EA, Lander ES, Venkatesh B, Fernald RD, Meyer A, Ponting CP, Streelman JT, Lindblad-Toh K, Seehausen O, Di Palma F. The genomic substrate for adaptive radiation in African cichlid fish. Nature. 2014 Sep 18;513(7518):375-81. doi: 10.1038/nature13726. Epub 2014 Sep 3. (IF :41,46) Chaudieu G, Olivier A, Thomas A, Bunel M, Albaric O, Lafont E, Quignon P, André C. Atrophie progressive de la rétine du Border Collie : étude rétrospective (1996-2012). PMCAC, Revue vétérinaire clinique. 09/2014; 49(3). Gillard M*, Cadieu E*, De Brito C, Abadie J, Vergier B, Devauchelle P, Degorce F, Dreano S, Primot A, Dorso L, Lagadic M, Galibert F, Hédan B, Galibert MD, and André C. Naturally occurring melanomas in dogs as relevant models for human melanomas. Pigment Cell & Melanoma Research 2014, 2014 Jan;27(1):90-102. (IF : 5,64) Plassais J, Guaguère E, Lagoutte L, Guillory AG, Dufaure de Citres C, Degorce-Rubiales F, Delverdier M, Vaysse A, Quignon P, Bleuart C, Hitte C, Fautrel A, Kaerle C, Bellaud P, Bensignor E, Queney G, Bourrat E, Thomas A* and André C*. A spontaneous KRT16 mutation in a dog breed: a model for human Focal Non Epidermolytic Palmoplantar Keratoderma (FNEPPK). J Invest Dermatol, 2014 Dec 18. (IF : 6,37)

2015 Azzouzi N, Barloy-Hubler F, Galibert F. Identification and characterization of cichlid TAAR genes and comparison with other teleost TAAR repertoires. BMC genomics. 2015 Apr 23;16:335. doi: 10.1186/s12864-015-1478-4. (IF :3,99) Broeckx B.J.G, Hitte C, Coopman F, Verhoeven G.E.C, De Keulenaer S, De Meester E, Derrien T, Alfoldi J, Lindblad- Toh K, Bosmans T, Gielen I, Van Bree H, Van Ryssen B, H. Saunders J, Van Nieuwerburgh F, Deforce D. Improved canine exome designs, featuring ncRNAs and increased coverage of protein coding genes. Scientific Reports, 2015. In press.

Edited books André C and Plassais J. Dogs in comparative pathology and genetics : examples of diseaes and genes shared between humans and dogs. Bull. Acad. Vet. France — 2012 - Tome 165 - N°3 : 215-224. Quignon P, Robin S, Galibert F. Canine olfactory genetics. In “The genetics of the dog”, 2nd edition, CAB International. 2012. Robin S, Quignon P, Galibert F. Génétique et évolution des récepteurs olfactifs chez les vertébrés. In “Odorat et Goût”, QUAE 2012. Cadieu E*, De Brito C*, Gillard M, Abadie J, Vergier B, Guillory AG, Devauchelle P, Degorce F, Lagoutte L, Hédan B, Galibert F, Galibert MD, and André C. Analyses comparées des mélanomes chez le chien et l’Homme. Bull. Acad. Vet. France — 2014

2. Patents (with licence) 1) International Patent November 17th 2009: Methods for diagnosing skin diseases Ref 03116-01 ANDRE et al. (Ref: VD/ACV BET 09P1294). European patent accepted in June 2014, US patent ongoing. 60

2) International Licence to ANTAGENE (Lyon France since April 2010).

3. Conferences (International & National)

Conferences: 2010 – Present Presentations of the research results at international scientific and veterinarians meetings (10 times / year at international meetings and 10 national meetings)

Selected papers: - National Veterinary AFVAC meetings (several presentations each year); - Assises de Génétique: 5th Strasbourg, Feb. 2010; 6th Marseille, Feb. 2012. Bordeaux 2014 - Canceropole Grand Ouest bisannual meetings, 2008, 2010, 2012, 2014; - European Society of Human Genetics, Amsterdam June 2011; - Internationl Lymphoma meeting, Lugano; 2013, 2015 - International Pigment Cell and Cancer IPCC, Bordeaux Sept. 2011; Lisbone Sept 2013, - Advanced on canine and feline genetics and genetic diseases, Baltimore, 2010; Visby (Sweeden) May 2012; Boston October 2013, Cambridge June 2015.

Invited speaker/keynote: - International Working Dog Congress, University of Pensylvania, Philidelphie, March 2010; - World Veterinary Cancer Congress, Paris March 2012; Vienna May 2014; - World Dermatology Veterinary Congress, Vancouver July 2012 - Académie Nationale Vérétinaire, Mars 2012, Fevrier 2014 - Académie Nationale de Médecine: Working groups on comparative oncology (dog – human): 2014 (Melanomas, lymphomas, gliomas) - International meeting of the American Society Melanoma Research, Philadelphie, November 2013 - Workshop INCa : Spontaneous canine models of cancers : sarcomas, lymphomas, melanomas, January 2014 - International Lymphoma meeting, Lugano; June 2015

- Presentations to breeders at National and European Kennel meetings (10 times /year). Bernese Mountain Dog, Dogue de Bordeaux, Berger des Pyrénées …

4. Funding

2007-2010 ARED DOGTUMOR, Région Bretagne 88,5 k€ 2008-2010 AKC Canine Health foundation US – Co-PI 50 k€ 2008-2010 RACP (Réunion des Amateurs de Chiens Pyrénéens) 15 k€ 2008-2011 Construction of a high-density Tilapia RH map- ANR - PI 500 k€ 2008-2011 French Guide Dogs Association - PI 500 k€ 2008-2012 7th PCRD European, LUPA – Cancer Melanoma WP leader 420 k€ 2010-2011 INCa, Biobank Cani-DNA - PI 200 k€ 2011 AKC Canine Health foundation US – Co-PI 15 k€ 2010-2011 Retina France Association, Post-doc (Estèle LAFONT) 30 k€ 2010-2012 Ligue Nationale contre le Cancer, Post-doc (Edouard CADIEU) 150 k€ 2010-2012 Eurostar European - Partner 60 k€ 2010-2013 Région Bretagne, ARED DERM-DOG (Jocelyn PLASSAIS) 90 k€ 2011-2012 Canceropole Grand Ouest - PI 32 k€ 2011-2013 Ligue Nationale Contre le Cancer 128k€ 2011-2013 IBISA 40 k€ 2012-2014 Aquipment Rennes Métropole (Pascale QUIGNON) 40 k€ 2012-2015 CNRS, PhD "Handicap" fellowship (Morgane BUNEL) 97 k€ 2012-2020 Investissements d’avenir (PIA1), CRB-Anim - Partner 1 800 k€ 2013-2015 INCa PLBio, rare tumors - PI 213 k€ 2013-2015 INCa PLBio, melanomas - Partner 127 k€ 2013-2015 INCa/INSERM, melanomas - PI 145 k€ 2013-2015 INCa/INSERM, lymphomas - Partner 103 k€ 2013-2015 Natural Environment Research Council (NERC) 3,4 k€ 2014-2015 AKC Canine Health foundation US – Co-PI 7 k€ 2015-2016 Canceropole Grand Ouest, spontaneous models of cancers - PI 70 k€ 2014-2016 CIFRE PhD fellowship with Biotrial (Ronan ULVE) - PI 90 k€ 2015-2017 FUI Lyon Biopole - Partner 200 k€

Funding Francis GALIBERT 2008-2010 DARPA - Etats unis 283 k€ 2009-2012 ANR - Programme Génomique Animale 150 k€ 2009-2010 INRA 25,6 k€ 2011-2015 DPI/CNRS 185 k€

61

5. Training Ecoles, Faculté de Médecine, IUT, BTS 2010-2013 - Amandine OLIVIER, Thèse vétérinaire (1 to 2 weeks/year) 2010 - Mélanie PAULMERY, 1e année BTS Bioanalyses et contrôles, Le Mans (2 months) 2011 - Morgane BUNEL, 4e année INSA de (3,5 months) 2012 - Chloé AZÉ, DUT Bioinformatique Rennes (2,5 months) 2013 - François LE TORTOREC, 4e année de médecine de Rennes (2 weeks) - Tifaine BOUFFARD, 5e année de médecine de Rennes (2 weeks) Licence 2010 - Jeanne GUIHOT, L3 biologie de Rennes (6 weeks) 2012 - Alix DUBOIS, L2 biologie de Rennes (4 weeks) - Rozenn RIOU, L3 biologie de Rennes (6 weeks) 2013 - Sophie BOISSEAU, L3 biologie de Rennes (6 weeks) 2014 - Claire DIETSCH, L1 biologie de Guingamp (1 month) 2015 - Erwan LE NEVE, L3 biologie de Rennes (6 weeks) - Kathleen SANTAMARIA, L3 biologie de Rennes (6 weeks) Master 2010 - Manuella LEVEQUE, M1 Sciences Moléculaires et Cellulaires de Rennes (2 months) 2011 - Ronan ULVÉ, M1 Sciences Moléculaires et Cellulaires de Rennes (2 months) - Kevin DRUET, M2 Bioinformatique de Rennes (6 months) - Natacha GUÉRIN, M2 European Master in Animal Breeding and Genetics de Agro ParisTech de Paris (6 months) 2012 - Mathilde LAINE, M1 Bioinformatique de Rennes (2 months) - Hillel JEAN-BAPTISTE-ADOLPHE, M2 Bioinformatique de Rennes (6 months) - Ambre-Aurore JOSSELIN, M2 Biologie Cellulaire Génétique Microbiologie Physiologie de Rennes (7 months) 2013 - Adrien SUDRAT, M1 Bioinformatique de Rennes (2 months) - Floriane ETHIS DE CORNY, M2 Bioinformatique de Rennes (6 months) - Mélanie RAULT, M2 biologie Biologie Cellulaire Génétique Microbiologie Physiologie de Rennes (5 months) 2014 - Alexandre NICAISE, M2 Bioinformatique de Rennes (6 months) 2015 - Charles VAN GOETHEM, M1 Bioinformatique de Rennes (4 months) - Geoffroy MALLARET, M1Génétique et Physiologie, Université Blaise Pascal de Clermont-Ferrrand (2 months) - Solenne CORREARD, M2 Génétique Biologie cellulaire et Pathologies de Lyon (6 months 3 weeks) PhD 21/11/2011: Marc GILLARD – Dir. Thèse Catherine ANDRE 16/12/2011: Amaury VAYSSE – Dir. Thèse Christophe HITTE 13/12/2013: Naoual SADEQUI – Dir. Thèse Francis GALIBERT 01/12/2014: Jocelyn PLASSAIS – Dir. Thèse Catherine ANDRE Since 11/2012: Morgane BUNEL – Dir. Thèse Catherine ANDRE & Pascale QUIGNON Since 07/2013: Ronan ULVÉ – Dir. Thèse Catherine ANDRE & Benoît HEDAN Since 10/2013: Mélanie RAULT – Dir. Thèse Christophe HITTE & Catherine ANDRE Starting 10/2015: Solenne CORREARD – Dir. Thèse Pascale QUIGNON Starting 10/2015: Céline LE BEGUEC – Dir. Thèse Christophe HITTE & Thomas DERRIEN Post-doctorants 2005-2011: Naima BENBERNOU, post-doctoral fellow 6 years & 3,5 months – Direction Francis GALIBERT 2006-2010: Mickaelle RAKOTOMANGA, post-doctoral fellow 4 years & 10 months – Direction Francis GALIBERT 2006-2011: Stéphanie ROBIN, post-doctoral fellow 5 years & 5 months – Direction Francis GALIBERT 2007-2011: Richard GUYON, post-doctoral fellow 4 years & 5 months – Direction Francis GALIBERT 2009-2011: Estèle LAFONT, post-doctoral fellow 2 years – Direction Catherine ANDRE 2009-2010: Matthieu LE GALLO, post-doctoral fellow 1 year & 7 months – Direction Catherine ANDRE 2009-2010/2011-2015: Edouard CADIEU, post-doctoral fellow 6 years & 6 months – Direction Catherine ANDRE 2010-2011: Thomas DERRIEN, post-doctoral fellow 10 months – Direction Christophe HITTE 2010-2012: Anaïs GRALL, post-doctoral fellow 1 year & 9 months – Direction Catherine ANDRE 2010-2012: Benoît HEDAN, post-doctoral fellow 2 years & 10,5 months – Direction Catherine ANDRE 2014-2015: Naoual SADEQUI, post-doctoral fellow 1 year & 2 months - Direction Francis GALIBERT 2014-2015: Aline PRIMOT, post-doctoral fellow 1 year – Direction Catherine ANDRE 2015: Valentin WUCHER, post-doctoral fellow 11 months - Direction Christophe HITTE CDD 2009-2012+ 2012-2015: Laëtitia LAGOUTTE - Engineer CNRS, contract 6 years - Direction Catherine ANDRE 2012-2015: Clotilde DE BRITO – Veterinary CNRS, contrat 3 years & 1 months - Direction Catherine ANDRE 2013-2014: Mathieu BAHIN - Engineer CNRS, contract 1 year & 2 months - Direction Christophe HITTE 2014-2016: Anne-Sophie GUILLORY – Veterinary CNRS, contrat 2 years - Direction Catherine ANDRE

62

Team 2 " Kidney cancer: molecular basis of tumorigenesis" Leaders: Yannick ARLOT-BONNEMAINS & Cécile VIGNEAU

63

2.1. Team presentation

Most of the cancers arise through the accumulation of multiple genetic alterations. The von Hippel–Lindau (VHL) tumor suppressor gene was the first gene identified for hereditary RCC that is now known to be involved in 60-70% of sporadic ccRCC. VHL inactivation has been established as an early and requisite step of ccRCC pathogenesis. Biallelic inactivation in the proximal renal tubular epithelial cells has been documented in early premalignant cysts of VHL patients and is also observed in the majority of sporadic ccRCC, establishing VHL as the critical gatekeeper of the renal epithelium integrity (Iliopoulos O et al., Proc. Natl. Acad. Sci USA. (1998) 95: 11661-11666). The VHL gene products (pVHL) have been implicated in multiple biological processes including cell growth and differentiation, extracellular matrix assembly, cell motility, invasion and physiological response to hypoxia. The understanding of the molecular mechanisms that regulate the function of the different forms of pVHL as an E3 ligase as well as characterizing their non-canonical functions in order to delineate pVHL role(s) in the process of tumorigenesis should therefore help to clarify the antitumor effects of pVHL and may provide useful informations concerning the identification of new molecular therapeutic targets.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

ARLOT-BONNEMAINS Yannick DR2 CNRS RIOUX-LECLERCQ Nathalie PU-PH1 UR1-CHU BELAUD-ROTUREAU M.-A. PU-PH2 UR1-CHU VIGNEAU Cécile PU-PH1 UR1-CHU CHESNEL Franck CR1 CNRS COUTURIER Anne IE2 CNRS LE GOFF Cathy MCU UR1 JOUAN Florence TCS UR1 LE GOFF Xavier CR1 CNRS

2.2.2.2. Temporary staff

HASCOET Pauline PhD student (2012-2016) PLADYS Adélaïde PhD student (2013-2016) KAMMERER-JACQUET Solène-Florence PhD student (2013-2016)

64

2.2.3. Achievements

2.2.3.1. Scientific achievements

MARTIN et al. Plos One 2013. We described and characterised for the first time the relationships between pVHL and the oncogenic Aurora-A protein kinase. We demonstrated that the functional interaction between Aurora-A and pVHL may participate in cell cycle regulation and in tumour initiation/progression through signalling pathway(s) that need to be further investigated.

In addition, we recently identified other proteins as putative new interactors of pVHL172, pVHL213 or both.

DUGAY et al. IJC 2014. We identified the polarity protein PAR-3 as a prognostic marker in renal cell carcinoma and showed functional studies through PARD3 knock-down experiments. We reported that PAR-3 protein overexpression in renal cell carcinoma tissue samples is correlated with poor survival. Functional studies on PAR3 led to the discovery of a role of this protein in the cellular architecture and polarity of the cell during transformation of renal proximal tubular cells.

CHESNEL et al. BJC 2015 - Patent N° 14305925.1 -1402-reference : BEP :130403EPDP Juin 2014. We generated and validated a specific pVHL monoclonal antibody which constitutes a powerful tool to better delineate the respective tumor-suppressive functions of the pVHL isoforms in the context of tumor development. Thus, this antibody can have potential utility in the cancer research field. The results are likely to spawn new research avenues on the role of VHL172 in the development, or prevention, of renal tumours.

DOLLEY HITZE et al 2013, Verhoest et al 2013. Differential analyses of patients who did or did not respond to anti-VEGF therapy are ongoing to identify biomarkers of response to therapy. Since we previously showed that Angiotensin II receptor type 1 (AT1R) expression in ccRCC is correlated to Furhman grade, we evaluated in an in vivo model, the synergic effect of AT1R inhibitor (telmisartan) in association with anti-angiogenic therapies. On sarcomatoid tumors, this association showed a beneficial effect on tumor necrosis. Complementary experiments are in progress on non sarcomatoid tumors.

2.2.3.2. Scientific dissemination and influence

Yannick ARLOT-BONNEMAINS: - Scientific leader of the BiChe Workshops (Biology and Chemistry) in the Biology and Health Federative Research Structure of Rennes - Member of the International Advisory Committee of the Ph.D program in “Endocrinological, Metabolic Andrologic and Reproductive Biotechnology “University la Sapienza - Rome 2014-2015 - Referent person for international relationships in the IGDR.

Xavier LE GOFF: Scientific leader for Signaling and CANcer workshops- Biology and Health Federative Research Structure of Rennes President of the Research Committee (Life Sciences, Agronomy and Health Doctoral school, Univ Rennes 1).

Cathy LE GOFF: Teaching leader for Licence (2nd year) of Sciences de la Vie et de la Terre (50 students, starting 2015) -Teaching leader of the Licence 2 Cell Biology module (300 students) at University of Rennes 1.

Franck CHESNEL: Member (elected) of the Life Sciences UFR Council - Vice-president of the "Career development and international mobility" committee of the VAS (Life Sciences, Agronomy and Health) Doctoral School (Univ of Rennes 1)

2.2.3.3. Interaction with the economic, social and cultural environment

Franck CHESNEL: - Participation to "Des élèves invitent des chercheurs" (organised by Rectorat d’Ille & Vilaine): Presentation of the careers in research in biology - CNRS: University programm courses objectives, staff...: 7H in 3 high schools in march/april 2010 - Participation to "Carrefour des métiers en collèges" (Half a day every year – collège La Binquenais Rennes): 2011 ; 2012, 2014 et 2015. -Supervision of pupils within the Institute (3-5 days each) as part of their compulsory internship exploration: tutoring of 5 pupils (level: 8th Gr) from 2010-2015 - Doctoral school VAS: organization of 1/ visits in local private companies (R&D departments) & 2/workshops with phD coming from industry and phD students from VAS.

65

Xavier LE GOFF & Cathy LE GOFF: Presentation of the careers in research in biology - CNRS: University programm courses objectives, staff... in a high school: 2012, 2013, 2015.

2.3. Projects, scientific strategies & perspectives (5 years)

Permanent staff (people who arrive in the team in Jan. 2017 are in red).

ABADIE Caroline PU CHU LE GOFF Xavier CR1 CNRS ARLOT-BONNEMAINS Yannick DR2 CNRS COUTURIER Anne IE2 CNRS CHESNEL Franck CR1 CNRS JOUAN Florence TCS UR1 LE GOFF Cathy MCU UR1

Caroline ABADIE (pediatric oncologist) is the head to the PREDIR Western center dedicated to the clinical follow up of VHL patients. Dr Patricia FERGELOT MD,PHD,HDR Molecular Genetic – CHU Bordeaux – will participate to the sceintifc discussion with the group as a consultant in oncology.

The team has been studying the biological functions of VHL in kidney tumorigenesis and plan to investigate the functions of the VHL protein isoforms and the impact of their mutations in VHL diseases including Renal cell Carcinoma.

The Von Hippel Lindau (VHL) disease is a rare autosomal-dominant hereditary neoplastic disease associated with germline loss-of-function mutations of the VHL gene. Patients affected by the VHL disease may develop both benign and malignant tumors including clear-cell renal cell carcinoma (ccRCC), retinal angioma, cerebellar and spinal hemangioblastoma, pheochromocytoma and pancreatic cancers. Patients with VHL disease usually harbour a single mutant allele, and tumour development depends on the spontaneous inactivation or loss of the second wild-type allele. Early evidence that the VHL gene is a TSG (tumor suppressor gene) came from studies of loss of heterozygosity (LOH) that showed that inactivation of both VHL alleles is a crucial event in the development of neoplasms in VHL disease and sporadic non- hereditary ccRCC.

In human, the VHL gene is located at position 3p25.3 and encodes two mRNA variants. The variant 1 encodes two protein isoforms, pVHL213 and pVHL160, that have been extensively documented in the literature. Variant 2 is produced by alternative splicing of exon 2 and encodes a pVHL isoform of 172 amino acids with a theoretical molecular weight of 19 kDa (pVHL172).

Studies about the expression, structure and function of pVHL172 and tumor induction in mice strongly suggest the involvement of this particular pVHL isoform in epithelial-mesenchymal transition (EMT) of cancer cells whereas the longer - pVHL213 – isoform is known to exert a tumor suppressing activity (Hascoet et al., 2015 in preparation). Moreover we detected both mRNA encoding pVHL213 or 66

pVHL172 in tumor tissues (ccRCC, breast) but with a variable ratio (unpublished data). Thus, it appears that an imbalance in the expression of the different pVHL213/172 isoforms may be crucial in cell malignant transformation. We will pursue the investigation of the pVHL172 function(s) in tumorigenesis.

Specific aims of the present research proposal are as follows: - to unravel the regulatory mechanisms driving the alternative splicing of the VHL pre-mRNA (sequences and associated proteins) leading to pVHL172 expression - to identify specific pVHL172 interacting proteins - to understand the molecular and cellular mechanisms underlying the functions of the pVHL isoforms in cells and induced tumors.

1- Analysis of the regulatory mechanisms driving the mRNA splicing of pVHL

Gene expression and alternative splicing, highly regulated and cell-type specific processes, have been found to be globally altered in cancer cells. Cancer-specific differences in alternative splicing offer an immense potential to improve the diagnosis and the prognosis of cancer. Cancer-specific changes in splicing profiles can result from mutations that are affecting splice sites and splicing control elements, or from alterations in the expression of proteins that control splicing decisions. Studies performed by the team highlighted the expression of two mRNA variants (variant 1 and variant 2) encoding respectively the isoform pVHL213 and pVHL172 in a ratio which fluctuates as a function of the pathological features in kidney, breast or thyroid. By screening for pathogenic genetic alterations in the ccRCC data base (CRB- Rennes), we identified 6 mutations at the intron1/exon2 junction and 7 mutations in the whole exon 2 of the VHL gene. We want to elucidate: (1) How the mutations at the intron/exon boundaries can affect the expression of the two variants? ; (2) How exonic sequence changes can alter pre-mRNA splicing directly disturbing natural splice sites (for example by creating 3’ or 5’ splice sites by introducing de novo sites or by activating or inhibiting pre-existing exon splicing enhancer or exonic splicing silencer? (3) How splicing is differentially regulated in normal and tumor cells?

In order to evaluate the impact of these mutations, we will perform functional assays based on the comparative analysis of the splicing pattern of a minigene containing WT or mutant VHL exon 2 flanked with 150-bp intronic regions. All mutant versions of VHL intron-exon boundaries or exon 2 will be prepared and analysed by RT-PCR for their expression in human transfected cells.

We will characterise the tissue specific splicing program for vhl by focusing on the molecular mechanisms that alter the splicing decisions in normal vs cancer cells. To investigate cancer cell specific alternative splicing, the association of proteins on the splicing sites of the premRNA will be evaluated in different types of cell lines (normal and tumoral) from kidney, breast, pancreas (tumoral) tissues. This work will be supported by the collaboration with L. Paillard (EGD Team; IGDR) and A. Martins (INSERM U1079; Rouen). In parallel, we will build up a comprehensive repertoire of potential splicing mutations in dedicated VHL international databases.

2- Identification of pVHL partners & post-translational modifications and analyses of VHL mutants

A search for new pVHLs partners has been undertaken through a comparative proteomic analysis (in collaboration with J.P. GAGNÉ/G. POIRIER, Quebec, Canada) with cells stably expressing pVHL172 vs pVHL 213 with the double objective of dissecting the mechanisms underlying pVHL172 -facilitating EMT and gaining better insight in the functions of pVHL172/pVHL213. About fourty proteins have been identified as putative interactors of pVHL172, pVHL213 or both. Identified protein partners of pVHL172 involved in cell adhesion and invasion will then be assessed (i) in vitro by biochemical approaches (pull- down, thermophoresis) after production and purification of the recombinant proteins; (ii) in cellulo through co-localization analyses by immunocytochemistry on fixed cells or bimolecular fluorescence complementation on live cells during cell cycle and migration.

In a previous study, we demonstrated Aurora-A as a novel interactor of pVHL213 that phosphorylates it on Ser72 (Martin et al., 2013). Our objectives are now to understand (i) the role of this post-translational modification on pVHLs function(s) and (ii) whether some mutations associated with VHL syndrome that do not impact canonical functions of pVHL213 may affect its phosphorylation (and subsequent function) by Aurora A.

We recently identified two aggregation-prone mutations in pVHL (Le Goff et al., submitted to Plos genet.). Mutations, which are predicted to alter pVHL stability or aggregation, will be first selected by in- 67

depth modelling studies together with the establishment of a comprehensive structural model of pVHL isoforms (collaboration with O. DELALANDE, IGDR). We will prioritize uncharacterized disease-associated mutations, especially located in exon 2 (leaving VHL172 intact) and/or at the vicinity of post- translationally modified residues. The effect of these mutations on pVHL folding will be assayed first in vitro using recombinant proteins and in cellulo. How the interaction with partners impacts pVHL folding/stability will be investigated by different approaches.

3- Molecular and cellular mechanisms underlying the functions of the pVHL isoforms in cells and induced tumors

Mutations in the vhl gene cause Von Hippel Lindau disease, a cancer predisposing syndrome characterized by a variety of benign and malignant neoplasms. The best documented functions of VHL is its E3 ubiquitin ligase activity that targets the alpha subunit of the hypoxia inducible factor (HIF-α) However VHL possesses multiple HIF-independent functions but it is not clearly established how the different isoforms as well as mutations of VHL contribute to the initation and or progression of tumor. Increased understanding may be relevant clinically either in terms of prognosis or in therapy selection.). We have already established cell lines stably expressing different isoforms of pVHL and generated tumor in mice. The tumor initiation and progression differ in term of kinetics of growth, dimensions and histological aspect (Hascoet et al., in preparation). Analysis of the resulting induced tumors will allow to characterize the role of this particular isoforms morphologically and biochemically. The tumors induced in mice will be recovered and RNA extracted for performing a transcriptomic analysis focused on the study of genes involved in cell cycle and epithelial-mesenchymal transition (Agilent). These studies will be initiated in parallel on tumors (derived either from 786-O or 786-O-pVHL172) as well as on initial cell lines, before xenograft. These analyzes will be performed on genomics platform (BIOSIT), the observed changes will be validated by RT-qPCR and Western blot and the results analyzed through the Pathway Studio Web software. s.

As well, specific mutations on VHL sequence will be selected in accordance to the clinical database of patients suffering from VHL disease (Dr ABADIE, CHU-Rennes) and the effect of these mutations of will be analysed on cell behaviour as well as on tumor initiation, and progression. Ultimately we will identify the biochemical pathways involved that could reveal new therapeutic target.

Conclusion

Our challenge is to analyse how the alternative splicing of the VHL pre-mRNA contributes to tumorigenesis by producing the pVHL172 spliced isoform and to understand the signals and mechanisms that govern VHL splicing regulation in order to evidence possible dysregulatory events.

The data and understanding of the balance pVHL213/172 in the function(s) of pVHL that we will collect in the fundamental approach will help to characterise more precisely the involvement of pVHL213/172 in tumorigenesis of VHL disease.

2.4. Collaboration

2.4.1. Within the IGDR

Olivier DELALANDE (VHL -Modelisation and docking with interactors) - Denis CHRETIEN (VHL and microtubules interactions) - Luc PAILLARD (VHL- splicing analysis) - Damien COUDREUSE (VHL homeostasis).

2.4.2. Other collaborations

Rennes Franck CAMEREL, FOURMIGUE Marc, University of Rennes (chemistry) (Angiogenesis inhibitors) – Caroline ABADIE, CHU Rennes (VHL-disease) - Fabrice MAHE (mathematical approaches).

France Amam GASSAMA, IGR, Paris, (cell polarity and PAR proteins) – Odile FILHOL-COCHET, CEA, (Kindey cancer inhbitors) – Jean-Jacques PATARD, Kremlin Bicetre Paris (kidney cancer) - Patricia FERGELOT, CHU Bordeaux (sequencage VHL).

68

Europe Ignacio PEREZ DE CASTRO, CNIO Madrid, Spain (VHL and Post translational modification) - Andrea KLOTZBUCHER, Proqinase Freiburg, Germany (VHL and kinases) - Salvatore W. ULISSE, University La Sapeinza Rome, Italy (Thyroid disease) – Per a SUNNERHAGEN, Sweden (protein quality control mutants).

Out of Europe Jean-Philippe GAGNE, CHUL, Quebec, Canada (VHL partners and isoforms) – Daniel G. CYR, Institut Armand Frappier Montreal, Canada (cell polarity).

2.5. SWOT Analysis STRENGTHS Strong tools (specific antibodies, stable cell lines, patient cancer cell lines) National and international collaborations (VHL alliance partnerships ) 3 full-time researchers (strong involvement and highly experienced) Strong skills in Multiple model organisms (tissue culture, mice, Yeast, Xenopus) Geneticists for VHL disease in CHU Rennes WEAKNESSES Absence of secure findings No publications in high impact factor journals yet OPPORTUNITIES Appropriate scientific local environment with: - Structuralists - expertise in modelling and docking - RNA expertise (splicing in particular) - Geneticists for VHL disease in CHU Rennes Multifactorial and rare disease Pioneer in VHL172 study THREATS The scarcity of phD students and post-doctorals trainees

69

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

1.1. International publications 2010-2012 before the creation of the team Kidney Cancer: Molecular Basis of Tumorigenesis

Within the team Cell Cycle (Claude PRIGENT) until Jan 1st 2012

2010 Baldini E, Arlot-Bonnemains Y, Mottolese M, Sentinelli S, Antoniani B, Sorrenti S, Salducci M, Comini E, Ulisse S, D'Armiento M Deregulation of Aurora kinase gene expression in human testicular germ cell tumours. Andrologia. 2010 Aug;42(4):260-7. (IF 1.17) Bouter A, Buisine N, Le Grand A, Mouchel N, Chesnel F, Le Goff C, Le Tilly V, Wolff J, Sire O Control of vitellogenin genes expression by sequences derived from transposable elements in rainbow trout. Biochim Biophys Acta. 2010 Aug;1799(8):546-54. (IF 4.6) Gohin M, Bobe J, Chesnel F Comparative transcriptomic analysis of follicle-enclosed oocyte maturational and developmental competence acquisition in two non-mammalian vertebrates. BMC Genomics. 2010 Jan 8;11:18. (IF 3.9) Ulisse S, Arlot-Bonnemains Y, Baldini E, Morrone S, Carocci S, Di Luigi L, D'Armiento M. Inhibition of the aurora kinases suppresses in vitro NT2-D1 cell growth and tumorigenicity. J Endocrinol. 2010 Feb;204(2):135-42. (IF 3.7)

2011 Baldini E, Arlot-Bonnemains Y, Sorrenti S, Mian C, Pelizzo MR, De Antoni E, Palermo S, Morrone S, Barollo S, Nesca A, Moretti CG, D'Armiento M, Ulisse S.Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT. BMC Cancer. 2011 Sep 26;11:411. (IF 3.3) Gabillard JC, Ulisse S, Baldini E, Sorrenti S, Cremet JY, Coccaro C, Prigent C, D'Armiento M, Arlot-Bonnemains Y. Aurora-C interacts with and phosphorylates the transforming acidic coiled-coil 1 protein. Biochem Biophys Res Commun. 2011 May 20;408(4):647-53. (IF 2.3) Gohin M, Bodinier P, Fostier A, Chesnel F, Bobe J. Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation. Mol Reprod Dev. 2011 Jul;78(7):510-8. (IF 2.7) Gohin M, Bodinier P, Fostier A, Bobe J, Chesnel F. Aromatase expression in Xenopus oocytes: a three cell-type model for the ovarian estradiol synthesis. J Mol Endocrinol. 2011 Sep 7;47(2):241-50. (IF 3.7)

Within the team Division and Cell Polarity (Jean-Pierre TASSAN) until Jan 1st 2012

2010 Cadou A, Couturier A, Le Goff C, Soto T, Miklos I, Sipiczki M, Xie L, Paulson JR, Cansado J, Le Goff X Kin1 is a plasma membrane-associated kinase that regulates the cell surface in fission yeast. Mol Microbiol. 2010 Sep;77(5):1186-202. (IF 5.02)

Within the team Renal Carcinoma (Jean-Jacques PATARD & Cécile VIGNEAU) until Jan 1st 2012

2010 Bensalah K, Fleureau J, Rolland D, Rioux-Leclercq N, Senhadji L, Lavastre O, Guillé F, Patard JJ, de Crevoisier R [Optical spectroscopy: a new approach to assess urological tumors]. Prog Urol. 2010 Jul;20(7):477-82. (IF 0.77) Bensalah K, Fleureau J, Rolland D, Lavastre O, Rioux-Leclercq N, Guillé F, Patard JJ, Senhadji L, de Crevoisier R.Raman spectroscopy: a novel experimental approach to evaluating renal tumours. Eur Urol. 2010 Oct;58(4):602-8. (IF 13.9) Bigot P, Lughezzani G, Bensalah K, Rioux-Leclercq N, Guillé F, Lobel B, Moulinoux JP, Catros-Quemener V, Cipolla B, Azzouzi AR, Karakiewicz P, Patard JJ. [Prognostic value of erythrocyte polyamines levels in renal cell carcinoma. Prospective study in 418 cases]. Prog Urol. 2010 Apr;20(4):272-8. (IF 0.77) Dolley-Hitze T, Jouan F, Martin B, Mottier S, Edeline J, Moranne O, Le Pogamp P, Belaud-Rotureau MA, Patard JJ, Rioux-Leclercq N, Vigneau C. Angiotensin-2 receptors (AT1-R and AT2-R), new prognostic factors for renal clear- cell carcinoma? Br J Cancer. 2010 Nov 23;103(11):1698-705.(IF 5.2) Edeline J, Vigneau C, Patard JJ, Rioux-Leclercq N. [Signalling pathways in renal-cell carcinoma: from the molecular biology to the future therapy]. Bull Cancer. 2010;97:5-15. (IF :0.63)

70

Lughezzani G, Karakiewicz PI, Bigot P, Perrotte P, Crépel M, Rioux-Leclercq N, Catros-Quemener V, Moulinoux JP, Bouet F, Cipolla B, Patard JJ. The prognostic value of erythrocyte polyamines in the preoperative evaluation of patients with renal cell carcinoma. Eur J Cancer. 2010 Jul;46(10):1927-35 (IF 4.82) Masson D, Rioux-Leclercq N, Fergelot P, Jouan F, Mottier S, Théoleyre S, Bach-Ngohou K, Patard JJ, Denis MG. Loss of expression of TIMP3 in clear cell renal cell carcinoma. Eur J Cancer. 2010 May;46(8):1430-7. (IF 4.82) Mathieu R, Patard JJ, Stock N, Rioux-Leclercq N, Guillé F, Fergelot P, Bensalah K. [Study of the expression of Aurora kinases in renal cell carcinoma]. Prog Urol. 2010 Dec;20(13):1200-5. (IF 0.77) Paparel P, Long JA, Baumert H, Meyer V, Escudier B, Grenier N, Hetet JF, Rioux-Leclercq N, Lang H, Poissonier L, Soulie M, Patard JJ.[Current role of lymph node dissection in renal cell carcinoma: review of the literature by the Oncology Committee of the French Association of Urology (CCAFU)]. Prog Urol. 2010 May;22(6):313-7. (IF 0.77) Patard JJ, Escudier B, Paparel P, Neuzillet Y, Long JA, Baumert H, Correas JM, Lang H, Poissonnier L, Rioux-Leclercq N, Soulié M; (CCAFU).. [Progress and summary of recent congress: ASCO-GU, EAU, AUA, ASCO about the medical management of locally advanced or metastatic kidney cancer]. Prog Urol 2010 Mar;20 Suppl 1:S11-5. (IF 0.77) Verine J, Lehmann-Che J, Soliman H, Feugeas JP, Vidal JS, Mongiat-Artus P, Belhadj S, Philippe J, Lesage M, Wittmer E, Chanel S, Couvelard A, Ferlicot S, Rioux-Leclercq N, Vignaud JM, Janin A, Germain S.Determination of angptl4 mRNA as a diagnostic marker of primary and metastatic clear cell renal-cell carcinoma. PLoS One. 2010 Apr 29;5(4):e10421. (IF 3.53)

2011 Eimer S, Belaud-Rotureau MA, Airiau K, Jeanneteau M, Laharanne E, Véron N, Vital A, Loiseau H, Merlio JP, Belloc F Autophagy inhibition cooperates with erlotinib to induce glioblastoma cell death. Cancer Biol. Ther. 2011; 11: 1017- 1027 (IF 3.6) Jaillard S, Loget P, Lucas J, Dubourg C, Le Bouar G, Demurger F, Bertorello I, David V, Poulain P, Odent S, Belaud- Rotureau MA. Terminal 6.9 Mb deletion of chromosome 15q, associated with a structurally abnormal X chromosome in a patient with congenital diaphragmatic hernia and heart defect. Eur. J. Med. Genet. 2011; 54: 186-188 (IF 3.5) Jaillard S, Andrieux J, Plessis G, Krepischi AC, Lucas J, David V, Le Brun M, Bertola DR, David A, Belaud-Rotureau MA, Mosser J, Lazaro L, Treguier C, Rosenberg C, Odent S, Dubourg C. 5q12.1 deletion: delineation of a phenotype including mental retardation and ocular defects . Am. J. Med. Genet. A. 2011; 155A: 725-7 (IF 2.05) Perrin C, Patard JJ, Jouan F, Collet N, Théoleyre S, Edeline J, Zerrouki S, Laguerre B, Belaud Roturaud MA, Rioux- Leclercq N -, Vigneau C.-[The neutrophil gelatinase-associated lipocalin, or LCN 2, marker of aggressiveness in clear cell renal cell carcinoma]. Prog Urol. 2011 Nov;21(12):851-8. (IF 0.77)

1.2. International publications 2012-2015 of the team Kidney Cancer: Molecular Basis of Tumorigenesis

2012 Charlier C, Montfort J, Chabrol O, Brisard D, Nguyen T, Le Cam A, Richard-Parpaillon L, Moreews F, Pontarotti P, Uzbekova S, Chesnel F, Bobe J. Oocyte-somatic cells interactions, lessons from evolution. BMC Genomics. 2012 Oct 19;13:560.. (IF 4.04) Edeline J, Mottier S, Vigneau C, Jouan F, Perrin C, Zerrouki S, Fergelot P, Patard JJ, Rioux-Leclercq N. Description of 2 angiogenic phenotypes in clear cell renal cell carcinoma. Hum Pathol. 2012 ov;43(11):1982-90. (IF 3.02) Eimer S, Dugay F, Airiau K, Avril T, Quillien V, Belaud-Rotureau MA, Belloc F. Cyclopamine cooperates with EGFR inhibition to deplete stem-like cancer cells in glioblastoma-derived spheroid cultures. Neuro Oncol. 2012; 14:1441- 1451 (IF 6.1) Jaglarz MK, Bazile F, Laskowska K, Polanski Z, Chesnel F, Borsuk E, Kloc M, Kubiak JZ. Association of TCTP with centrosome and microtubules. Biochem Res Int. 2012;2012:541906.. (IF 1.5) Leyme A, Bourd-Boittin K, Bonnier D, Falconer A, Arlot-Bonnemains Y, Théret N. Identification of ILK as a new partner of the ADAM12 disintegrin and metalloprotease in cell adhesion and survival. Mol Biol Cell. 2012 Sep;23(17):3461-72. (IF 4.6) Martin B, Edeline J, Patard JJ, Oger E, Jouan F, Boulanger G, Zerrouki S, Vigneau C, Rioux-Leclercq N. J Combination of Temsirolimus and tyrosine kinase inhibitors in renal carcinoma and endothelial cell lines. Cancer Res Clin Oncol. 2012 Jun;138(6):907-16. (IF 3.0) Pinot M, Steiner V, Dehapiot B, Yoo BK, Chesnel F, Blanchoin L, Kervrann C, Gueroui Z. Confinement induces actin flow in a meiotic cytoplasm. Proc Natl Acad Sci U S A. 2012 Jul 17;109(29):11705-10 (IF 9.8) T. Thi Phan, S. Abbour, F. Jouan Y. Arlot-Bonnemains,M. Baudy-Floc’h. Analogs of RGD as anti-angiogenic compounds. European Peptide Society, 2012. P482-483 G. Kokotos, V. Constantinou-Kokotou, J. Matsoukas (Editors) (IF 2.0) Verhoest G, Patard JJ, Fergelot P, Jouan F, Zerrouki S, Dreano S, Mottier S, Rioux-Leclercq N,. Denis M. Paraffin- embedded tissue is less accurate than frozen section analysis for determining VHL mutational status in sporadic renal cell carcinoma. Urologic Oncology: Seminars and Original Investigations 2012;30(4):469-75 (IF 3.3) 71

2013 Alekseeva L, Rault L, Almeida S, Legembre P, Edmond V, Azevedo V, Miyoshi A, Even S, Taieb F, Arlot-Bonnemains Y, Le Loir Y, Berkova N. Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency. PLoS One. 2013 May 23;8(5):e63279. (IF 3.6) Dagher J, Dugay F, Verhoest G, Cabillic F, Jaillard S, Henry C, Arlot-Bonnemains Y, Bensalah K, Oger E, Vigneau C, Rioux-Leclercq N, Belaud-Rotureau MA. Histologic prognostic factors associated with chromosomal imbalances in a contemporary series of 89 clear cell renal cell carcinomas. Hum Pathol. 2013 Oct;44(10):2106-15. (IF :2.8) Dolley-Hitze T, Verhoest G, Jouan F, Le Pogamp P, Arlot-Bonnemains Y, Oger E, Belaud-Rotureau MA, Rioux- Leclercq N, Vigneau C. Angiotensin-2 type 1 receptors (AT1R) and cancers]. Nephrol Ther. 2013 Apr;9(2):85-91. (IF 0.43) Cadou A, Couturier A, Le Goff C, Xie L, Paulson JR, Le Goff X. The Kin1 kinase and the calcineurin phosphatase cooperate to link actin ring assembly and septum synthesis in fission yeast. Biol Cell. 2013 Mar;105(3):129-48 (IF 3.87) Fergelot P, Bernhard JC, Soulet F, Kilarski WW, Léon C, Courtois N, Deminière C, Herbert JM, Antczak P, Falciani F, Rioux-Leclercq N, Patard JJ, Ferrière JM, Ravaud A, Hagedorn M, Bikfalvi A. The experimental renal cell carcinoma model in the chick embryo. Angiogenesis. 2013 Jan;16(1):181-94. (IF 4.4) Fievet A, Belaud-Rotureau MA, Dugay F, Abadie C, Henry C, Taque S, Andrieux J, Guyetant S, Robert M, Dubourg C, Edan C, Rioux-Leclercq N, Odent S, Jaillard S Involvement of germline DDX1-MYCN duplication in inherited nephroblastoma. Eur J Med Genet. 2013; 56:643-647 (IF 5.6) Launay E, Henry C, Meyer C, Chappé C, Taque S, Boulland ML, Ben Abdelali R, Dugay F, Marschalek R, Bastard C, Fest T, Gandemer V, Belaud-Rotureau MA. MLL-SEPT5 fusion transcript in infant acute myeloid leukemia with t(11;22)(q23;q11) Leuk Lymphoma. 2013; Aug 20. (IF 2.6) Martin B, Chesnel F, Delcros JG, Jouan F, Couturier A, Dugay F, Le Goff X, Patard JJ, Fergelot P, Vigneau C, Rioux- Leclerq N, Arlot-Bonnemains Y Identification of pVHL as a novel substrate for Aurora-A in clear cell renal cell carcinoma (ccRCC). PLoS One. 2013 Jun 13;8(6):e67071 (IF 3.53)

2014 Baldini E, Tuccilli C, Prinzi N, Sorrenti S, Antonelli A, Gnessi L, Morrone S, Moretti C, Bononi M, Arlot-Bonnemains Y, D'Armiento M, Ulisse S. Effects of selective inhibitors of Aurora kinases on anaplastic thyroid carcinoma cell lines. Endocr Relat Cancer. 2014 Oct;21(5):797-811. (IF 5.26) Blanchard M, Dubourg C, Pasquier L, Odent S, Lucas J, Quélin C, Launay E, Henry C, Belaud-Rotureau MA, Dugay F, Jaillard S. Postnatal diagnosis of 9q interstitial imbalances involving PTCH1, resulting from a familial intrachromosomal insertion. Eur J Med Genet. 2014; 57:195-199 (IF 1.79) Bouleau A, Desvignes T, Traverso JM, Nguyen T, Chesnel F, Fauvel C, Bobe J. Maternally inherited npm2 mRNA is crucial for egg developmental competence in zebrafish. Biol Reprod. 2014 Aug;91(2):43 (IF 3.94) Brisard D, Chesnel F, Elis S, Desmarchais A, Sánchez-Lazo L, Chasles M, Maillard V, Uzbekova S. Tribbles expression in cumulus cells is related to oocyte maturation and fatty acid metabolism. J Ovarian Res. 2014 Apr 26;7:44. (IF 2.04) Cabillic F, Gros A, Dugay F, Begueret H, Mesturoux L, Chiforeanu DC, Dufrenot L, Jauffret V, Dachary D, Corre R, Lespagnol A, Soler G, Dagher J, Catros V, Le Calve M, Merlio JP, Belaud-Rotureau MA. Parallel FISH and immunohistochemical studies of ALK status in 3244 non-small-cell lung cancers reveal major discordances. J Thorac Oncol. 2014 Mar;9(3):295-306. (IF 5.8) Dagher J, Dugay F, Rioux-Leclercq N, Verhoest G, Oger E, Bensalah K, Cabillic F, Jouan F, Kammerer-Jacquet SF, Fergelot P, Vigneau C, Arlot-Bonnemains Y, Belaud-Rotureau MA Cytoplasmic PAR-3 protein expression is associated with adverse prognostic factors in clear cell renal cell carcinoma and independently impacts survival. Hum Pathol. 2014 Aug;45(8):1639-46. (IF:2.8) Dugay F, Dagher J, Verhoest G, Henry C, Jaillard S, Arlot-Bonnemains Y, Bensalah K, Vigneau C, Rioux-Leclercq N, Belaud-Rotureau MA. [Cytogenetics profiles of renal carcinoma]. Morphologie. 2014 Mar;98(320):1-7. (IF 0.7) Dugay F, Le Goff X, Rioux-Leclerq N, Chesnel F, Jouan F, Henry C, Cabillic F, Verhoest G, Vigneau C, Belaud- Rotureau MA* and Arlot-Bonnemains Y*. (* corresponding author) Overexpression of the polarity protein PAR-3 in clear cell renal cell carcinoma is associated with poor prognosis. Int J Cancer. 2014 May 1;134(9):2051-60. (IF 5.07) Mebrouk K, Chotard F, Le Goff-Gaillard C, Arlot-Bonnemains Y, Fourmigué M, Camerel F Water-soluble nickel- bis(dithiolene) complexes as photothermal agents. Chem Commun (Camb). 2014 Nov 21.(IF 6.7) Parker D, Belaud-Rotureau MA. Micro-cost Analysis of ALK Rearrangement Testing by FISH to Determine Eligibility for Crizotinib Therapy: Implications for Cost Effectiveness of Testing and Treatment. Clinical Medicine Insights: Oncology. 2014 Dec 8;8:145-52 (no IF) Penquerc'h M, Picot D, Vigneau C. [Magnesium: a supercation misunderstood? Overview and survey of GPs in "Ille-et- Vilaine"]. Nephrol Ther. 2014 Feb;10(1):25-34. (IF 0.45)

72

Quintin J, Le Péron C, Palierne G, Bizot M, Cunha S, Sérandour AA, Avner S, Henry C, Percevault F, Belaud-Rotureau MA, Huet S, Watrin E, Eeckhoute J, Legagneux V, Salbert G, Métivier R.Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities. Mol Cell Biol. 2014 Jul;34(13):2418-36 (IF 5.03) Rioux-Leclercq N [Rare renal tumors. Pretest].Ann Pathol. 2014 Apr;34(2):131-3. (IF 0.24) Rioux-Leclercq N [Rare renal tumors. Introduction]. Ann Pathol. 2014 Apr;34(2):130. (IF 0.24) Verhoest G, Dolley-Hitze T, Jouan F, Belaud-Rotureau MA, Oger E, Lavenu A, Bensalah K, Arlot-Bonnemains Y, Collet N, Rioux-Leclercq N,Vigneau C.Sunitinib combined with angiotensin-2 type-1 receptor antagonists induces more necrosis: a murine xenograft model of renal cell carcinoma. Biomed Res Int. 2014;2014:901371. (IF 2.7) Verhoest G, Dolley-Hitze T, Jouan F, Bensalah K, Arlot-Bonnemains Y, Dugay F, Belaud-Rotureau MA, Rioux- Leclercq N, Vigneau C [Renin-angiotensin system and urological cancers]. Prog Urol. 2014 Feb;24(2):73-9. (IF 0.77) Vigneau C, Lorcy N, Dolley-Hitze T, Jouan F, Arlot-Bonnemains Y, Laguerre B, Verhoest G, Goujon JM, Belaud- Rotureau MA, Rioux-Leclercq N. All anti-vascular endothelial growth factor drugs can induce 'pre-eclampsia-like syndrome': a RARe study. Nephrol Dial Transplant. 2014 Feb;29(2):325-32. (IF 3.4)

2015 Chesnel F, Hascoet P, GagneJP, Couturier A, Jouan F, Poirier G, Le Goff C, Vigneau C, Danger Y, Verite F , Le Goff X, & Arlot-Bonnemains The Von Hippel Lindau tumor suppressor gene: Uncovering the expression of the pVHL172 . Br J Cancer. 2015 Jun 2. (IF 5.2)

2. Patents (with licence)

2014 - HASCOET Pauline, CHESNEL Franck, LE GOFF Xavier, COUTURIER Anne & ARLOT-BONNEMAINS Yannick Production d’anticorps monoclonaux dirigés contre la séquence protéique 172 de la proteine VHL humaine. Cabinet Plasseraud 52 Rue de la Victoire Paris – Brevet N° 14305925.1 -1402-reference : BEP :130403EPDP Juin 2014

2015 - JOUAN Florence & ARLOT-BONNEMAINS Yannick – Technologie de culture organotypique de tissu tumoral - Cabinet Plasseraud 52 Rue de la Victoire Paris – Dépôt le 27 Mai 2015

3. Conferences (actual team members)

- International 2012 ARLOT-BONNEMAINS Yannick: Cell cycle and kidney cancer : a new partner for Aurora-A 10th international VHL meeting Conference January 26-29 2012 Houston Tx (USA) (invited speaker) 2013 ARLOT-BONNEMAINS Yannick: University La Sapienza –Service of thyroide f-disease – Rome –Italy - Molecular investigation of the role of VHL- (invited by Pr D’Armiento) ARLOT-BONNEMAINS Yannick: AFCAS Universite de Laval Quebec –Canada Interaction of VHL and Aurora-A Mai (Invited by Huot J) 2014 ARLOT-BONNEMAINS Yannick: Aurora-A and VHL in kidney cancer 11th international VHL meeting Conference October 26-29 2014 Madrid –Spain (invited speaker) 2015 ARLOT-BONNEMAINS Yannick: Institut A Frappier Montreal Canada- Different molecular role of VHL-(invited by C.Dozois)

- National 2011 BELAUD-ROTUREAU Marc-Antoine: Efficacité des antagonistes des récepteurs de type 1 de l'angiotensine II en association aux inhibiteurs de tyrosine kinase dans un modèle murin de xénogreffe de carcinome rénal à cellules claires. Congrès de l’Association française d’Urologie 2011 2012 RIOUX-LECLERCQ Nathalie: Telmisartan potientites antiangiogenic effects of sunitinib in a xenograft model of renal cell carcinoma. 49eme congrès ERA-EDTA, Paris 2013 BELAUD-ROTUREAU Marc-Antoine: Profils moléculaires et phénotypiques des carcinomes à cellules claires du rein

73

présentant une résistance primaire aux thérapies ciblées. Ann. Pathol 2013,33S, COSFP1104, S33-S62. BELAUD-ROTUREAU Marc-Antoine: Surexpression de la protéine PAR-3 : facteur de mauvais pronostic dans le carcinome rénal à cellules claires (ccRCC). Congrès Français d'Urologie Paris 2013 RIOUX-LECLERCQ Nathalie: Profils moléculaires et phénotypiques des carcinomes à cellules claires du rein présentant une résistance primaire aux thérapies ciblées. Carrefour de Pathologie Paris, 18-22 Novembre 2013 2014 BELAUD-ROTUREAU Marc-Antoine: JOUAN F, RIOUX-LECLERCQ N, KAMMERER-JACQUET S. Le carcinome à cellules claires (CRCC) sans altération du gène de Von Hippel Lindau (VHL) : une entité anatomoclinique à part ? Carrefour Pathologie 2014, CNIT Paris 17-21

4. Funding

Before the creation of the team

2010-2013 ANR Genanimal 120 K€

After the création of the team Jan 1st 2012

2007-2010 CNRS – DREI 2 k€ 2007-2010 Région Bretagne – ARED 47 k€ 2007-2010 ANR Programme Jeunes chercheuses et jeunes chercheurs 16 k€ 2008-2011 Université de Rennes 1 12,5 k€ 2009-2012 ANR - Programme Génomique Animale 98,3 k€ 2009-2010 Région Bretagne 16 k€ 2009-2011 Région Bretagne – SAD 50 k€ 2010-2012 Université européenne de Bretagne – UEB 12 k€ 2010-2012 Ligue Nationale Contre le Cancer 88 k€ 2011 Institut Fédératif de Recherche 140 7 k€ 2011-2012 Université de Rennes1 8,8 k€ 2012 BIOSIT- (fabrication d’Anticorps) 10 K€ 2012 SF Nephrologie (étude antiangiogenique) 30 K€ 2012 Contrat doctoral Ministère (Pauline HASCOET) 90 K€ 2012 Ligue contre le cancer 20 k€ 2013 Ligue contre le cancer- (variant 172 de VHL) 25 K€ 2013 Ligue contre le cancer- (antimitotique) 20 K€ 2013 BIOSIT- (culture oragnotypiques) 10 K€ 2014 BIOSIT- (Séquencage VHL) 3 K€ 2014 Ligue contre le cancer- (variant 172 : suite) 25 K€ 2014 Ligue contre le cancer- (réponse ou résistance) 30 K€ 2014 CORECT-(thérapies ciblées) 38 K€ 2015 Défis émergents- (outils thérapeutiques) 10 K€ 2015 4e année ARC (Pauline HASCOET) 15 K€ 2015 Université de Rennes 1 1,5 k€

5. Training

Before the creation of the team Licence 2011 - DENGERMANN Julien - L3 Sciences et Vie de la terre, Université de Vannes (Encadrement Franck CHESNEL) Expression et purification de la protéine recombinante pVHL chez E. coli Master 2011 - JEZEQUEL Kévin - M1 Sciences cellulaires et moléculaires du vivant, Université Rennes1 (encadrement Xavier LE GOFF) Etude fonctionnelle de variants du suppresseur de tumeur VHL dans un modèle levure - LERAY H. & AUFFRET J. M1 UE11 (encadrement Franck CHESNEL) Etude de l'influence de la protéine pVHL humaine sur la maturation ovocytaire chez Xenopus laevis 2011 - PHAN Thi Trang – M2 International Hanoi Vietnam (encadrement Yannick ARLOT-BONNEMAINS & Michèle BAUDY-FLOCH ICR Rennes) Synthesis of analogs of RGD as antiangiogenic compounds

After the création of the team Jan 1st 2012 Licence 2014 - PIGNIER Jean-Baptiste - L3 Sciences cellulaires et moléculaires du vivant, Université Rennes 1 (Encadrement Franck CHESNEL) Etude de l’impact de la mutation L158P de pVHL sur la structure et la phosphorylation par Aurora-A

74

- HANCSOVSZKI Renata - BSc (L3), Université de Debrecen (Hongrie) (Encadrement Xavier LE GOFF) Expression and aggregation of human pVHL in fission yeast Master 2012 - DIGUE Enora - M1 Sciences cellulaires et moléculaires du vivant, Université Rennes1 (encadrement Franck CHESNEL) Etude de l’expression des isoformes de VHL dans différentes lignées cellulaires humaines - DONNOU Céline & FAOUCHER Marie - M1 UE11 (encadrement Franck CHESNEL) E Phosphorylation de VHL par Aurora A : mutagenèse dirigée de VHL172 (sur 2 résidus d'intérêt) et production des protéines mutées correspondantes - LEHURE Kévin – M2 Sciences cellulaires et moléculaires du vivant, Université Rennes1- (encadrement Franck CHESNEL & Xavier LE GOFF) Étude des différentes isoformes protéiques du suppresseur de tumeur Von Hippel Lindau (VHL) 2013 - AUVRET Bastien - M1 Sciences cellulaires et moléculaires du vivant, Université Rennes1 (encadrement Yannick ARLOT-BONNEMAINS) Etude du rôle de la phosphorylation de pVHL par Aurora-A dans le cancer du rein - PICHARD Camille & WATTECAMPS Guilhem - M1 UE11 (encadrement Cathy LE GOFF) Etude de l’effet cytotoxique de deux inhibiteurs de l’angiogenèse sur des cellules endothéliale - TERRASSE Marianne - M2 Sciences, Santé et Applications, Paris Diderot (encadrement Cécile VIGNEAU - co encadrement de Franck CHESNEL et Florence JOUAN) Etude in vitro des mécanismes moléculaires des effets secondaires rénaux des thérapies ciblées antiangiogéniques 2014 - BOSSEBOEUF E. - M1 Sciences cellulaires et moléculaires du vivant, Université Rennes 1 (Encadrement Nathalie RIOUX-LECLERCQ) - LE MEUR Gwenn - M1 Nantes (encadrement Yannick ARLOT-BONNEMAINS) Analyse phénotypique des cellules tumorales de rein exprimant les différentes isoformes de VHL : mise au point du test de clonogénicité pour criblage pharmacologique - SCHEWIN Coline - M1 Sciences cellulaires et moléculaires du vivant, Université Rennes1 (encadrement Franck CHESNEL et Olivier DELALANDE) Phosphorylation de pVHL par Aurora- A : Analyse de l’implication de la Ser 111 par une double approche de mutagenèse dirigée et de modélisation - GARNIER Matilde & PIHAN Anne-Sophie - M1 UE11 (encadrement Franck CHESNEL) Caractérisation de différentes lignéestumorales rénales de statut VHL variable (variant 1 ou 2) soumises à une hypoxie chimique - DAGHER Julien - M2 Sciences Santé et Applications-, Paris Diderot (encadrement Nathalie RIOUX-LECLERCQ) Comparaison des profils histologiques immuno-histochimiques cytogénétiques et moléculaires des carcinomes rénaux à cellules claires primitifs et de leur métastases PhD 01/2010-04/2011: FALCONNER Anaïs - Dir. Thèse : Yannick ARLOT-BONNEMAINS, stop thesis. 20/12/2012: DOLLEY HITZE Thibault – PhD Université Rennes 1, Expression et rôle du récepteur à l’angiotensine-2 de type-1 par les carcinomes rénaux à cellules claires – Dir. Thèse : Cécile VIGNEAU 16/06/2014: VERHOEST Grégory - PhD Université Rennes 1, Développement et mise au point de modèles murins de xénogreffe de carcinome rénal à cellules claires et évaluation de la réponse de l’association d’un antagoniste des récepteurs à l’angiotensin-II au sunitinib – Dir. Thèse : Cécile VIGNEAU 17/12/2014: DUGAY Frédéric - PhD-Université Rennes 1, PAR-3 et carcinome à cellules claires : Rôle dans la tumorigenèse - Dir. Thèse : Marc-Antoine BELAUD ROTUREAU & Yannick ARLOT-BONNEMAINS Since 10/2012: HASCOET Pauline - PhD-Université Rennes 1, Etude fonctionnelle du rôle de pVHL172 dans la tumorigenèse Dir Thèse : Xavier LE GOFF & Franck CHESNEL Since 10/2013: KAMMERER-JACQUET Solène-Florence - PhD-Université Rennes 1, Etude des profils histologiques, immuno-histochimiques, cytogénétiques et moléculaires des carcinomes rénaux à cellules claires répondant et non répondant aux thérapies anti-angiogéniques – Dir. Thèse : Nathalie RIOUX-LECLERCQ Since 12/2013: PLADYS Adélaïde - PhD-Université Rennes 1, Les pratiques d'indication et des bénéfices de la dialyse quotidienne, en France et au niveau international – Dir. Thèse : Cécile VIGNEAU Training of technical staff of the team COUTURIER Anne, IE-CNRS: 2012- Atelier INSERM Biochimie de protéines - 2012 Stage radio-compétence et radio- protection –Université Rennes1 - 2012 Microscopie photonique en Biologie Université Rennes 1 - 2013- Ateliers thématiques Anglais JOUAN Florence, TCN-UR1: 2013 Ateliers thématiques Anglais –Université Rennes 1 - 2014- Formation à la chirurgie sur le petit animal "Ecole Vétérinaire Nantes"

75

Team 3 "Tubulin and interacting proteins" Leader: Denis CHRETIEN

77

2.1. Team presentation

Microtubules are highly dynamic polymers involved in fundamental processes such as cell motility, compartmentalization, intracellular trafficking and cell division. While their dynamic properties are regulated by an ensemble of stabilizing and destabilizing proteins throughout the cell cycle, microtubules assembled from purified tubulin can alternate between growing and shrinking phases, a unique behavior known as dynamic instability. In this overall framework, our team studies the molecular mechanisms at the origin of microtubule dynamic instability, and how this behavior is regulated by microtubule associated proteins (MAPs). We focus on a particular class of MAPs, the plus-end tracking proteins (+TIPs), that associate with microtubule growing ends where they regulate their dynamic properties and their interaction with intracellular targets. Toward this aim, we develop original strategies, which range from molecular biology, biochemistry, video-light and cryo-electron microscopies. We are particularly involved in the development of cryo-electron tomography, which include the design of original software and nanogold probes. Along this line, our team manages the cryo-electron microscope platform of MRic (Microscopy Rennes imaging center) on the campus of Beaulieu in Rennes.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

CHRETIEN Denis DR2 CNRS HEICHETTE Claire TCE UR1 DUCHESNE Laurence MCU UR1

2.2.2.2. Temporary staff

BAZILE Franck ATER UR1

2.2.2.3. Permanent staff who left the team during the evaluation period

ARNAL Isabelle CR2 CNRS until 10/2010 MONIER-TSCHOPE Solange CR1 INSERM until 03/2015

78

2.2.3. Achievements

2.2.3.1. Scientific achievements

Characterization at the structural level of the region recognized by the plus-end tracking protein EB1 at the tip of growing microtubules. This work was carried out in collaboration with three international teams (S. STEINMETZ, Switzerland, A. AKHMANOVA, Netherlands; R. TAMPÉ, Germany). Our study revealed the conformational changes that the tubulin dimer undergoes during assembly, in relation with its GTPase cycle (Guesdon, Bazile et al., submitted).

Development of cryo-electron tomography (Coquelle et al., 2011), in association with gold nanoparticles conjugated to individual proteins (Duchesne et al., 2012, Guesdon, Bazile et al., submitted). A special emphasis was given on the development of dual-axis cryo-electron tomography (Guesdon et al., 2013), which provides a significant improvement in cryo-electron tomogram quality.

Development of image analysis software devoted to cryo-electron microscope images, in collaboration with C. KERVRANN (Inria, Rennes): TubuleJ (deposit to the Program Protection Agency in 2010), and CryoSeg (Kervrann et al., 2014).

Collaborations with local (R. GIET, IGDR, Gallaud et al, 2014) and National teams (S. MARCO, Institut Curie, Orsay, Guichard et al., 2010; F. LIVOLANT, laboratoire de Physique des Solides, Orsay, project ANR "DNA"), to whom we provided our expertize in microtubule research and/or cryo-electron tomography.

2.2.3.2. Scientific dissemination and influence

Denis CHRETIEN has been member of the administration council of the French Society for Microscopies (SFµ, 2011-14), and was involved in the organization of the 13th and 14th Congress of the society as scientific advisory board member. From 2010 to 2014, he was member of the editorial board of Biology of the Cell, and as such co-editor of a special issue on microtubules.

Laurence DUCHESNE was invited to an EMBO workshop on "Morphogen gradients" (Oxford, 2013), following her publication in PLOS Biology. In 2014, she received an installation grant from Rennes Métropole to acquire new scientific equipment, namely a disc-scanning unit implemented on our video- microscope to follow microtubule and +TIP dynamics in real time.

2.2.3.3. Interaction with the economic, social and cultural environment

Our team is in charge of the Structural Electron Microscopy platform located on the campus of Beaulieu, which is part of the Microscopy Rennes imaging center (Biosit, UMS 3480 CNRS). The platform hosts two electron microscopes, one used for routine work (Philips CM12, 1988), and one dedicated to cryo-electron microscopy (Tecnai G2 Sphera, 200 kV, LaB6, 2004). This equipment is essentially used by our team and that of R. GILLET (IGDR), but we also provide services to local, regional or national groups. However, our functioning as a platform is limited by the lack of an engineer dedicated to the platform, and most of the duties are performed by D. CHRETIEN. There is a high demand from the scientific community, and also from private companies. It will thus be essential that we can recruit an engineer on the platform, in order to satisfy this demand.

We organized three courses on cryo-electron tomography under the auspice of the French Society for Microscopies, in 2012, 2013 and 2014 (the next one is planned in 2015). We have trained 12 persons at the national level, originating from the academic (doctorates, engineers, researchers) and non-academic (JEOL society) domains.

2.3. Projects, scientific strategies & perspectives (5 years)

Molecular basis of microtubule dynamic instability We now have in hands unique tools to investigate the structure and dynamics of the GTP-cap at microtubule growing ends. The precise coupling between tubulin polymerization and GTP-hydrolysis remains unclear, and several models have been proposed (coupled, vectorial, stochastic...). Having the possibility to measure the GTP-cap length as a function of microtubule growth rate should allow us to sort out these different models, and also to link more precisely GTP-hydrolysis to tubulin conformational 79

changes during assembly. Additionally, we plan to investigate how the GTP-cap re-organizes in response to regulatory proteins and drugs, which either stabilize or destabilize microtubules. At the structural level, our results indicate that tubulin must undergo a "curved-to-straight" conformational change, which contrasts with the widespread model of the inverse event. Our working hypothesis is that this conformational change stimulates the GTPase activity of tubulin, giving rise to GDP-Pi intermediates in the straightest regions of the microtubule lattice. The inorganic phosphate would be released stochastically, which would account for the long tails of EB1-gold comets that we observe by cryo-ET. To address this question, we plan to investigate in deeper details the end structure of microtubules assembled in the - presence of GTP and GDP-Pi analogues such as GTPγS and GDP-BeF3 , respectively. In parallel to these experimental studies, we will develop theoretical models of microtubule assembly that will take into account the conformational changes and mechanical properties of tubulin and microtubules. This work has already been started in collaboration with P. FRANÇOIS and G. BROUHARD from the McGill Institute (Montreal, Canada).

Regulation of microtubule dynamics by associated proteins We plan to focus on two MAPs, doublecortin (DCX) and the alpha-tubulin acetyltransferase (αTAT1). DCX is involved in neurogenesis during embryonic development, and mutations in the gene that encodes this protein are involved in several cerebral deseases such as the lissencephally that is characterized by the development of a smooth brain. This protein has been shown to bind in-between protofilaments, to favor the formation of 13 protofilament microtubules commonly found in cells, and is the only protein known to stimulate microtubule assembly in the absence of GTP. More recently, the group of G. BROUHARD showed that DCX binds with high affinity to microtubule ends, and more specifically to curved lattices, and thus presumably to the outwardly curved sheets present at microtubule growing ends. In this project, we plan to investigate this hypothesis by conjugating DCX with gold nanoparticles and analyze its localization at microtubule ends. In turn, like EB1 this protein could constitute a powerful tool to address the conformational changes that the tubulin dimer undergoes during assembly in relation with its GTPase cycle. This work will also be performed in collaboration with the teams of G. BROUHARD and P. FRANÇOIS. αTAT1 is the only protein known to interact with a site located inside the microtubule lumen. This protein acetylates a lysine present on a flexible loop close to microtubule wall openings. However, it remains unclear whether αTAT1 diffuses inside the microtubule lumen or binds to the external surface of microtubules and recruits this loop through wall openings, or even penetrates inside the lumen through larger holes. In collaboration with C. JANKE (Institut Curie, Orsay) and G. MONTAGNAC (Institut Gustave Roussy, Paris), we have started to investigate this issue. αTAT1 has been conjugated with gold nanoparticles, and we are now examining its localization inside microtubules reassembled in vitro from HeLa tubulin, or present in ghost cells whose membranes have been removed using mild detergents. While the function of αTAT1 remains unclear, since mutations or knockout experiments did not reveal any visible phenotypes in mice, this protein may be part of a new and yet unknown family of MAPs that interact specifically with sites located inside the microtubule lumen.

Scientific environment Our team was originally planned to join the campus of Villejean, together with the two other teams still located in Beaulieu. Unfortunately, there seems to be little perspective due to the lack of space in Villejean. An alternative is that, in the next years, an efficient bi-site organization is established so that we can interact more closely with other teams from the IGDR. During the last years, we developed further cryo-EM methods, and would warmly welcome other teams that could benefit from our instrumentation, and who might implement new strategies such as correlative light-electron microscopy approaches. Such a project would strengthen methodological developments that take place at the IGDR on light and electron microscopy techniques. At the instrumental level, we would strongly benefit to acquire a more modern cryo-electron microscope equipped with a field emission gun and a direct electron CCD camera. An engineer devoted to our current - and hopefully future - instrumentation is also desperately needed. It is only at this price that we will be able to develop further our thematic and experimental approaches in Rennes.

2.4. Collaboration

80

2.4.1. Within the IGDR

Régis GIET (Ensconsin) - Jean-François HUBERT (actin-dystrophin interaction) - Reynald GILLET (HSP90-Tau fiber interaction).

2.4.2. Other collaborations

Rennes Christelle LOPEZ (INRA STLO, milk sphyngomielin vesicles).

National Amélie LEFORESTIER (Laboratory of Solid Physics, Orsay, DNA structure in viruses) - Carsten JANKE (Institut Curie, Orsay, alpha-tubulin acetyl transferase) - Guillaume MONTAGNAC (Institut Gustave Roussy, Villejuif, alpha-tubulin acetyl transferase) - René-Marc MÈGE (Institut Jacques Monod, Paris, cadherins) - Antoine GUICHET (Institut Jacques Monod, Paris, microtubule associated proteins) - Isabelle ARNAL (Neurosciences Institute, Grenoble, Tau-microtubule interaction).

International Michel STEINMETZ (Paul Scherrer Institute, Villigen, Switzerland, EB1-microtubule interactions) - Anna AKHMANOVA (Utrecht University, The Netherlands, EB1-microtubule interactions) - Eisa Inc. (Japan and USA, Eribulin-microtubule interaction)- Robert TAMPÉ (Institute of Biochemistry, Frankfurt, Germany, functionalized gold-nanoprobes) - David G. FERNIG (Liverpool University, UK, FGF2-heparan sulfate interaction) - Gary BROUHARD and Paul FRANÇOIS (McGill University, Montreal, Canada, DCX-microtubule interaction).

2.5. SWOT Analysis STRENGTHS Hot topics Original methodological approaches Network of local, national and international collaborations WEAKNESSES Size of the team Out-dated electron microscope Lack of an EM engineer Bi-site localization OPPORTUNITIES Publication in high-ranked journals National and/or international grants Recruitment of a permanent researcher Recruitment of PhD and Post-docs THREATS Stay isolated on the campus of Beaulieu Results of higher quality obtained by other teams using more recent equipment Lack of recent grant and recruitment

81

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Guichard P., Chrétien D., Marco S., and A.M. Tassin. Procentriole assembly revealed by cryo-electron tomography. The Embo Journal, 2010, 29:1565-72. (IF 10.2). Valiron O., Arnal I., Caudron N., and D. Job. GDP-tubulin incorporation into growing microtubules modulates polymer stability. The Journal of Biological Chemistry, 2010, 285:17507-13. (IF 4.9). Weis F., Moullintraffort L., Heichette C., Chrétien D., and C. Garnier. The 90-kDa heat shock protein HSP90 protects tubulin against thermal denaturation. The Journal of Biological Chemistry, 2010, 285:9525-34. (IF 4.9).

2011 Coquelle F., Blestel S., Heichette C., Arnal I., Kervrann C. and D. Chrétien. Cryo-electron tomography of microtubules assembled in vitro from purified components. Methods in Molecular Biology, 2011, 777:193-208. (IF 1.29)

2012 *Duchesne L., Octeau V., Bearon R.N., Beckett A., Prior I., Tampé R., Lounis B., and D.G. Fernig. Transport of Fibroblast Growth Factor 2 in the Pericellular Matrix Is Controlled by the Spatial Distribution of Its Binding Sites in Heparan Sulfate. PLoS Biology, 2012, 10, e1001361. (IF 12.8).

2013 Guesdon A., Blestel S., Kervrann C. and D. Chrétien. Single versus dual-axis cryo-electron tomography of microtubules assembled in vitro: Limits and perspectives. Journal of Structural Biology, 2013, 1:169-178. (IF 3.4).

2014 Gallaud E., Caous R., Pascal A., Bazile F., Gagné J-P., Huet S., Poirier G. G., Chrétien D., Richard-Parpaillon L., and R. Giet. Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells. The Journal of Cell Biology, 2014, 204:1111-21. (IF 10.4). Kervrann C., Blestel S., and D. Chrétien. Conditional Random Fields for tubulin-microtubule segmentation in cryo- electron tomography. Proceedings of the IEEE International Conference on Image Processing (ICIP 2014), Paris, 2014, pages 1-4. (IF 3.11)

2015 Strale P.O., Duchesne L., Peyret G. Montel L., Nguyen T., Png E, Tampé R., Troyanovsky S., Hénon S., Ladoux B., and R.M. Mège. The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity. The Journal of Cell Biology, 2015, 210:333-346. (IF 10.4)

2. Patents (with licence)

Blestel S., Guesdon A., Kervrann C. and D. Chrétien. TubuleJ. IDDN.FR.001.240023.000.S.P.2011.000.21000

3. Conferences (actual team members)

- International 2010 EMBO Meeting, Barcelona 2010 September 4-7. • Fisch C.R, B. Desforges, J. Jerber, N. Lebreton, S. Blestel, A. Guesdon, C. Heichette, C. Kervrann, D. Chrétien, P. Dupuis-Williams. Ultrastructural analysis of wild-type and mutant axonemes in Paramecium by cryo-electron tomography. Poster. Inserm Workshop "Microtubules and cell migration", Saint-Raphael 2010 October 27-29. • Chrétien D. Structure of microtubule assemblies by cryo-electron microscopy. Invited speaker. • Guesdon A., Blestel S., Heichette C., Kervrann C. and D. Chrétien Three dimensional reconstruction of 14 protofilaments GMPCPP-microtubules using TubuleJ. Poster. • Coquelle F.M., Blestel S., Kinoshita K., Vitre B., Heichette C., Kervrann C., Arnal I. and D. Chrétien. Effects of XMAP215 on Microtubule Dynamics and Structure and its Visualization by Electron Cryo- Tomography. Poster.

82

2011 6th International Congress on Electron Tomography, Heidelberg, 2011 May 5-8. • Guesdon A., Coquelle F., Vitre B., Angevin M., Heichette C., Arnal I. and D. Chrétien. Cryo-electron tomography of macromolecular assemblies: from quantum dots to 3D crystals of proteins. Poster. 2012 EMBO Conference Series. Microtubules: Structure, Regulation and Functions, Heidelberg, 2012, May 23- 26. • Guesdon A., Buey R.M., Duchesne L., Steinmetz M.O. and D. Chrétien. Interaction of EB1 conjugated to gold nanoparticles with dynamic microtubules: a cryo-electron tomographic study. Poster. 2013 EMBO Workshop "Morphogen gradients", Oxford, 2013 June 26-29. • Duchesne L. Transport of Fibroblast Growth Factor 2 in the Pericellular Matrix Is Controlled by the Spatial Distribution of Its Binding Sites in Heparan Sulfate. Invited speaker. 2014 EMBO Conference Series. Microtubules: Structure, Regulation and Functions, Heidelberg, 2014 May 28-31. • Guesdon A. Cryo-electron tomography of EB1-gold comets at growing microtubule ends. Selected oral presentation. • Guesdon A., Bazile F., Buey R.M., Mohan R., Monier S., Heichette C., Angevin M., Duchesne L., Akhmanova A., Steinmetz M.O. and D. Chrétien. Cryo-electron tomography of EB1-gold comets at growing microtubule ends. Poster. 2015 Jacques Monod Conference, Actin and Microtubule Cytoskeleton in Cell Motility and Morphogenesis: An Integrated View, Roscoff, 2015 May 26-30. • Chrétien D. EB1 reveals the GTP-cap architecture of microtubule growing ends. Selected oral presentation.

- National 2011 12th Congress of the French Society of Microscopies, Strasbourg, 2011 June 27 - July 1. • Guesdon A., Blestel S., Kervrann C. and D. Chrétien. How does GTP-hydrolysis destabilizes the microtubule lattice?. Poster. • Coquelle F., Guesdon A., Angevin M., Chenuel T., Chrétien M., Heichette C., Blestel S., Kervrann C., Arnal I. and D. Chrétien. Interaction of +TIPs with microtubule ends. Poster. • Pollet E., Fisch C., Guesdon A., Heichette C., Dupuis-Williams P., Kervrann C., Leforestier A. and D. Chrétien. Structure of Paramecium trichocysts. Poster. • Guesdon A., Coquelle F., Blestel S., Pollet E., Vitre B., Angevin M., Heichette C., Arnal I., Kervrann C. and D. Chrétien. Cryo-electron tomography of macromolecular assemblies: from quantum dots to 3D crystals of proteins. Poster. Institut Charles Sardon, Strasbourg, 2011 January 25. • Chrétien D. Molecular mechanisms at the origin of microtubule dynamic instability. Invited speaker. 2012 1st Symposium of the IGDR Cell & Developmental Biology Department, Rennes, 2012 October 25. • Chrétien D. Team Tubulin and Interacting Proteins., oral presentation. • Guesdon A. Cryo-electron tomography of microtubules: toward a correlative approach. Oral presentation. • Chrétien D., Monier S., Duchesne L., Heichette C., Angevin M., Bazile F., Guesdon A. and A. Filliol. Team Tubulin and Interacting proteins: methodological approaches and developments. Poster. • Guesdon A., Buey R.M., Duchesne L., Steinmetz M.O. and D. Chrétien. Interaction of EB1 conjugated to gold nanoparticles with dynamic microtubules: a cryo-electron tomographic study. Poster. 2013 13th Congress of the French Society of Microscopies, Nantes, 2013 July 2-5. • Guesdon A., Blestel S., Kervrann C. and D. Chrétien. Dual-axis cryo-electron tomography of vitrified specimens. Poster. • Duchesne L., Guesdon A., Heichette C., Octeau V., Bearon R.N., Beckett A., Prior I.A., Lounis B., Fernig D.G. and D. Chrétien. New generation of gold nanoparticle probes for quantitative imaging by photothermal microscopy and cryo-electron tomography. Poster. 1st meeting of the French Microtubule Network, Marseille, 2013 July 1-2. • Chrétien D. 3D structure of tubulin sheets at growing microtubule ends. Invited speaker. 2014 Congrès Gen2Bio, Saint-Malo, 2014 April 3. • Chrétien D. Presentation of the TEM platform: Cryo-electron microscopy and 3D reconstructions. Oral presentation. 83

2015 2nd meeting of the French Microtubule Network, Grenoble, 2015 July 6-7. • Bazile F. EB1 reveals the GTP-cap architecture of microtubule growing ends. Selected oral presentation.

4. Funding

2007-2011 ANR Programme Physique et Chimie du Vivant 102,4 k€ 2008-2012 ANR Programme Physique et Chimie du Vivant 217,4 k€ 2010-2015 Association for Cancer Research (ARC) (Coll. A. GUICHET, IJM Paris - R. GIET, IGDR) 131 k€ 2011 University of Rennes 1, Emerging scientific challenges – (Coll. C. KERVRANN, INRIA Rennes) 15 k€ 2012 Federative Research Institute (IFR 140 GFAS), Innovative scientific projects 10 k€ 2012-2014 Association for Cancer Research (ARC), Post-Doctoral fellowship (Franck BAZILE) 90 k€ 2013 Thematic Research Network SIScom Bretagne – Partner 8 k€ (Coord. C. KERVRANN, INRIA Rennes - coll. J. PECREAUX; IGRD) 2013-2015 National Agency for Research (ANR). Programme Blanc SVSE 5 DNA - Partner 15 k€ (Coord. F. LIVOLANT, Laboratoire de Physique des Solides, Orsay) 2014 Rennes Métropole, acquisition of equipment (Laurence DUCHESNE) 40 k€ 2014 Région Bretagne & Rennes Métropole, acquisition of equipment (MRic-TEM platform) 30 k€

5. Training Licence 2013 - Aurélie TILLY, L3 Biochemis try, 6 weeks. 2015 - Kevin BEVANT, L3 Biology, 6 weeks. IUT 2013 - Floriane ALMIRE, IUT Laval, 4 months. Master 2010 - Emilie PROVOST, M1 Biology UR1, 4 months. 2011 - Thomas CHESNUEL, M1 Biology, UR1, 4 months. - Emeline POLLET, M1 Modelisation of Biological Systems, UR1, 4 months. 2012 - Aveline FILLIOL, M1 Biology, UR1, 4 months. 2013 - Marie LIAMIN, M1 Biology, UR1, 2 months. 2014 - Mo ZHANG, M1 Insa Rouen, 1 month. 2015 - Alexandre THOMAS, M1 Biology, UR1, 2 months. PhD 10/2008-10/2010: Sophie BLESTEL, PhD in Informatics, Irisa, Rennes - Direction Charles KERVRANN (Inria) & Denis CHRETIEN, stop thesis - Currently School Professor. 20/06/2013: Audrey GUESDON, PhD in Biology - Direction Denis CHRETIEN - Currently Post-doc at the EMBL, Heidelberg. Post-doctorants 2005-2011: Frédéric COQUELLE, CNRS fellowship (2005-07) and ANR project (2007-11). Recruited as Assistant Professor at the University of Paris 11. 2012-2013: Franck BAZILE, ARC fellowship 2 years. Currently Temporary Assistant Professor in the team. CDD 2011-2012: ANGEVIN Morgane, Engineer Assistant CNRS, contract 1 year & 10,5 months.

84

Team 4 "Architecture and evolution of eukaryotic genetic circuits" (SyntheCell) Leader: Damien COUDREUSE

85

2.1. Team presentation

The SyntheCell team was established in January 2012 with funding from the ATIP-AVENIR program of the CNRS/INSERM. Since January 2013, the team is supported by a Starting Grant from the European Research Council under the 7th Framework Program.

The team is taking a synthetic biology approach using the fission yeast Schizosaccharomyces pombe to understand some key aspects of the organization and evolution of the cell cycle control network. Using our model of yeast cells operating with minimal cell cycle control circuits, we are investigating the importance of specific CDK activity dynamics on cell cycle progression, the role of the architecture of cell cycle control in buffering the variability inherent to all biological processes, and the rationale for the evolution of complex cell cycle regulation in eukaryotes. In addition, we use these synthetic cells as starting points for laboratory evolution experiments, assessing the mechanisms that allow the evolution of rudimentary regulatory networks.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

COUDREUSE Damien CR1 CNRS GRISCOM Laurent IR2 CNRS CALLENS Céline IE2 CNRS

2.2.2.2. Temporary staff

DIXON Sarah Post-doc CASTILHO COELHO Nelson IE DOMINGO SANANES Maria Rosa Post-doc BABIC Julien PhD student (2014-2017) MUNOZ GARCIA Javier Post-doc BAIDI Feriel PhD student (2012-2016) RADHAKRISHNAN BALASUBRAMANIAM Vasanthakrishnan Post-doc

2.2.3. Achievements

2.2.3.1. Scientific achievements

Major scientific achievements

Mitotic catastrophe: insights from synthetic yeasts In present-day eukaryotes, the cell division cycle is controlled by a complex network of inter- acting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk) families, and the Anaphase Promoting Complex (APC). Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control 86

DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model’s predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities. Gérard C, Tyson JJ, Coudreuse D*, Novák B Cell cycle control by a minimal CDK network PLoS Comput Biol. 2015 Feb 6;11(2):e1004056 - * Corresponding Author

A microfluidic platform for controlling the cellular environment in microscopy Modulating the environmental parameters of growing cells dynamically, precisely and rapidly during live-cell imaging experiments is particularly difficult. Recently, the emergence of microfluidic technologies has paved the way towards new and powerful devices that will allow researchers to subject cells to various treatments while monitoring in real-time their responses. However, the most commonly used polymer in the microfluidic field, PDMS, is known strongly absorb small compounds, making this approach challenging for a vast number of applications. In particular, the small molecule inhibitor we use in our projects to control the activity of the synthetic CDK modules is incompatible with PDMS, and represents a class of drugs that has been widely used for understanding the functions of a broad range of kinases. We have developed a novel, PDMS-free microfluidic chip and showed that it is biocompatible without any of the drawbacks of PDMS. Beyond the critical advantage of this device for the projects of the team, it will be of general interest for all laboratories that want to use microfluidic technologies in combination with the treatment of cells with small molecules. A manuscript describing our method is being finalised and will be submitted before the summer of 2015.

An automated culture system for laboratory evolution experiments using fission yeast Laboratory evolution experiments represent one of the major aspects of the team’s projects. However, in eukaryotes, such an approach has mostly been used in budding yeast, and the tools developed in this organism were found to be incompatible with fission yeast, which is larger and shows a different density and physiology. We implemented and adapted the experimental evolution setup that was developed for S. cerevisiae by the laboratory of Dr. Maitreya DUNHAM (University of Washington, Seattle, USA). This device is now functional and routinely used in the laboratory, allowing the simultaneous and controlled growth of multiple fission yeast strains for hundreds of generations. This tool development was critical for our evolution project and may be of general interest for the fission yeast community. A manuscript describing the technique is being prepared and should be submitted for publication in the coming months.

2.2.3.2. Scientific dissemination and influence

The group leader has been invited to present the work of the team at several international conferences, including the 2014 Jacques Monod Conference “Cell cycle: bridging scales in cell division” and the 2014 symposium “Views into Nuclear Function”, Patras, Greece. The results of the first studies and resource development made by the team are published or nearly ready for submission. The team has received strong financial support from various funding organisations, including the 7th Framework program from the European Union (Marie Curie Career Integration Grant, ERC Starting Grant), the CNRS (ATIP- AVENIR program), and the local government of the Brittany Region. The team has attracted talented international post-doctoral fellows of very different scientific backgrounds who have been trained in some prestigious universities.

2.2.3.3. Interaction with the economic, social and cultural environment

Together with another team from the IGDR, the team is involved in a collaboration with Cherry Biotech, a startup company that develops microfluidic tools for the life sciences. This work has led to the optimisation and validation of a microfluidic temperature control system that allows for fast and precise temperature shifts during microscopy experiments, as well at maintenance of constant temperature of the

87

sample, even at below ambient temperature. The ongoing project, which has received significant funding from the local government of Brittany, aims at developing a powerful microfluidic platform to simultaneously, dynamically, rapidly and precisely control most of the common environmental parameters in which cells are grown while under microscopic observation. This device will be compatible with most model systems and represents a real technological advance in the field.

2.3. Projects, scientific strategies & perspectives (5 years)

Research interests Our knowledge of the functioning of eukaryotic cells has emerged from thorough investigation of the molecular mechanisms driving cellular events. However, the complexity of the underlying regulatory networks has made it difficult to understand the core control of essential functions. In the same way that model organisms were chosen for their ease of manipulation or simplicity, model pathways need to be developed to decipher the design principles of regulatory circuits. Since the pioneering isolation of cyclins and CDKs, significant advances have been made in the identification and description of the multiple layers of control that ensure proper progression through the eukaryotic cell division cycle. However, our recent work has opened novel and exciting avenues of research toward understanding the core principles of this essential function and the rationale behind evolving its complex regulatory network (Coudreuse and Nurse, Nature 2010; Gerard et al. Plos Comp. Biol., 2015). Using fission yeast, we demonstrated the possibility to replace the cell cycle machinery by a simple synthetic system in vivo and proposed a novel paradigm for the core cell cycle engine. This established the foundation for the projects that are currently being pursued in the team. First, we are interested in how the architecture of the cell cycle network impacts the division cycle at different levels, from its integrity to its cell-to-cell robustness. In particular, we aim to understand the importance of the dynamic changes in CDK activity throughout the cell cycle, and to investigate the advantage of the naturally occurring dynamics over other types of modulations that may also be apparently compatible with cellular life. Furthermore, we are studying what role the precise topology of the cell cycle control circuit plays in maintaining the homogeneity of cell cycle progression in a population. Second, we use our synthetic systems as models to investigate the rationale for the evolution of complexity in cell cycle control. Why cells regulate biological events the way they do is a fundamental and challenging question that has remained poorly explored. Finally, our minimal strains serve as starting points for laboratory evolution experiments that aim at assessing how cell proliferation can evolve in adverse growth conditions when cells initially operate with basic control circuits. These studies, relying on synthetic rewiring of cell cycle control, will shed light on fundamental aspects of this essential process and bring new perspectives to our understanding of its evolution. As cell cycle progression is a foundation of cellular life, our work will have important implications for multicellular organisms as well as pathological situations.

Research plans for the coming five years A large part of our initial efforts have been focused on developing the experimental and technological approaches that are essential to our projects. We are now building on this work to investigate the biological questions described above. To this end, we recently recruited a number of new lab members, with a PhD student, a technician and three post-docs joining the team between September 2014 and March 2015. Methods / Technology development

Microfluidics: Our synthetic cell cycle systems allow us to precisely control the cell cycle machinery through chemical genetics, optogenetics and the use of temperature-sensitive mutants. To perform such alterations while imaging cells, we have established a microfluidic fabrication platform. Furthermore, we have developed new cell chambers based on alternative materials that circumvent major issues encountered in the microfluidic field (manuscript in preparation). This also led us to establish a collaboration with a company specialized in microfluidics for microscopy, with whom we have optimised a dynamic temperature control system for microfluidic devices. We are now working with them on a novel generation of microchips that allow for a much more versatile control of the cellular environment, a project that has received strong financial support from the government of the Brittany region.

Experimental evolution with fission yeast: We have established a powerful system that allows for automated control of multiple cultures of 88

fission yeast cells in different conditions. This system is based on the “ministats” developed by Dr. Maitreya Dunham (University of Washington, Seattle, USA) for S. cerevisiae, to which we applied a number of modifications for the use of fission yeast. We can now automatically maintain cultures at a constant optical density for more than 400 generations (manuscript in preparation). We have also started developing a novel and more powerful device, with automated measurements of the optical density of each population and computer-controlled dilution. Biological projects Taking advantage of the unique systems we have developed, from synthetic fission yeast strains to technological devices, we are investigating four main questions:

1) How do the dynamics of the CDK system contribute to efficient cellular reproduction? We previously showed that a simple oscillation between two activity thresholds is sufficient to drive the cell cycle. However, the importance of how these changes dynamically occur remains elusive. To investigate this, we are using novel synthetic cell cycle circuits that we have constructed in which we can switch from endogenous regulation to artificial control of CDK activity levels using chemical genetics. This synthetic module operates without any interference from the known endogenous regulatory pathways. Coupled to our microfluidic platform, this allows us to impose disctinct and precise activity dynamics and assess the functional relationship between specific patterns of CDK activity and the different steps in cell cycle progression at the single cell and population levels. We will also compare the impact of different dynamics that may appear interchangeable in laboratory conditions on the long-term integrity of cell cycle progression and the capacity of cells to respond to challenging environments.

2) How is cell cycle reproducibility achieved? A population of fission yeast cells shows a high homogeneity in cell cycle progression from cell to cell, despite the fact that cells are growing independently from each other. This suggests that the robustness of the cell cycle is a cell-autonomous property that may be built in its regulatory circuit. To investigate this, we are coupling a mathematical modeling approach to molecular and spatial rewiring of the CDK network, assessing what aspects of the architecture of the system have an impact on the maintenance of population homogeneity. Such a study is also relevant to tissue organization in higher eukaryotes, where the buffering of cell-to-cell variability in cell cycle progression is key.

3) What is the rationale behind the evolution of complexity in cell cycle regulation despite the apparent dispensability of entire branches of the endogenous CDK control network? This is a fundamental question that arose from the absence of phenotype in fission yeast cells operating with simplified synthetic control circuits. We are comparing seemingly optimal minimal strains with wild type cells in different conditions, over short and long periods of time, to understand what advantages are associated with a complex CDK circuit and what costs may be associated with alternative systems that appear to function even more efficiently than those in wild type cells. Understanding why “modern” cells operate the way they do is a fascinating question that has remained mostly unstudied.

4) What are the principles of the evolution of regulatory circuits in response to extrinsic or intrinsic limitations? We are interested in deciphering key principles associated with the evolution of rudimentary eukaryotic cells in challenging conditions, for which our minimal fission yeast strains serve as models. To this end, we are taking an unprecedented approach that uses these synthetic circuits as starting points for laboratory evolution experiments. The advantage of our system compared to existing experimental evolution studies is that it relies on the control of an essential function, cell cycle progression, by a basic core network. By selecting clones for their “evolved” capacity to overcome growth conditions that are initially deleterious, we hope to uncover evolutionary pathways that may also be relevant for other cellular processes.

Based on simplified cell cycle circuits, this work will bring different views and more conceptual insights into the above questions. In addition to elucidating some of the principles of cell cycle organization and evolution, our studies will have implications for multicellular systems, with cell cycle regulation being a key component of developmental and differentiation processes.

2.4. Collaboration

89

2.4.1. Within the IGDR

Dr. Pei-Yun Jenny WU, Genome Duplication and Maintenance, IGDR, Rennes, France

2.4.2. Other collaborations

Rennes Dr. Jeremy CRAMER, Cherry Biotech, Rennes, France.

France Dr. Gilles CHARVIN, IGBMC, Strasbourg, France - Dr. Emmanuel ROY, LPN, Paris, France.

Europe Dr. Bela NOVAK, University of Oxford, Oxford, UK - Dr. Zsolt SZILAGYI, Institute of Biomedicine, Gothenburg, Sweden - Dr. Jürg BÄHLER, UCL, London, UK.

2.5. SWOT Analysis STRENGTHS Unique model of fission yeast cells operating with synthetic cell cycle circuits. Successful development of powerful technologies. Wide range of interdisciplinary expertise within the team. Fruitful collaborations (academic and industrial). International team members. Funding acquired for the next 3 years. WEAKNESSES Risky and exploratory projects. Long initial phase of resource development. Difficulty to extend the stay of post-doctoral fellows, while positions at the CNRS are extremely difficult to obtain for a new team. OPPORTUNITIES Synthetic strategy initially developed by the group leader. Strong collaboration with a team in the UK who is a worldwide reference in mathematical modeling of the eukaryotic cell cycle. Productive collaboration with a company developing microfluidic tools for biology. Strong support from the city of Rennes and local government of the Brittany region. THREATS Group leader has heavy administrative tasks and finds it difficult to spend more time in supervising the research projects. The length of the post-doctoral and PhD training periods are too short, especially in a starting team with risky and long-term projects. Most grants are for short periods of time (2-4 years), which is a handicap for ambitious long-term projects.

90

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2012 Novak B, Krasinska L, Coudreuse D, Fisher D. Phosphorylation network dynamics in the control of cell cycle transitions. J Cell Sci. 2012 Oct 15;125(Pt 20):4703-11 (IF: 5.3)

2015 Gérard C, Tyson JJ, Coudreuse D*, Novák B - Cell cycle control by a minimal CDK network. PLoS Comput Biol. 2015 Feb 6;11(2):e1004056 - * Corresponding Author (IF: 4.8)

2. Patents (with licence)

N/A

3. Conferences (actual team members)

- International

2011 COUDREUSE D., NURSE P.: Driving the cell cycle with a minimal CDK control network. HFSPO Awardee meeting, Montreal, Canada 2011 (invited speaker) COUDREUSE D., NURSE P.: Driving the cell cycle with a minimal CDK control network. International Pombe meeting, Boston, USA 2011 (invited speaker)

2012 COUDREUSE D., NURSE P.: Driving the cell cycle with a minimal CDK control network. University of Warwick, Warwick, UK, 2012 (invited speaker) COUDREUSE D., NURSE P.: Unraveling the simplicity and modularity of cellular reproduction. Department of Biochemistry, University of Oxford, Oxford, UK, 2012 (invited speaker)

2013 COUDREUSE D.: Synthetic cell cycles: simplicity and evolution of cell proliferation. Hubrecht Institute, Utrecht, The Netherlands, 2013 (invited speaker)

2014 COUDREUSE D.: Operation and regulation of synthetic cell cycles. Jacques Monod conference “Cell cycle: bridging scales in cell division, Roscoff, France, 2014 (invited speaker) COUDREUSE D.: Operation and regulation of synthetic cell cycles. Views into Nuclear Functions symposium, Patras, Greece, 2014 (invited speaker)

2015 COUDREUSE D.: Beyond complexity: insights from simplifying the cell cycle in fission yeast. Virginia Tech, Blacksburg, USA, 2015 (invited speaker)

- National

2011 COUDREUSE D., NURSE P.: Unraveling the simplicity and modularity of cellular reproduction. IGBMC, Strasbourg, France, 2011 (invited speaker)

2012

COUDREUSE D.: Biologie synthétique, Recherche fondamentale. “Perspectives en biologie de synthèse” symposium, Paris, France, 2012 (invited speaker)

2013 COUDREUSE D., NURSE P.: An alternative look at cell proliferation: towards a modular model of cell cycle control 91

Institut Jacques Monod, Paris, France, 2013 (invited speaker)

2014 COUDREUSE D.: Operation and regulation of synthetic cell cycles. “Biologie synthétique et systémique” symposium, Toulouse, France, 2014 (invited speaker)

2015 COUDREUSE D.: Beyond complexity: insights from simplifying the cell cycle in fission yeast. Laboratory of Photonics and Nanostructures, Paris, France, 2015 (invited speaker)

4. Funding

2012 Institut Fédératif de Recherche 140 - Génétique fonctionnelle, Agronomie et Santé 10 k€ 2012 CE-Communauté européenne 25 k€ 2012 Alliance Nationale pour les Sciences de la Vie et de la Santé (AVIESAN)-Itmo CANCER 80 k€ 2012 City of Rennes, equipment 75 k€ 2012-2013 Brittany Region, equipment 50 k€ + consumables 50 k€ 100 k€ 2012-2014 CNRS ATIP grant* 180 k€ + salary for a post-doctoral fellow for 2 years (Chen TONG)* 92 k€ 2012-2016 Marie Curie Career Integration Grant* 100 k€ 2013-2018 ERC Starting Grant 1 700 k€ 2014-2016 Brittany Region, PhD grant (Feriel BAIDI) 90 k€ 2015-2016 Brittany Region, R&D program 70 k€ 2015-2017 Ministery of Defense & Cherry Biotech, PhD grant (Julien BABIC) 99 k€

* (ended after one year due to the ERC starting grant)

5. Training PhD Since 12/2012: Feriel BAIDI – Direction Damien COUDREUSE Since 10/2014: Julien BABIC – Direction Damien COUDREUSE Post-doctorants 2013-2014: Tong CHEN, post-doctoral fellow 1 year & 3 months 2013-2016: Javier MUNOZ GARCIA, post-doctoral fellow 3 years 2015-2016: Sarah DIXON, post-doctoral fellow 1 year 2014-2016: Maria Rosa DOMINGO SANANES, post-doctoral fellow 2 years 2014-2015: Vasanthakrishnan RADHAKRISHNAN BALASUBRAMANIAM, post-doctoral fellow 1 year CDD 2014-2015: Nelson CASTILHO COELHO – Engineer, contract 1 year

92

Team 5 "Genetics of pathologies related to development" Leader: Véronique DAVID

93

2.1. Team presentation

Our team works on deciphering the genetics and physiopathology of a rare disease, Holoprosencephaly (HPE). HPE is a compound disease that arises from multiple altered interactions of several signalling pathways during early formation of the forebrain. Our study focuses both on the identification of additional genes involved in HPE and on the understanding of their biological interactions.This work needs the geneticists to cooperate with developmental biologists to succeed in establishing the diagnosis for this rare disease.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

DAVID Véronique PU-PH1 DUPÉ Valérie CR1 INSERM de TAYRAC Marie MCU-PH ODENT Sylvie PU-PH1 DUBOURG Christèle MCU-PH CARRÉ Wilfrid IR CHU (20%)

2.2.2.2. Temporary staff

WARE Michelle Post-Doc HAMDI-ROZÉ Houda PhD student (2015-2018) MOUDEN Charlotte PhD student (2013- 2016)

2.2.2.3. Permanent staff who left the team during the contract

BENDAVID Claude PU-PH2 UR1-CHU until 12/2012 GUERRIER Daniel CR1 INSERM until 12/2014 PELLERIN Isabelle PU2 UR1 until 10/2014 GICQUEL Isabelle IE2 UR1 until 06/2012

2.2.3. Achievements

94

2.2.3.1. Scientific achievements

Our team works on deciphering the genetics and physiopathology of a rare disease, holoprosencephaly (HPE) that arises from defects taking place during early brain development. HPE (1/250 conceptuses, 1/10000 newborns) is caused by a failure to form the midline of the forebrain and face. The aetiology of HPE has pointed to a strong genetic component complicated by locus heterogeneity. It is a compound disease that arises from complex interactions between numerous developmental signalling pathways. A molecular basis has been identified for only 30% of the HPE cases. A major common effect of these mutations is the impairment of Sonic Hedgehog (SHH) and/or the Nodal signalling pathway, leading to disruption of the rostroventral region of the embryo. Fifteen genes have been involved in HPE, SHH, ZIC2, SIX3 and TGIF1 being the four major genes. Mutations are inherited in 70% of cases, from one of the parents, who are asymptomatic or carry a microform of HPE, suggesting the implication of a second anomaly arising de novo or inherited from the other parent.

Our main goal was to identify new genes that would explain the emergence of abnormal events during formation of the forebrain and rely these genes to signalling pathways implicated in brain development.

Since 1997, we have set up a large collection of 2000 HPE patients and relatives. The CHRU of Rennes has been labelled "Reference National Centre for Rare Disorders" on the topic "developmental anomalies and malformative syndromes" managed by Pr S. ODENT.

Human Molecular Genetics 2011. 20, 1122-31. IF: 7,64 (V. DUPÉ – V. DAVID). In order to identify new genes we performed a pangenomic CGH-array screening on a cohort of 260 HPE patients. We described a new candidate gene in redundant 6qter deletions, DELTA Like 1 (DLL1) which is a ligand of NOTCH. We showed that DLL1 was co-expressed in the developing chick forebrain with Fgf8. By treating chick embryos with a pharmacological inhibitor, we demonstrated that DLL1 interacts with FGF signaling pathway. These various findings implicate DLL1 in early patterning of the forebrain and identify NOTCH as a new signaling pathway involved in HPE.

Neural Development 2013. Dec 23;8:25. IF: 3,55; Frontiers in Neuroanatomy 2014. Dec 18;8:158 IF: 4,2; Frontiers in Neuroanatomy 2014 Dec 2;8:140 IF: 4,2 (M. WARE – V. DUPÉ). The role of Notch during hypothalamus formation along with its downstream effectors remained poorly defined. We have transiently blocked Notch activity with a specific inhibitor in chick embryos and used global gene expression analysis (transcriptomic approach) to provide evidence that Notch signalling modulates the generation of neurons in the early developing hypothalamus by lateral inhibition. We have also identified novel targets of Notch signalling, such as Transgelin 3 (Tagln3) and Chromogranin A (Chga), which were expressed in hypothalamic neuronal nuclei. We demonstrated that inhibition of Notch signalling during early development of the hypothalamus enhances expression of several new markers. These genes must be considered as important new targets of the Notch/proneural network. We described in detail the expression pattern of Tagln3, Chga and Contactin 2 (Cntn2) during early chick and mouse embryogenesis and demonstrated that Tagln3, Chga and Cntn2 represent strong new candidates to contribute to the sequential progression of vertebrate neurogenesis These results have been recently validated in the mouse embryos mutant for Rbpj, the effector of NOTCH signalling (Ware et al., in preparation). Importantly, we have observed that the Notch pathway is implicated in maintaining Shh expression in the chick ventral forebrain. It suggests that NOTCH signalling would be implicated in HPE appearance through the regulation of the major gene of the disease, SHH. We are currently testing this hypothesis in the mouse embryos.

Disease Models Mechanisms 2013. 6, 537-43. IF: 5,5 (V. DUPÉ – V. DAVID). In parallel, in order to elucidate molecular mechanisms leading to HPE, we have set up an ex ovo chick embryo culture system in the presence of pharmacological inhibitors known to block the different signalling pathways involved in forebrain development. Using this strategy we have been able to generate animal models for HPE. We have shown that NODAL and SHH dose-dependent double inhibition can promote an HPE-like phenotype in chick embryos. It support the view that genetic heterogeneity is important in the etiology of HPE and may contribute to the phenotypic variability. Journal of Medical Genetics 2011 11, 752-60. IF: 5,6 (C. DUBOURG – S. ODENT). We reported the clinical and molecular data of the largest European series in HPE encompassing 645 probands. New phenotype-genotype correlations were found based on statistical analyses. As previously reported, only 26% of the patients have a mutation in the four main genes involved in HPE (SHH, ZIC2, SIX3 and TGIF). In this series, fifteen probands carry a double genetic alteration, either with one gene mutation and one rearrangement, or two rearrangements, which highlights the multigenic model of HPE. We proposed an 95

algorithm based on these new phenotype-genotype correlations that helps to define molecular analysis strategy and genetic counselling in HPE. PLoS One. 2015 Feb 6;10(2) IF: 3,73 (C. DUBOURG – V. DAVID). The recent development of massive sequencing (NGS) allows the simultaneous sequencing of many genes as a routine procedure and facilitates the diagnosis approach in disorders governed by multigenic inheritance such as HPE. We have developed a new diagnostic strategy based on the sequencing of a panel of 20 known and candidate HPE genes using the Ampliseq method on Ion Torrent device -Applied Biosystem- (Dubourg et al., in preparation). In the coming years, our strategy will likely evolve to systematic exome sequencing. We set up homozygosity mapping on 9 inbred families. In one family, we formally identified a homozygous missense mutation in STIL, a gene implicated in centriole formation. Collaboration with Regis GIET (CPC Team at the IGDR) has allowed us to validate the deleterious effect of this mutation. This work has shown for the first time that HPE could be due to a recessive mode of inheritance. Exome sequencing was also performed in 13 family trios with severe HPE patients carrying a mutation in SHH, SIX3 or TGIF, the main HPE genes. These mutations were inherited from one of the parents, who were asymptomatic or presented a microform of the disease. Sequencing was performed for the affected offspring and the two parents, in order to find a potential inherited or de novo mutation in new candidate genes. Several cases of digenism were revealed (Mouden et al., in preparation)

2.2.3.2. Scientific dissemination and influence

- Welcome of foreign students: • Bruno FAULIN GAMBA : Oct 2012- April 2013 ; University of San Paolo, Brazil • Chun Hin LEE : 3 months 2013, Erasmus student at School of Biological Sciences, Portsmouth, UK

- Scientific societies: • Valérie DUPÉ: (2007-2013): Active member of the Société Française de Biologie du Développement (SFBD) • Michelle WARE: (2014-2015) Postdoctoral Representative for the British Society of Developmental Biology (BSDB) • Véronique DAVID, Sylvie ODENT, Christèle DUBOURG: ASHG American Society of Human Genetics; ESHG European Society of Human Genetics; SFGH French Society of Human Genetics; ANPGM National Association of Molecular biologist

- Organisation of scientific meetings: Sylvie ODENT: Scientific Organisation of the annual seminar of Genetics in the Ouest of France from 2012 (Le Croisic) ; Organisation of the 30rd "National seminar of clinical genetics genetic counselling" 2015, St Malo, France ; Genetic meetings in Necker – Enfants malades - Paris, every third Thursday of the month Valérie DUPÉ: (2007-2013): Contribution to the organisation of the annual seminar of "Société Française de Biologie du Développement (SFBD)"

- Invitation to participate to scientific meetings: Véronique DAVID: Seminars "Genomics and beyond" November 2014 and March 2015 – Indonesia ; Seminar to PhD school of Poitiers-Limoges 11-12 February 2015

- Post Doctoral fellows: Michelle WARE (British citizen) recruited on ANR Grant from 2013 to 2015

- Editorial committees: Véronique DAVID: Molecular Genetics and Genomic Medicine

- Nomination in national expertise committees Véronique DAVID: Appointed member of the National Committee of the Universities (CNU) Biochemistry (44-01) 2003-2010 and from 2013 ; 2010: Ecole des Neurosciences Paris- Appel d’offres Financement Post- Doctoral NERF ; 2012: Rapport d’expertise pour APREL (Appel à PRojets à destination des équipes Emergentes et Labellisées) CHU Limoges ; 2012: PHRC National ; 2013 and 2015: Expertise Technique pour le Service d’Accréditation Suisse Laboratoire de Génétique des Hôpitaux Universitaires de Genève ; 96

2014: expertise pour ABIGEN – Appel à projets du conseil régional du Limousin -Université se Limoges ; 2015: expertise pour l’Agence de la Biomédecine - AMP, diagnostic prénatal et diagnostic génétique Christèle DUBOURG: 2013, 3 expertises for Fondation Maladies Rares- High throughput sequencing and rare diseases Sylvie ODENT: Comité AERES 2013 - Visit of the research unit "Physiopathologie des Maladies Génétiques d’Expression Pédiatrique" Pr Serge AMSELEM, Université Paris 6 ; 2012 - PHRC National

2.2.3.3. Interaction with the economic, social and cultural environment

Our work concerns Human Health in the field of rare diseases and we directly interact with patients and their families. Holoprosencephaly affects one child out of 10,000 births and one out of 250 conceptuses.

Genetic counselling for this pathology is particularly complex and in practice the prenatal diagnosis is based mainly on ultrasound examination. To improve the genetic counselling for this pathology, it is absolutely required to characterize the molecular processes involved in the formation of the brain which are disrupted in HPE patients. Our recent data have reinforced our leader role in the field and allow us to be labelled "National Reference Centre for Rare Disorders" on the topic "developmental anomalies and malformative syndromes". Pr Sylvie ODENT manages this centre.

Sylvie ODENT participated to numerous events leading to the scientific dissemination: Marraine des oscars des entrepreneurs d’Ile et Vilaine en 2014, émission sur TV Rennes (février 2014), article dans Ouest France (février 2014), conférence grand public pour les Mardi Santé du CHU 2014 "Et si c’était une maladie génétique", Contribution texte et videos au site internet de l’Agence de la biomédecine sur la Génétique.

2.3. Projects, scientific strategies & perspectives (5 years)

The ultimate goal of our team is to improve diagnosis for HPE patients by increasing the panel of HPE genes. This will be possible only with a better understanding of the pathophysiology of the disease.

Until now, genes linked to HPE have been shown to play a role in major developmental pathways such as Shh, Nodal, Fgf and Notch. There is significant cross-regulation among these pathways, which has important implications for early brain development (Mercier et al., 2013, Ratié et al., 2013). It seems that HPE is due to combined failures of several genes necessary for proper forebrain development. Among these genes, Sonic Hedgehog (SHH) is a secreted molecule that plays a crucial role in the specification of ventral brain identity. In zebrafish, mutations in Cyclops (cyc), the gene that encodes for Nodal, results in brain defects similar to HPE. Cyc mutants express Shh in the notochord but lack specific expression of Shh in the rostroventral brain. Similarly in hypomorphic mice for the FGF signalling pathway, Shh expression is downregulated in the ventral brain and the mutants display a HPE phenotype. We have also observed that similar Shh down-regulation is observed in chick embryos when Notch is chemically inactivated by DAPT during forebrain development. Thus, it appears that a major common effect of the human mutations associated to HPE would be the impairment of SHH signalling leading to disruption of the rostroventral region of the embryo. This is comforted by our last publication showing that STIL known to be implicated in the primary cilia formation is a HPE gene. The importance of cilia in Hedgehog signalling transduction is well described.

Therefore, our goal for this new contract is to demonstrate that an accumulation of gene mutations leading to a decreased activity of SHH in the midline is necessary to develop HPE features. In this purpose, our team will go on using complementary strategies based on genetics and animal models.

1/ Genetics strategy and holoprosencephaly patients

To date, NGS data has been obtained for 14 HPE families, but as each family is very informative, only half of them have been explored. We first focused on the most evident candidate genes, known to have a role in brain development. We are currently working on the remaining candidate genes, by sequencing them in the other family members to check the familial segregation and by looking at their expression profile in chick and mouse embryos. Due to the genetic heterogeneity of this pathology it is now necessary to enrich our NGS data. More that 100 families have not yet been studied by high throughput sequencing. This work will be the 97

first large-scale genotype-driven strategy for this pathology. Their further sequencing will be powerful to get statistical results and show the involvement of the same gene variant in different families. For this purpose, we will set up a high-throughput pipeline for filtering and analysis of these sequencing data. W Carré, our engineer in Biocomputing, and Marie de TAYRAC who recently joined our team, will work in collaboration with the bioinformatics team of the Biogenouest platform to support this strategy.

Our recent results based on exome sequencing on consanguineous families and family trios revealed several cases of digenism implicating genes involved in the transmission of SHH signalling, such as DISP1, NDST1 (N-deacetylase-sulfotransferase) or LRP2. Deleterious variants were found in NDST1, a gene involved in heparan sulfate metabolism, that helps the binding of morphogens like SHH or FGF8 to their receptors and thus increases their local concentration. Another candidate gene is LRP2 (or Megalin) that acts as a co-receptor of PTCH1 for SHH, specifically in the forebrain during development. These results confirm our hypothesis that HPE would be linked to combined failures of several genes related to SHH signalling. However, the deleterious role of the mutation found in the HPE patient has to be validated. To do this we are going to set up functional tests to assess SHH activity. Cultured murine embryonic NIH3T3 fibroblasts are routinely used as a model system for Shh signalling. This cell line contains components and overall structure of the SHH signalling pathway and is able to form primary cilia. This model will be used for two objectives; the first one will be to test the impact of the mutation on the function of the protein. The second will allow us to test the function of the candidate genes that have not been well documented by evaluating a possible contribution of these candidate genes in the Shh signalling pathway.

In parallel, we have established a collaboration with a German team (U. Strahle, Karlsruhe) that is competent in sophisticated mutagenesis and screening strategy on a large scale using zebrafish. To investigate whether a candidate gene is implicated in HPE, we can use the CRISPR/Cas9 system, TALE nucleases or antisense morpholino knockdown methodology, in order to produce a knockdown zebrafish embryo. This will be followed by rescue experiments with either the mutated gene or wild-type mRNA.

It has been reported that DNA methylation is highly dynamic during mammalian development and participates in regulation of embryogenesis. The aberrant DNA methylation programming during development would cause the inheritance of epigenetic mutations that have been found to be associated with human diseases. We could study DNA methylation by high-throughput technologies in order to identify potentially novel developmental genes or regions regulated by DNA methylation via integrating methylomes and identifying differentially methylated genes during development.

2/ Animal models strategy and molecular mechanisms of holoprosencephaly

We have recently demonstrated that dysregulation of NOTCH pathway would confer susceptibility to the occurrence of human HPE. Although NOTCH signalling has been described to be important during the neurogenic phase of forebrain development (Ratié, 2013), no link to HPE has yet been observed. To determine whether a HPE phenotype is associated to Notch inactivation, transgenic mice will be used. As there is redundancy in the Notch receptor mutant mice, RBPj mutant mice are commonly used to study the role of Notch inhibition in mouse. However, the full knockout mouse is embryonic lethal before brain formation, therefore we created a conditional mutant mouse using the R26R-creETR2 mouse controlling knockdown with Tamoxifen injections. The conditional mouse will allow us to analyse the brain phenotype after specific inactivation of Notch signalling during ventral brain specification (around E8.5). We will also test the Shh activity in this mouse model, in order to validate the hypothesis that a down-regulation of SHH signalling is the major effect associated to HPE.

In order to evaluate the implication of the following signalling pathways, SHH, NODAL, FGF and NOTCH, during HPE appearance our appraoch using chick embryo cultures will be continued. In these experiments, we are going to use inhibitors to specifically inhibit these pathways. This culture method is quick, simple and has been proven to work in our hands (Dupé and Lumsden, 2001; Mercier et al., 2013). HPE-like phenotypes are relatively easy to produce by disturbances to the highly conserved process of gastrulation in a controlled time window (Mercier et al., 2013). The molecular reprogramming of the brain will be tested in these various conditions. For this, microdissection of the midline will be obtained and microarray approach (Agilent Gallus gene expression microarray) will be used in order to identify genes that are deregulated during these various conditions. Overall, this data will reveal the mechanism by which HPE can be produced through disruption of various molecular pathways.

Overall, such data will lend greater insight into the complex genetics that lead to phenotypic expression of HPE. Ultimately, we will design a gene network dedicated to HPE that will give a global view

98

of all genes potentially involved in HPE and their interconnections. In a context of exome sequencing of a multigenic pathology, it will represent an essential tool for diagnosis.

2.4. Collaboration

2.4.1. Within the IGDR

Regis GIET (Functional tests to validate STIL mutations) - Frédérique BARLOY-HUBLER (In silico study of proneural gene targets).

2.4.2. Other collaborations

National Pascale SAUGIER-VEBER – Inserm U1079, Rouen (Rhombencephalosynapsis) - Shahragim TAJBAKHSH – Pasteur Institute, Paris (Extra ocular muscle patterning and Retinoic acid)

International Max MUENKE, NIH, USA (10 collaborating papers on genetics of HPE) - George AMAN – NIH, USA (functional tests on Zebrafish Model (FAT1) - Lucilene RIBEIRO - Brazil (genetics and HPE) welcome of a PhD student (6 months) - Frank SCHUBERT, University Portsmouth, UK (Paper submitted: Ware M, Dupé V and Schubert F, 2015) - François GUILLEMOT, Francis Crick Institute, London, UK (Functional study of Ascl1 target genes).

2.5. SWOT Analysis STRENGTHS GPLD has accumulated a world-leading clinical resource and is the European reference centre for holoprosencephaly (HPE). Good publication record. Project supported by ANR funding (2013-2015). First group to develop exome sequencing on HPE families and to identify new genes using this strategy. Translational project from research to diagnosis, due to close collaboration between research team and hospital team. Group’expertise covers both human genetics, computational biology and developmental biology. Good collaboration between team members with complementary skills. Dynamic team welcoming one or more Master 2 students each year, these students have all continued their scientific careers by a PhD. Recruitment of a Post Doc who published 3 papers in 2 years. WEAKNESSES Size of the team (4 members have teaching, hospital and administration tasks besides their research interest). Difficulties to find grants to develop exome sequencing at a higher scale (all the HPE families), leading to weak statistical analysis. No technical help since July 2012 and difficulty of recruitment. Financial support for PhD from the Doctoral school (VAS) is low. OPPORTUNITIES E-Rare project (European Research Projects on Rare Diseases) would be the opportunity to enlarge the HPE recruitment to European families. Our 2015 proposal was not selected but it will be submitted again. Hospital grants to develop exome sequencing. Collaboration with other bioinformatics engineers from Biosit to enhance our data analyses strength. Possibility to recruit students from the medical school. Tools and skills are numerous in the unit and help to develop functional tests. THREATS Less and less grants available.

99

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Bendavid C, Dupé V, Rochard L, Gicquel I, Dubourg C and David V. Holoprosencephaly: an update on cytogenetic abnormalities. Am J Hum Genet C Semin Med Genet 2010 Jan 26:154C(1):86-92. (IF: 4,06) Dubourg C, David V, Gropman A, Mercier S, Muenke M, Odent S, Pineda-Alvarez DE, Roessler E. Clinical utility gene card for: Holoprosencephaly. Eur J Hum Genet. 2011 Jan;19(1):preceeding 118-20. (IF : 4,23) Jaillard S, Andrieux J, Plessis G, Krepischi A, Lucas J, David V, Le Brun M, Bertola D, David A, Belaud-Rotureau MA, Mosser J, Lazaro L, Tréguier C, Rosenberg C, Odent S, and Dubourg C. Deletion 5q12 : Delineation of a phenotype including mental retardation and ocular defects. Am. J. Med Genet. Part A 2010, 1 – 7. (IF : 2,05) Pineda-Alvarez DE, Dubourg C, David V, Roessler E, Muenke M. Current recommandations for the molecular evaluation of newly diagnosed holoprosencephaly patients. Am J Med Genet C Semin Med Genet 2010 Jan 26:154C(1):93-101. (IF : 3,54) Solomon BD, Lacbawan F, Mercier S, Clegg NJ, Delgado MR, RosenbaumK, Dubourg C, David V, Olney AH, Wehner LE, Hehr U, Bale S, Paulussen A, Smeets HJ, Hardisty E, Tyki-Szymanska A, Pronicka E, Clemens M, McPherson E, Hennekam RC, Haln J, Stashinko E, Levey E, Wieczorek D, Roeder E, Schell-Apacik CC, Booth CW, Thomas RL, Kenwrich S, Keaton A, Balog JZ, Hadley D, Zhou N, Long R, Velez JI, Pineda-Alvarez DE, Odent S, Roessler E, Muenke M. Mutations in ZIC2 in human holoprosencephaly : Description of a novel ZIC2-Specific phenotype and comprehensive analysis of 157 individuals. J Med Genet. 2010 Aug;47(8):513-24 (IF: 5,63) Solomon BD, Mercier S, Velez JI, Pineda-Alvarez DE, Wyllie A, Zhou N, Dubourg C, David V, Odent S, Roessler E, Muenke M. Analysis of genotype-phenotype correlations in human holoprosencephaly. Am J Med Genet C Semin Med Genet 2010 Jan 26:154C(1):133-141. (IF : 3,54)

2011 Dubourg C, Sanlaville D, Doco-Fenzy M, Le Caignec C, Missirian C, Jaillard S, Schluth-Bolard C, Landais E, Boute O, Philip N, Toutain A, David A, Edery P, Moncla A, Martin-Coignard D, Vincent-Delorme C, Mortemousque I, Duban-Bedu B, Drunat S, Beri M, Mosser J, Odent S, David V, Andrieux J. Clinical and molecular characterization of 17q21.31 microdeletion syndrome in 14 French patients with mental retardation. Eur J Med Genet. 2011 Mar-Apr;54(2):144-51. (IF: 1,68) Dupé V, Rochard L, Mercier S, Le Pétillon Y, Gicquel I, Bendavid C, Bourrouillou G, Usha K, Thauvin-Robinet C, Bohan T, Odent S, Dubourg C and David V (2011). NOTCH, a new signaling pathway implicated in holoprosencephaly. Human Molecular Genetics. 20, 1122-31. (IF: 7,64) Mercier S, Dubourg C, Garcelon N, Campillo-Gimenez B, Gicquel I, Belleguic M, Ratié L, Pasquier L, Loget P, Bendavid C, Jaillard S, Rochard L , Quélin C, Sinteff J-P, Dupé V, David V and Odent S (2011). New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases. Journal of Medical Genetics. 11, 752-60. (IF: 5,6) Morcel K, Watrin T, Pasquier L, Rochard L, Le Caignec C, Dubourg C, Loget P, Paniel BJ, Odent S, David V, Pellerin I, Bendavid C, Guerrier D. Utero-vaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome) associated with deletions in known DiGeorge or DiGeorge-like loci. Orphanet J Rare Dis. 2011 Mar 15;6:9. (IF: 3,96)

2012 Morcel K, Watrin T, Jaffre F, Deschamps S, Omilli F, Pellerin I, Levêque J and Guerrier D (2012). Involvement of ITIH5, a candidate gene for congenital uterovaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome), in female genital tract development. Gene Expr. 15:207-214. (IF: 1.7) Solomon BD, Bear KA, Wyllie A, Keaton AA, Dubourg C, David V, Mercier S, Odent S, Hehr U, Paulussen A, Clegg NJ, Delgado MR, Bale SJ, Lacbawan F, Ardinger HH, Aylsworth AS, Bhengu NL, Braddock S, Brookhyser K, Burton B, Gaspar H, Grix A, Horovitz D, Kanetzke E, Kayserili H, Lev D, Nikkel SM, Norton M, Roberts R, Saal H, Schaefer GB, Schneider A, Smith EK, Sowry E, Spence MA, Shalev SA, Steiner CE, Thompson EM, Winder TL, Balog JZ, Hadley DW, Zhou N, Pineda-Alvarez DE, Roessler E, Muenke M. Genotypic and phenotypic analysis of 396 individuals with mutations in Sonic Hedgehog. J Med Genet. 2012 Jul;49(7):473-9. (IF: 5,6)

2013 Démurger F, Pasquier L, Dubourg C, Dupé V, Gicquel I, Evain C, Ratié L, Jaillard S, Beri M, Leheup B, Lespinasse J, Martin-Coignard D, Mercier S, Quelin C, Loget P, Marcorelles P, Laquerrière A, Bendavid C, Odent S, David V (2013). Array-CGH analysis suggests genetic heterogeneity in Rhombencephalosynapsis. Molecular Syndromology. 4: 267-272. (IF: NA) Mercier S, David V, Ratié L, Gicquel I, Odent S and Dupé V (2013). NODAL and SHH dose-dependent double inhibition promotes an HPE-like phenotype in chick embryo. Disease Models Mechanisms. 6, 537-43. (IF: 5,5) 100

Ratié L, Ware M, Barloy-Hubler F, Romé H, Gicquel I, Dubourg C, David V, Dupé V (2013). Novel genes upregulated when NOTCH signalling is disrupted during hypothalamic development. Neural Development. 8:25. (IF: 3,55)

2014 Dubourg C, Bonnet-Brilhault F, Toutain A, Mignot C, Jacquette A, Dieux A, Gérard-Banluet M, Beaumont-Epinette M, Julia S, Isidor B, Rossi M, Odent S, Bendavid C, Barthélémy C, Verloes A, David V. Identification of nine new RAI1 truncating mutations in Smith-Magenis syndrome patients without 17p11.2 deletions. Molecular Syndromology 2014 Feb;5(2):57-64. (IF: NA) Ratié L, Ware M, Jagline H, David V, Dupé V (2014). Dynamic expression of Notch-dependent neurogenic markers in the embryonic nervous system of the chick. Frontiers in Neuroanatomy. (IF : 4,2) Ware M, Hamdi-Rozé H, Dupé V (2014). Notch signalling and proneural genes work together to control the neural building blocks for the initial scaffold in the hypothalamus. Frontiers in Neuroanatomy. (IF : 4,2)

2015 Mouden C, de Tayrac M, Dubourg C, Rosé S, Carré W, Hamdi-Rozé H, Akloul L, Héron B, Odent S, Dupé V, Giet R, David V (2015). Homozygous STIL mutation causes holoprosencephaly and microcephaly in two siblings. PLOS ONE . (IF: 3,73)

Collaborating papers

2011 Blanchard A, Roubertie A, Simonetta-Moreau M, Ea V, Coquart C, Frederic MY, Gallouedec G, Adenis JP, Benatru I, Borg M, Burbaud P, Calvas P, Cif L, Damier P, Destee A, Faivre L, Guyant-Marechal L, Janik P, Janoura S, Kreisler A, Lusakowska A, Odent S, Potulska-Chromik A, Rudzińska M, Thobois S, Vuillaume I, Tranchant C, Tuffery-Giraud S, Coubes P, Sablonnière B, Claustres M, Collod-Béroud G. Mov Disord. Singular DYT6 phenotypes in association with new THAP1 frameshift mutations.2011 Aug 1;26(9):1775-7. Demars J, Rossignol S, Netchine I, Syin Lee K, Shmela M, Faivre L, Weill J, Odent S, Azzi S, Callier P, Lucas J, Dubourg C, Andrieux J, Le Bouc Y, El-Osta A, Gicquel C. (2011). New insights into the pathogenesis of Beckwith- Wiedemann and Silver-Russell syndromes: Contribution of small copy number variations to 11p15 imprinting defects. Hum Mutat. Oct ;32(10) :1171-82. Epub 2011 Sep 8. (IF 5,21) Ghoumid J, Andrieux J, Sablonnière B, Odent S, Philippe N, Zanlonghi X, Saugier-Veber P, Bardyn T, Manouvrier-Hanu S, Holder-Espinasse M. Duplication at chromosome 2q31.1-q31.2 in a family presenting syndactyly and nystagmus. Eur J Hum Genet. 2011 Nov;19(11):1198-201. (IF 4,31) Jacquemont S, Reymond A, Zufferey F, Harewood L, Walters RG, Kutalik Z, Martinet D, Shen Y, Valsesia A, Beckmann ND, Thorleifsson G, Belfiore M, Bouquillon S, Campion D, de Leeuw N, de Vries BB, Esko T, Fernandez BA, Fernández- Aranda F, Fernández-Real JM, Gratacòs M, Guilmatre A, Hoyer J, Jarvelin MR, Kooy RF, Kurg A, Le Caignec C, Männik K, Platt OS, Sanlaville D, Van Haelst MM, Villatoro Gomez S, Walha F, Wu BL, Yu Y, Aboura A, Addor MC, Alembik Y, Antonarakis SE, Arveiler B, Barth M, Bednarek N, Béna F, Bergmann S, Beri M, Bernardini L, Blaumeiser B, Bonneau D, Bottani A, Boute O, Brunner HG, Cailley D, Callier P, Chiesa J, Chrast J, Coin L, Coutton C, Cuisset JM, Cuvellier JC, David A, de Freminville B, Delobel B, Delrue MA, Demeer B, Descamps D, Didelot G, Dieterich K, Disciglio V, Doco- Fenzy M, Drunat S, Duban-Bedu B, Dubourg C, El-Sayed Moustafa JS, Elliott P, Faas BH, Faivre L, Faudet A, Fellmann F, Ferrarini A, Fisher R, Flori E, Forer L, Gaillard D, Gerard M, Gieger C, Gimelli S, Gimelli G, Grabe HJ, Guichet A, Guillin O, Hartikainen AL, Heron D, Hippolyte L, Holder M, Homuth G, Isidor B, Jaillard S, Jaros Z, Jiménez-Murcia S, Helas GJ, Jonveaux P, Kaksonen S, Keren B, Kloss-Brandstätter A, Knoers NV, Koolen DA, Kroisel PM, Kronenberg F, Labalme A, Landais E, Lapi E, Layet V, Legallic S, Leheup B, Leube B, Lewis S, Lucas J, MacDermot KD, Magnusson P, Marshall C, Mathieu-Dramard M, McCarthy MI, Meitinger T, Mencarelli MA, Merla G, Moerman A, Mooser V, Morice- Picard F, Mucciolo M, Nauck M, Ndiaye NC, Nordgren A, Pasquier L, Petit F, Pfundt R, Plessis G, Rajcan-Separovic E, Ramelli GP, Rauch A, Ravazzolo R, Reis A, Renieri A, Richart C, Ried JS, Rieubland C, Roberts W, Roetzer KM, Rooryck C, Rossi M, Saemundsen E, Satre V, Schurmann C, Sigurdsson E, Stavropoulos DJ, Stefansson H, Tengström C, Thorsteinsdóttir U, Tinahones FJ, Touraine R, Vallée L, van Binsbergen E, Van der Aa N, Vincent-Delorme C, Visvikis- Siest S, Vollenweider P, Völzke H, Vulto-van Silfhout AT, Waeber G, Wallgren-Pettersson C, Witwicki RM, Zwolinksi S, Andrieux J, Estivill X, Gusella JF, Gustafsson O, Metspalu A, Scherer SW, Stefansson K, Blakemore AI, Beckmann JS, Froguel P. (2011). Mirror extreme BMI phenotypes associated with gene dosage at the 16p11.2 locus. Nature, Aug 31;478(7367):97-102. (IF 38,5) Jaillard S, Loget P, Lucas J, Dubourg C, Le Bouar G, Demurger F, Bertorello I, David V, Poulain P, Odent S, Belaud- Rotureau MA. Terminal 6.9 Mb deletion of chromosome 15q, associated with a structurally abnormal X chromosome in a patient with congenital diaphragmatic hernia and heart defect. Eur J Med Genet. 2011 Mar-Apr;54(2):186-8. (IF 1,68) Le Goff C, Mahaut C, Wang LW, Allali S, Abhyankar A, Jensen S, Zylberberg L, Collod-Beroud G, Bonnet D, Alanay Y, Brady AF, Cordier MP, Devriendt K, Genevieve D, Kiper PÖ, Kitoh H, Krakow D, Lynch SA, Le Merrer M, Mégarbane A, Mortier G, Odent S, Polak M, Rohrbach M, Sillence D, Stolte-Dijkstra I, Superti-Furga A, Rimoin DL, Topouchian V, Unger S, Zabel B, Bole-Feysot C, Nitschke P, Handford P, Casanova JL, Boileau C, Apte SS, Munnich A, Cormier-Daire V. Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias. Am J Hum Genet. 2011 Jul 15;89(1):7-14. (IF 11,2) 101

Le Lan C, Mosser A, Ropert M, Detivaud L, Loustaud-Ratti V, Vital-Durand D, Roget L, Bardou-Jacquet E, Turlin B, David V, Loréal O, Deugnier Y, Brissot P, Jouanolle AM. Sex and acquired co-factors determine phenotypes of disease. Gastroenterology. 2011 Apr;140(4):1199-1207. (IF 12,82) Niculescu C, Indra G, Pfister V, Dupé V, Messadeq N, Metzger D, Chambon P, De Arcangelis A and Georges-Labouesse E (2011). Conditional ablation of integrin alpha-6 in mouse epidermis leads to skin fragility and inflammation. Eur. J. Cell. Biol. 90, 270-7. Odent S. Role of the geneticist in prenatal diagnosis. Rev Prat. 2011 Apr;61(4):528-30. Ryckewaert-D'Halluin A, Le Bouar G, Odent S, Milon J, D'Hervé D, Lucas J, Rouget F, Loget P, Poulain P, Le Gall E, Taque S. Prenat Diagn. Diagnosis of fetal urinary tract malformations: prenatal management and postnatal outcome. 2011 Nov;31(11):1013-20. (IF 2,68)

2012 Anheim M, Mariani LL, Calvas P, Cheuret E, Zagnoli F, Odent S, Seguela C, Marelli C, Fritsch M, Delaunoy JP, Brice A, Dürr A, Koenig M. Arch Neurol. 2012 Jul;69(7):912-6. Besnard T, Vaché C, Baux D, Larrieu L, Abadie C, Blanchet C, Odent S, Blanchet P, Calvas P, Hamel C, Dollfus H, Lina- Granade G, Lespinasse J, David A, Isidor B, Morin G, Malcolm S, Tuffery-Giraud S, Claustres M, Roux AF. Non-USH2A mutations in USH2 patients. Hum Mutat. 2012 Mar;33(3):504-10. (IF 5,21) Colin E, Touraine R, Levaillant JM, Pasquier L, Boussion F, Ferry M, Guichet A, Barth M, Mercier A, Gérard-Blanluet M, Odent S, Bonneau D. Binder phenotype in mothers affected with autoimmune disorders. J Matern Fetal Neonatal Med. 2012 Aug;25(8):1413-8 Coron F, Rousseau T, Jondeau G, Gautier E, Binquet C, Gouya L, Cusin V, Odent S, Dulac Y, Plauchu H, Collignon P, Delrue MA, Leheup B, Joly L, Huet F, Thevenon J, Mace G, Cassini C, Thauvin-Robinet C, Wolf JE, Hanna N, Sagot P, Boileau C, Faivre L. What do French patients and geneticists think about prenatal and preimplantation diagnoses in Marfan syndrome? Prenat Diagn. 2012 Dec;32(13):1318-23 (IF 2,68) Leuret O, Barth M, Kuster A, Eyer D, de Parscau L, Odent S, Gilbert-Dussardier B, Feillet F, Labarthe F. J Inherit Metab Dis. Efficacy and safety of BH4 before the age of 4 years in patients with mild phenylketonuria. 2012 Nov;35(6):975-81. Lopez E, Callier P, Cormier-Daire V, Lacombe D, Moncla A, Bottani A, Lambert S, Goldenberg A, Doray B, Odent S, Sanlaville D, Gueneau L, Duplomb L, Huet F, Aral B, Thauvin-Robinet C, Faivre L. Search for a gene responsible for Floating-Harbor syndrome on chromosome 12q15q21.1. Am J Med Genet A. 2012 Feb;158A(2):333-9. Perche O, Laudier B, Menuet A, Odent S, Laumonnier F, Briault S. FG syndrome: the FGS2 locus revisited. Am J Med Genet A. 2012 Jun;158A(6):1489-92 Quélin C, Loget P, Verloes A, Bazin A, Bessières B, Laquerrière A, Patrier S, Grigorescu R, Encha-Razavi F, Delahaye S, Jouannic JM, Carbonne B, D'Hervé D, Aubry MC, Macé G, Harvey T, Ville Y, Viot G, Joyé N, Odent S, Attié-Bitach T, Wolf C, Chevy F, Benlian P, Gonzales M. Phenotypic spectrum of fetal Smith-Lemli-Opitz syndrome. Eur J Med Genet. 2012 Feb;55(2):81-90. (IF 1,68) Thierry G, Bénéteau C, Pichon O, Flori E, Isidor B, Popelard F, Delrue MA, Dubosq-Bidot L, Thuresson AC, van Bon BW, Cailley D, Rooryck C, Paubel A, Metay C, Dusser A, Pasquier L, Béri M, Bonnet C, Jaillard S, Dubourg C, Tou B, Quéré MP, Soussi-Zander C, Toutain A, Lacombe D, Arveiler B, de Vries BB, Jonveaux P, David A, Le Caignec C. (2012). Molecular characterization of 1q44 microdeletion in 11 patients reveals three candidate genes for intellectual disability and seizures. Am J Med Genet A. Jul;158A(7):1633-40. Vauthier V, Jaillard S, Journel H, Dubourg C, Jockers R. (2012). Homozygous deletion of a 80 kb region comprising part of DNAJC6 and LEPR genes on chromosome 1p31.3 is associated with early onset obesity, mental retardation and epilepsy. Mol Genet Metab, Jul;106(3):345-50. Verloes A, Héron D, Billette de Villemeur T, Afenjar A, Baumann C, Bahi-Buisson N, Charles P, Faudet A, Jacquette A, Mignot C, Moutard ML, Passemard S, Rio M, Robel L, Rougeot C, Ville D, Burglen L, des Portes V; Réseau DéfiScience. [Diagnostic investigations for an unexplained developmental disability]. Arch Pediatr. 2012 Feb;19(2):194-207. Vuillaumier-Barrot S, Bouchet-Séraphin C, Chelbi M, Devisme L, Quentin S, Gazal S, Laquerrière A, Fallet-Bianco C, Loget P, Odent S, Carles D, Bazin A, Aziza J, Clemenson A, Guimiot F, Bonnière M, Monnot S, Bole-Feysot C, Bernard JP, Loeuillet L, Gonzales M, Socha K, Grandchamp B, Attié-Bitach T, Encha-Razavi F, Seta N. Identification of mutations in TMEM5 and ISPD as a cause of severe cobblestone lissencephaly. Am J Hum Genet. 2012 Dec 7;91(6):1135-43 Zufferey F, Sherr EH, Beckmann ND, Hanson E, Maillard AM, Hippolyte L, Macé A, Ferrari C, Kutalik Z, Andrieux J, Aylward E, Barker M, Bernier R, Bouquillon S, Conus P, Delobel B, Faucett WA, Goin-Kochel RP, Grant E, Harewood L, Hunter JV, Lebon S, Ledbetter DH, Martin CL, Männik K, Martinet D, Mukherjee P,Ramocki MB, Spence SJ, Steinman KJ, Tjernagel J, Spiro JE, Reymond A, Beckmann JS, Chung WK, Jacquemont S; Addor MC, Arveiler B, Belfiore M, Bena F, Bernardini L, Blanchet P, Bonneau D, Boute O, Callier P, Campion D, Chiesa J, Cordier MP, Cuisset JM, David A, de Leeuw N, de Vries B, Didelot G, Doco-Fenzy M, Bedu BD, Dubourg C, Dupuis-Girod S, Fagerberg CR, Faivre L, Fellmann F, Fernandez BA, Fisher R, Flori E, Goldenberg A, Heron D, Holder M, Hoyer J, Isidor B, Jaillard S, Jonveaux P, Joriot S, Journel H, Kooy F, le Caignec C, Leheup B, Lemaitre MP, Lewis S, Malan V, Mathieu-Dramard M, Metspalu A, Morice- Picard F, Mucciolo M, Oiglane-Shlik E, Ounap K, Pasquier L,Petit F, Philippe A, Plessis G, Prieur F, Puechberty J, 102

Rajcan-Separovic E, Rauch A, Renieri A, Rieubland C, Rooryck C, Rötzer KM, Ruiter M,Sanlaville D, Selmoni S, Shen Y, Siffredi V, Thonney J, Vallée L, van Binsbergen E, Van der Aa N, van Haelst MM, Vigneron J, Vincent-Delorme C,Vittoria D, Vulto-van Silfhout AT, Witwicki RM, Zwolinski SA, Bowe A, Beaudet AL, Brewton CM, Chu Z, Dempsey AG, Evans YL, Garza S, Kanne SM, Laakman AL, Lasala MW, Llorens AV, Marzano G, Moss TJ, Nowell KP, Proud MB, Chen Q, Vaughan R, Berman J, Blaskey L, Hines K, Kessler S, Khan SY, Qasmieh S, Bibb AL, Paal AM, Page PZ, Smith-Packard B, Buckner R, Burko J, Cavanagh AL, Cerban B, Snow AV, Snyder LG, Keehn RM, Miller DT, Miller FK, Olson JE, Triantafallou C, Visyak N, Atwell C, Benedetti M, Fischbach GD, Greenup M, Packer A, Bukshpun P,Cheong M, Dale C, Gobuty SE, Hinkley L, Jeremy RJ, Lee H, Luks TL, Marco EJ, Martin AJ, McGovern KE, Nagarajan SS, Owen J, Paul BM,Pojman NJ, Sinha T, Swarnakar V, Wakahiro M, Alupay H, Aaronson B, Ackerman S, Ankenman K, Elgin J, Gerdts J, Johnson K, Reilly B, Shaw D, Stevens A, Ward T, Wenegrat J (2012). A 600 kb deletion syndrome at 16p11.2 leads to energy imbalance and neuropsychiatric disorders. J Med Genet 2012 Oct;49(10):660-8. (IF 5,7)

2013 Béna F, Bruno DL, Eriksson M, van Ravenswaaij-Arts C, Stark Z, Dijkhuizen T, Gerkes E, Gimelli S, Ganesamoorthy D, Thuresson AC, Labalme A, Till M, Bilan F, Pasquier L, Kitzis A, Dubourg C, Rossi M Bottani A, Gagnebin M, Sanlaville D, Gilbert-Dussardier B, Guipponi M, van Haeringen A, Kriek M, Ruivenkamp C, Antonarakis SE, Anderlid BM, Slater HR, Schoumans J (2013). Molecular and clinical characterization of 25 individuals with exonic deletions of NRXN1 and comprehensive review of the literature. Am J Med Genet B Neuropsychiatr Genet. 162B(4):388-403. Bubien V, Bonnet F, Brouste V, Hoppe S, Barouk-Simonet E, David A, Edery P, Bottani A, Layet V, Caron O, Gilbert- Dussardier B, Delnatte C, Dugast C, Fricker JP, Bonneau D, Sevenet N, Longy M, Caux F; French Cowden Disease Network. High cumulative risks of cancer in patients with PTEN hamartoma tumour syndrome.J Med Genet. 2013 Apr;50(4):255-63. Callier P, Aral B, Hanna N, Lambert S, Dindy H, Ragon C, Payet M, Collod-Beroud G, Carmignac V, Delrue MA, Goizet C, Philip N, Busa T, Dulac Y, Missotte I, Sznajer Y, Toutain A, Francannet C, Megarbane A, Julia S, Edouard T, Sarda P, Amiel J, Lyonnet S, Cormier-Daire V, Gilbert B, Jacquette A, Heron D, Collignon P, Lacombe D, Morice-Picard F, Jouk PS, Cusin V, Willems M, Sarrazin E, Amarof K, Coubes C, Addor MC, Journel H, Colin E, Khau Van Kien P, Baumann C, Leheup B, Martin-Coignard D, Doco-Fenzy M, Goldenberg A, Plessis G, Thevenon J, Pasquier L, Odent S, Vabres P, Huet F, Marle N, Mosca-Boidron AL, Mugneret F, Gauthier S, Binquet C, Thauvin-Robinet C, Jondeau G, Boileau C, Faivre L. Systematic molecular and cytogenetic screening of 100 patients with marfanoid syndromes and intellectual disability. Clin Genet. 2013 Dec;84(6):507-21. Cottereau E, Mortemousque I, Moizard MP, Bürglen L, Lacombe D, Gilbert-Dussardier B, Sigaudy S, Boute O, David A, Faivre L, Amiel J, Robertson R, Viana Ramos F, Bieth E, Odent S, Demeer B, Mathieu M, Gaillard D, Van Maldergem L, Baujat G, Maystadt I, Héron D, Verloes A, Philip N, Cormier-Daire V, Frouté MF, Pinson L, Blanchet P, Sarda P, Willems M, Jacquinet A, Ratbi I, Van Den Ende J, Lackmy-Port Lis M, Goldenberg A, Bonneau D, Rossignol S, Toutain A. Phenotypic spectrum of Simpson-Golabi-Behmel syndrome in a series of 42 cases with a mutation in GPC3 and review of the literature. Am J Med Genet C Semin Med Genet. 2013 May;163C(2):92-105. Courcet JB, Faivre L, Michot C, Burguet A, Perez-Martin S, Alix E, Amiel J, Baumann C, Cordier MP, Cormier-Daire V, Delrue MA, Gilbert-Dussardier B, Goldenberg A, Jacquemont ML, Jaquette A, Kayirangwa H, Lacombe D, Le Merrer M, Toutain A, Odent S, Moncla A, Pelet A, Philip N, Pinson L, Poisson S, Kim-Han le QS, Roume J, Sanchez E, Willems M, Till M, Vincent-Delorme C, Mousson C, Vinault S, Binquet C, Huet F, Sarda P, Salomon R, Lyonnet S, Sanlaville D, Geneviève D. Clinical and molecular spectrum of renal malformations in Kabuki syndrome.J Pediatr. 2013 Sep;163(3):742-6. Dieterich K, Quijano-Roy S, Monnier N, Zhou J, Fauré J, Smirnow DA, Carlier R, Laroche C, Marcorelles P, Mercier S, Mégarbané A, Odent S, Romero N, Sternberg D, Marty I, Estournet B, Jouk PS, Melki J, Lunardi J. The neuronal endopeptidase ECEL1 is associated with a distinct form of recessive distal arthrogryposis. Hum Mol Genet. 2013 Apr 15;22(8):1483-92. Fievet A, Belaud-Rotureau MA, Dugay F, Abadie C, Henry C, Taque S, Andrieux J, Guyetant S, Robert M, Dubourg C, Edan C, Rioux-Leclercq N, Odent S, Jaillard S. (2013). Involvement of germline DDX1-MYCN duplication in inherited nephroblastoma. Eur J Med Genet 56(12):643-647. (IF 1,68) Le Goff C, Mahaut C, Bottani A, Doray B, Goldenberg A, Moncla A, Odent S, Nitschke P, Munnich A, Faivre L, Cormier- Daire V. Not all floating-harbor syndrome cases are due to mutations in exon 34 of SRCAP. Hum Mutat. 2013 Jan;34(1):88-92. (IF 5,21) Michot C, Corsini C, Sanlaville D, Baumann C, Toutain A, Philip N, Busa T, Holder M, Faivre L, Odent S, Delrue MA, Till M, Jacquemont ML, Cordier MP, Goldenberg A, Sanchez E, Alix E, Poisson S, Kayirangwa H, Lacombe D, Gilbert- Dussardier B, Pelet A, Roume J, Jacquette A, Isidor B, Giuliano F, Burglen L, Fradin M, Schaefer E, Alembick Y, Doray B, Moncla A, Héron D, Willems M, Pinson L, Le Quan Sang KH, Le Merrer M, Cormier-Daire V, Sarda P, Amiel J, Lyonnet S, Geneviève D. Finger creases lend a hand Kabuki syndrome. Eur J Med Genet. 2013 Oct;56(10):556-60. (IF 1,68) Philippe A, Malan V, Jacquemont ML, Boddaert N, Bonnefont JP, Odent S, Munnich A, Colleaux L, Cormier-Daire V. Xq25 duplications encompassing GRIA3 and STAG2 genes in two families convey recognizable X-linked intellectual disability with distinctive facial appearance. Am J Med Genet A. 2013 Jun;161A(6):1370-5. Thauvin-Robinet C, Auclair M, Duplomb L, Caron-Debarle M, Avila M, St-Onge J, Le Merrer M, Le Luyer B, Héron D, Mathieu-Dramard M, Bitoun P, Petit JM, Odent S, Amiel J, Picot D, Carmignac V, Thevenon J, Callier P, Laville M, Reznik Y, Fagour C, Nunes ML, Capeau J, Lascols O, Huet F, Faivre L, Vigouroux C, Rivière JB. PIK3R1 mutations cause syndromic insulin resistance with lipoatrophy. Am J Hum Genet. 2013 Jul 11;93(1):141-9. (IF 11,2) 103

Thauvin-Robinet C, Munck A, Huet F, de Becdelièvre A, Jimenez C, Lalau G, Gautier E, Rollet J, Flori J, Nové- Josserand R, Soufir JC, Haloun A, Hubert D, Houssin E, Bellis G, Rault G, David A, Janny L, Chiron R, Rives N, Hairion D, Collignon P, Valeri A, Karsenty G, Rossi A, Audrézet MP, Férec C, Leclerc J, Georges Md, Claustres M, Bienvenu T, Gérard B, Boisseau P, Cabet-Bey F, Cheillan D, Feldmann D, Clavel C, Bieth E, Iron A, Simon-Bouy B, Izard V, Steffann J, Viville S, Costa C, Drouineaud V, Fauque P, Binquet C, Bonithon-Kopp C, Morris MA, Faivre L, Goossens M, Roussey M, Girodon E; collaborating working group on p.Arg117His. CFTR p.Arg117His associated with CBAVD and other CFTR- related disorders. J Med Genet. 2013 Apr;50(4):220-7.

2014 Barbier M, Gross MS, Aubart M, Hanna N, Kessler K, Guo DC, Tosolini L, Ho-Tin-Noe B, Regalado E, Varret M, Abifadel M, Milleron O, Odent S, Dupuis-Girod S, Faivre L, Edouard T, Dulac Y, Busa T, Gouya L, Milewicz DM, Jondeau G, Boileau C. MFAP5 loss-of-function mutations underscore the involvement of matrix alteration in the pathogenesis of familial thoracic aortic aneurysms and dissections. Am J Hum Genet. 2014 Dec 4;95(6):736-43. (IF 11,2) Baruteau J, Heissat S, Broué P, Collardeau-Frachon S, Bouvier R, Fabre M, Debiec H, Ronco P, Uzan M, Narcy P, Cordier MP, Lachaux A, Lamireau T, Elleau C, Filet JP, Mitanchez D, Dupuy MP, Salaün JF, Odent S, Davison J, Debray D, Guigonis V (2014). Transient neonatal liver disease after maternal antenatal intravenous Ig infusions in gestational alloimmune liver disease associated with neonatal haemochromatosis. J Pediatr Gastroenterol Nutr. (5):629-35. (IF 2,19) Blanchard M, Dubourg C, Pasquier L, Odent S, Lucas J, Quélin C, Launay E, Henry C, Belaud-Rotureau MA, Dugay F, Jaillard S (2014). Postnatal diagnosis of 9q interstitial imbalances involving PTCH1, resulting from a familial intrachromosomal insertion. Eur J Med Genet Jan 31. (IF 1,68) Chassaing N, Causse A, Vigouroux A, Delahaye A, Alessandri JL, Boespflug-Tanguy O, Boute-Benejean O, Dollfus H, Duban-Bedu B, Gilbert-Dussardier B, Giuliano F, Gonzales M, Holder-Espinasse M, Isidor B, Jacquemont ML, Lacombe D, Martin-Coignard D, Mathieu-Dramard M, Odent S, Picone O, Pinson L, Quelin C, Sigaudy S, Toutain A, Thauvin- Robinet C, Kaplan J, Calvas P. Molecular findings and clinical data in a cohort of 150 patients with anophthalmia/microphthalmia. Clin Genet. 2014 (IF 3,94) Coutton C, Poreau B, Devillard F, Durand C, Odent S, Rozel C, Vieville G, Amblard F, Jouk PS, Satre V. Currarino Syndrome and HPE microform associated with 2.7- MB deletion in 7q36.3 excluding SHH gene. Mol Syndromol. 2014 Jan;5(1):25-31. Delahaye A, El Khattabi L, Guimiot F, Pipiras E, Andrieux J, Baumann C, Bouquillon S, Carella M, Delezoide AL, Delobel B, Demurger F, Dessuant H, Drunat S, Dubourg C, Dupont C, Faivre L, Holder-Espinasse M, Jaillard S, Journel H, Lyonnet S, Malan V, Masurel A, Marle N, Missirian C, Moerman A, Moncla A, Odent S, Orazio P, Palumbo P, Ravel A, Romana S, Tabet AC, Valduga M, Vermelle M, Dupont JM, Verloes A, Benzacken B. (2014). Incomplete penetrance and phenotypic variability of 6q16 deletions including SIM1. Eur J Hum Genet Nov 5. (IF 4,31) Démurger F, Ichkou A, Mougou-Zerelli S, Le Merrer M, Goudefroye G, Delezoide AL, Quélin C, Manouvrier S, Baujat G, Fradin M, Pasquier L, Megarbané A, Faivre L, Baumann C, Nampoothiri S, Roume J, Isidor B, Lacombe D, Delrue MA, Mercier S, Philip N, Schaefer E, Holder M, Krause A, Laffargue F, Sinico M, Amram D, André G, Liquier A, Rossi M, Amiel J, Giuliano F, Boute O, Dieux-Coeslier A, Jacquemont ML, Afenjar A, Van Maldergem L, Lackmy-Port-Lis M, Vincent-Delorme C, Chauvet ML, Cormier-Daire V, Devisme L, Geneviève D, Munnich A, Viot G, Raoul O, Romana S, Gonzales M, Encha-Razavi F, Odent S, Vekemans M, Attie-Bitach T. Eur J Hum Genet. 2014 Apr 16. (IF 4,31) Gruchy N, Bigot N, Jeanne-Pasquier C, Read MH, Odent S, Galera P, Leporrier N. Involvement and alteration of the Sonic Hedgehog pathway is associated with decreased cholesterol level in trisomy 18 and SLO amniocytes. Mol Genet Metab. 2014 Jun;112(2):177-82. (IF 2,83) Jaillard S, Bashamboo A, Pasquier L, Belaud-Rotureau MA, McElreavey K, Odent S, Ravel C (2014). Gene dosage effects in 46, XY DSD: usefulness of CGH technologies for diagnosis. Reprod Genet. (IF 1,82) Khattabi LE, Guimiot F, Pipiras E, Andrieux J, Baumann C, Bouquillon S, Delezoide AL, Delobel B, Demurger F, Dessuant H, Drunat S, Dubourg C, Dupont C, Faivre L, Holder-Espinasse M, Jaillard S, Journel H, Lyonnet S, Malan V, Masurel A, Marle N, Missirian C, Moerman A, Moncla A, Odent S, Palumbo O, Palumbo P, Ravel A, Romana S, Tabet AC, Valduga M, Vermelle M, Carella M, Dupont JM, Verloes A, Benzacken B, Delahaye A (2014). Incomplete penetrance and phenotypic variability of 6q16 deletions including SIM1. Eur J Hum Genet. (IF 4,31) Klein C, Le Goff C, Topouchian V, Odent S, Violas P, Glorion C, Cormier-Daire V. Orthopedics management of acromicric dysplasia: follow up of nine patients. Am J Med Genet A. 2014 Feb;164A(2):331-7. Laquérriere A, Maluenda J, Camus A, Fontenas L, Dieterich K, Nolent F, Zhou J, Monnier N, Latour P, Gentil D, Héron D, Desguerres I, Landrieu P, Beneteau C, Delaporte B, Bellesme C, Baumann C, Capri Y, Goldenberg A, Lyonnet S, Bonneau D, Estournet B, Quijano-Roy S, Francannet C, Odent S, Saint-Frison MH, Sigaudy S, Figarella-Branger D, Gelot A, Mussini JM, Lacroix C, Drouin-Garraud V, Malinge MC, Attié-Bitach T, Bessieres B, Bonniere M, Encha-Razavi F, Beaufrère AM, Khung-Savatovsky S, Perez MJ, Vasiljevic A, Mercier S, Roume J, Trestard L, Saugier-Veber P, Cordier MP, Layet V, Legendre M, Vigouroux-Castera A, Lunardi J, Bayes M, Jouk PS, Rigonnot L, Granier M, Sternberg D, Warszawski J, Gut I, Gonzales M, Tawk M, Melki J. Mutations in CNTNAP1 and ADCY6 are responsible for severe arthrogryposis multiplex congenita with axoglial defects. Hum Mol Genet. 2014 May 1;23(9):2279-89. (IF 7,69) Laverdure N, Dabadie A, Alex-Cordier MP, Odent S, Lachaux A. Maternal uniparental disomy 14 revealed by alpha 1 antitrypsin deficiency. Clin Res Hepatol Gastroenterol. 2014 Oct;38(5):604-6. (IF 0,77)

104

Luscan A, Laurendeau I, Malan V, Francannet C, Odent S, Giuliano F, Lacombe D, Touraine R, Vidaud M, Pasmant E, Cormier-Daire V. Mutations in SETD2 cause a novel overgrowth condition. J Med Genet. 2014 Aug;51(8):512-7. (IF 5,7) Manes G, Guillaumie T, Vos WL, Devos A, Audo I, Zeitz C, Marquette V, Zanlonghi X, Defoort-Dhellemmes S, Puech B, Mohand Said S, Sahel JA, Odent S, Dollfus H, Kaplan J, Dufier JL, Le Meur G, Weber M, Faivre L, Behar Cohen F, Béroud C, Picot MC, Verdier C, Sénéchal A, Baudoin C, Bocquet B, Findlay JB, Meunier I, Dhaenens CM, Hamel CP (2014). Am J . Ophthalmol. Quélin C, Loget P, D'Hervé D, Fradin M, Milon J, Ferry M, Body-Bechou D, Tréguier C, Garcia Hoyos M, Odent S. Prenat Diagn. 2014 Oct 9. (IF 2,68) Riant F, Odent S, Cecillon M, Pasquier L, de Baracé C, Carney MP, Tournier-Lasserve E. Deep intronic KRIT1 mutation in a family with clinically silent multiple cerebral cavernous malformations. Clin Genet. 2014 Dec;86(6):585-8. (IF 3,94) Romani M, Mancini F, Micalizzi A, Poretti A, Miccinilli E, Accorsi P, Avola E, Bertini E, Borgatti R, Romaniello R, Ceylaner S, Coppola G, D'Arrigo S, Giordano L, Janecke AR, Lituania M, Ludwig K, Martorell L, Mazza T, Odent S, Pinelli L, Poo P, Santucci M, Signorini S, Simonati A, Spiegel R, Stanzial F, Steinlin M, Tabarki B, Wolf NI, Zibordi F, Boltshauser E, Valente EM (2014). Oral-facial-digital syndrome type VI: is C5orf42 really the major gene? Hum Genet. (IF 4,63) Tardy-Guidollet V, Menassa R, Costa JM, David M, Bouvattier-Morel C, Baumann C, Houang M, Lorenzini F, Philip N, Odent S, Guichet A, Morel Y. New management strategy of pregnancies at risk of congenital adrenal hyperplasia using fetal sex determination in maternal serum: French cohort of 258 cases (2002-2011). J Clin Endocrinol Metab. 2014 Apr;99(4):1180-8. Thevenon J, Milh M,Feillet F, St-Onge J, Duffourd Y, Jugé C, Roubertie A, Héron D, Mignot C, Raffo E, Isidor B, Wahlen S, Sanlaville D, Villeneuve N, Darmency-Stamboul V, Toutain A, Lefebvre M, Chouchane M, Huet F, Lafon A, de Saint Martin A, Lesca G, El Chehadeh S, Thauvin-Robinet C, Masurel-Paulet A, Odent S, Villard L, Philippe C, Faivre L, Rivière JB. Am J Hum Genet. 2014 Jul 3;95(1):113-20. (IF 11,2) Vulto-van Silfhout AT, Nakagawa T, Bahi-Buisson N, Haas SA, Hu H, Bienek M, Vissers LE, Gilissen C, Tzschach A, Busche A, Müsebeck J, Rump P, Mathijssen IB, Avela K, Somer M, Doagu F, Philips AK, Rauch A, Baumer A, Voesenek K, Poirier K, Vigneron J, Amram D, Odent S, Nawara M, Obersztyn E, Lenart J, Charzewska A, Lebrun N, Fischer U, Nillesen WM, Yntema HG, Järvelä I, Ropers HH, de Vries BB, Brunner HG, van Bokhoven H, Raymond FL, Willemsen MA, Chelly J, Xiong Y, Barkovich AJ, Kalscheuer VM, Kleefstra T, de Brouwer AP (2014). Variants in CUL4B are Associated with Cerebral Malformations. Hum Mutat. (IF 5,21)

2015 El Khattabi L, Jaillard S, Andrieux J, Pasquier L, Perrin L, Capri Y, Benmansour A, Toutain A, Marcorelles P, Vincent- Delorme C, Journel H, Henry C, De Baracé C, Devisme L, Dubourg C, Demurger F, Lucas J, Belaud-Rotureau MA, Amiel J, Malan V, De Blois MC, De Pontual L, Pipiras E, Tabet AC, Aboura A, Verloes A. Molecular delineation of 9p mosaic tetrasomy (isochromosome 9p): report of 11 new cases and literature review. Accepted par American journal of Medical Genetics. Fares-Taie L, Gerber S, Tawara A, Ramirez-Miranda A, Douet JY, Verdin H, Guilloux A, Zenteno JC, Kondo H, Moisset H, Passet B, Yamamoto K, Iwai M, Tanaka T, Nakamura Y, Kimura W, Bole-Feysot C, Vilotte M, Odent S, Vilotte JL, Munnich A, Regnier A, Chassaing N, De Baere E, Raymond-Letron I, Kaplan J, Calvas P, Roche O, Rozet JM. Submicroscopic deletions at 13q32.1 cause congenital microcoria. Am J Hum Genet. 2015 Apr 2;96(4):631-9. doi: 10.1016/j.ajhg.2015.01.014. Epub 2015 Mar 12. (IF 11,2) Ware M, Dupé V, Schubert F (2015). Evolutionary conservation of the early axon scaffold in the vertebrate brain. Dev. Dyn. (accepted with minor revisions).

2. Patents (with licence)

2012 : "ITIH5 as a diagnostic marker of uterine development and functional defects". European Patent # 13709136.9- 1405. North America # USP201261610590. Patent holders: D. GUERRIER and K. MORCEL.

3. Conferences (actual team members)

- International

2010 Odent S, Dubourg C, Mercier S, Belleguic M, Pasquier L, Viot G, Layet V, Moncla A, Journel H, Attia-Sobol J, Roume J, Blesson S, Désir J, Bendavid C, Loget P, Lucas J, David V. Limits of "molecular" prenatal diagnosis in the context of Holoprosencephaly: the French experience. American Society of Human Genetics (ASHG), Washington, USA, (2-6 November, 2010).

2011 Mercier S, Gicquel I, Dubourg C, Odent S, David V et Dupé V. Advances in phenotype-genotype correlations in 105

Holoprosencephaly: about a European series of 645 probands. European Human Genetics Conference, Amsterdam, The Nederlands, (28-31 Mai 2011). Oral Ratié L, Gicquel I, Mercier S, Dubourg C, Odent S, David V et Dupé V. Holoprosencephaly and Notch signalling pathway: transcriptomic approach using chick model. European Human Genetics Conference 2011, Amsterdam, The Nederlands, (28-31 Mai 2011). Poster David V. Four cases of deletion in 6qTer target a new candidate gene for holoprosencephaly International meeting on Craniofacial Anomalies: Clinical Phenotype, Genes Related and New Perspecives. Bauru, Brazil (27-30 avril, 2011). Oral David V, Ratié L, Gicquel I, Mercier S, Dubourg C, Odent S, et Dupé V. Notch signaling pathway and Holoprosencephaly: A transcriptomic approach using chick model. 12Th ICHG meeting, Montréal 12-15 October 2011. Poster

2012 Ratié L, Gicquel, David V and Dupé V. A transcriptomic approach to study NOTCH pathway function during hypothalamus formation. Conference Jacques Monod, emergence and evolution of developmental patterns, Roscoff, France (25-29 Avril 2012). Poster David V, Dubourg C, Dupé V, Ratié L, Mercier S, Odent S. Holoprosencephaly (HPE): Identification of new candidate genes by high throughput sequencing of family trios. Poster. American Society of Human Genetics (ASHG), San Francisco, USA, (6-10 November, 2012). Mercier M, de Tayrac M, Mosser J, Loget P, Rozel C, Jaillard S, Milon J, Riffaud L, le Bouar G, Poulain P, Martin E. Dubourg C, David V. A new dominant frontonasal dysplasia with major posterior cranial defect. Poster. American Society of Human Genetics (ASHG), San Francisco, USA, (6-10 November, 2012).

2013 Ratié L, David V, Hubler F, Gicquel I, et Dupé V. Notch signalling governs neuronal differentiation in the developing hypothalamus. Joint meeting of the British Societies for Cell Biology and Developmental Biology, Warwick University, Coventry, UK (17-20 Mars 2013). Poster de Tayrac M, Babron M-C, Dubourg C, Mouden C, Bouvet R, Gazal S, Sahbatou M, Ratié L, Dupé V, Mosser J, Odent S and David V. New candidate genes in holoprosencephaly: results from homozygosity mapping in six inbred families. European Human Genetics Conference, Paris, France, (8-11 juin 2013). Poster

2014 Lainé A, Francis S, Smith C, Ware M, Dupé V et Schubert FR. Molecular control of neurogenesis in the embryonic brain. Final TC2N meeting, Gent, U.K (June, 2014). Poster Ware M, Ratié L, David V et Dupé V. Expression of novel NOTCH target genes in the developing chick embryo. FENS Forum 2014 in Milan, Italy, July 5-9, 2014. Poster Mouden C, Dubourg C, Rose S, Viot G, Heron B, Carre W, de Tayrac M, Dupé V, Odent S and David V. New insights in holoprosencephaly inheritance: exome sequencing in families reveals new double-hit and recessive cases. The 64th annual meeting of the American Society of Human Genetics (ASHG), San Diego, USA (18-22 October 2014). Poster

2015 Schubert F, Lainé A, Ware M, Dupé V. Development of the early axon scaffold in the vertebrate brain. BSDB 2015. British Societies for Cell Biology (BSCB) and Developmental Biology (BSDB) joint spring meeting at Warwick University, Coventry, UK (12-15 April 2015). Poster Ware M, Hamdi-Rosé H, David V, Dupé V. Regulation of novel Notch targets by proneural transcription factors in the developing brain. BSDB 2015. British Societies for Cell Biology (BSCB) and Developmental Biology (BSDB) joint spring meeting at Warwick University, Coventry, UK (12-15 April 2015). Poster

- National

2010 Mercier S, Dubourg C, Gicquel I, Belleguic M, Pasquier L, Bendavid C, Loget P, Sinteff JP, Jaillard S, Rochard L, Dupé V, David V et Odent S. Actualités sur les corrélations génotype-phénotype dans l’Holoprosencéphalie : à propos d’une série européenne de 532 proposants. 5ième assises de génétique Humaine et médicale, Strasbourg (28-30 janvier 2010). Poster Rochard, L, Dupé V, Bendavid C, Dubourg C, Gicquel I, Mercier S, Odent S, Mosser J, Bohan T et David V. Trois cas de délétion en 6qter ciblent un nouveau gène candidat pour l’holoprosencéphalie. 5ième assises de génétique Humaine et médicale, Strasbourg (28-30 janvier 2010). Best poster award winner. Dupé V, Rochard L, Mercier S, Le Pétillon Y, Gicquel I, Bendavid C, Dubourg C, Odent S, Bohan T and David V. Four cases of deletion in 6qter target a new candidate gene for holoprosencephaly. Colloque annuel de la Société Française de Biologie du Développement, Paris, France (26-28 Mai 2010). 106

2011 David V. Notch signaling pathway and holoprosencephaly : a transcriptomic approach using chick model. 3ième Jeudi de Necker. Septembre 2011. Oral

2012 Ratié L, Dupé V, Gicquel I, Mercier S, Dubourg C, Odent S et David V. Notch, une nouvelle voie de signalisation impliquée dans l’holoprosencéphalie. 6ième assises de génétique Humaine et médicale, Marseille (2-4 février 2012). Oral Ratié L, Gicquel, David V and Dupé V. A transcriptomic approach to study NOTCH pathway function during hypothalamus formation. Meeting SBCF/SFBD “when development meets cell biology” Montpellier, france, (16-19 Octobre 2012). Poster

2014 Mouden C., De Tayrac M. ; Dubourg C., Carré W., Babron M.C., Héron-Longe B, Pinson L, Marlin S, Chassaing N, Ratié L, Dupé V, Odent S et David V. Holoprosencéphalie : Recherche de nouveaux gènes candidats par séquençage exomique de régions homozygotes dans des familles consanguines ; 7èmes Assises de Génétique Humaine et Médicale ; 29-31/01/2014 ; Bordeaux. Poster

4. Funding

2007-2010 Région Bretagne 29 k€ 2008-2010 National Institutes of Health –NIH 5,6 k€ 2010 ARC 14 k€ 2010 CHU de Poitiers 7 k€ 2012 Fondation-maladies-rares 3 k€ 2012-2013 Communauté d'agglomération Rennes Métropole 4,8 k€ 2012-2013 GIS Maladies rares, Plateforme-mutations (V. DAVID) 33 k€ 2013-2015 Fondation GROUPAMA 28 k€ 2013-2015 ANR Blanche (V. DUPÉ) 242 k€ 2012-2014 Agence de la biomédecine (V. DAVID) 33,5 k€ 2014 CHU de Rennes 32,4 k€

5. Training Teaching First year of medical studies (PACES Première année communes aux études de santé): Véronique DAVID Teaching Responsible for the competition Forewoman of the jury Master1 Sciences Cellulaires et Moléculaires du Vivant SCMV: Responsability and teaching Véronique DAVID: UE1 Fundamental methodologic basis cellular and molecular biology UE11 Internship in a laboratory (2 students/year) UE 13 Human molecular genetics Christèle DUBOURG: UE 13 Human molecular genetics UE3 Master 1 MGE Valérie DUPE: University Rennes1 (UFR SVE), MGE, “Chick model”. Genetics teaching: Véronique DAVID: Teaching of Genetics in L3 medical studies (University of Rennes 1) Christèle DUBOURG: Practical teaching of Genetics in L3 medical studies (University of Rennes 1) Sylvie ODENT: Responsibility with Pr Alain Verloes of DIU Dysmorphology (Paris7) Responsibility and teaching of Genetics in L3 medical studies (University of Rennes 1) Licence 2013 - Julien LE FRIEC – L3 (6 weeks) 2014 - Lee CHUN HIN – BSc Biology (3 months) Master 2010 - Yannick LE DANTEC – M1 (2 months) - Anabelle DECHANCE – M1 UE11 (2 weeks) - Yann LE PÉTILLON – M2 Study of the role of DLL1 during forebrain development (5 months) 2011 - Céline CADUDAL et Marine DAUVERGNE - M1 UE11 (2 weeks) - Florence DEMURGER - M2 Rhombencephalosynapsis: search for an ethiology (5 months) - Marie-Catherine VOLTZENLOGEL – M2 (5 months) 2012 - Marie MESGOUEZ – M1 UE11 (2 weeks) 107

- Hélène ROMÉ - M2 Expression study of new HPE genes in chick embryos (5 months) - Denis VELIC - M2 Search for new candidate genes for holoprosencephaly- using inbreed families (9 months) 2013 - Léa BOURGUIGNON – M1 (2 months) - Charlotte MOUDEN – M2 Search for new candidate genes for holoprosencephaly- using NGS (5 months) 2014 – Joanne FLEJEO & Claire LAISNE LE FEVRE - M1 UE11 (2 weeks) - Hélène JAGLINE – M2 Study of Notch function during holoprosencephaly appearance using chick embryos (5 months) - Houda HAMDI – M2 Study of the molecular basis of holoprosencephaly using mouse embryos (9 months) - Sophie ROSE – M2 Search for new HPE genes by homozygosity mapping (6 months) 2015 - Jeanne LE GUEVEL & Marie LE ROY – M1 UE11 (2 weeks) - Sophie BOISSEAU - M2 Search for new HPE genes by high-throughput sequencing (5 months) - Julien LE FRIEC - M2 Study of the function of Notch signalling during hypothalamus development in mouse (5 months) - Kevin MARCHE –5th year Pharmacist studies - Search for new HPE genes by high-throughput sequencing in trio families (4 months) PhD 24/01/2011: Lucie ROCHARD – Description of new candidate genes for Holoprosencephaly – Dir. Thèse Véronique DAVID 24/01/2012: Sandra MERCIER – Holoprosencphaly study using Human and Animal models – Dir. Thèse Valérie DUPE 19/12/2013: Leslie RATIE – Study of the Notch function during early forebrain development – Dir. Thèse Valérie DUPE Since 10/2013: Charlotte MOUDEN – Dir. Thèse Véronique DAVID Starting 10/2015: Houda HAMDI-ROZE – Dir. Thèse Valérie DUPE Post-doctorants 2013-2015: Michelle WARE (ANR), post-doctoral fellow 2 years & 3 months

108

Team 6 "Gene expression and oncogenesis" Leader: Marie-Dominique GALIBERT

109

2.1. Team presentation

Cancer is one of the most common cause of death world wide in the 20th century and the number of new cases is still increasing. The pressure to understand the development of different cancers on a molecular level is instrumental. It is important for early diagnosis and for the identification of effective drug treatments. At a molecular level, precise gene expression programs and signal transduction pathways orchestrate initiation and progression of cancer. In this competitive context, the GEO team focuses on understanding the genetics mechanisms implicated in the transcriptional regulations that support tumour initiation and development. Two cancer diseases are under study:

- The UV-dependent skin cancers with melanoma being one of the most aggressive skin cancer with poor prognosis at the metastatic stage. Understanding what are the molecular changes in response to UV-irradiation and DNA-photolesions as the identification of molecular pathways for drug target design are thus a real challenge.

- The ETV6/RUNX1-positive leukemia, the most frequent childhood B-lineage acute lymphoblastic leukemia (B-ALL). Most treatment protocols result in a good outcome, although some patients suffer very late relapse, which cannot be anticipated at the diagnosis. Understanding the onset on the disease, as well as anticipating and treating the late relapse are thus real challenges with therapeutic stake.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

BOUSSEMART Lise MCU-PH2 UR1-CHU GANDEMER Virginie PU-PH2 UR1-CHU CORRE Sébastien CR1 INSERM GILOT David MCU UR1 DUPUY Alain PU-PH2 UR1-CHU TROADEC Marie-Bérengère CR1 CNRS GALIBERT Marie-Dominique PU-PH2 UR1-CHU RIO Anne-Gaelle TCS CNRS

110

2.2.2.2. Temporary staff

MOUCHET Nicolas Post-doc GAUTRON Arthur PhD student (2015/2018) BACHELOT Laura AI JACOBCZYK Hélene PhD student (2015/2018) DEBAIZE Lydie PhD student (2014/2017) MIGAULT Mélodie PhD student (2013/2016) DONNOU Emmanuelle PhD student (2013/2016) GAUDICHON Jérémy PhD student (2015/2018)

2.2.3. Achievements

2.2.3.1. Scientific achievements

Role of the USF1 transcription factor in directing the response of the skin to UV irradiation and in melanoma cells. We have demonstrated that USF1 is required in vivo to maintain genome integrity in response to UV-induced DNA-damage. USF1 regulates the expression of two actors of the UV-dependent DNA repair machinery Nucleotide Excision Repair machinery, HR23A and CSA. HR23A and CSA are implicated in the recognition of UV-induced DNA-damage. USF1 mediates the increase of the level of the p53 protein in response to UV-induce DNA damage. In vivo loss of USF1 promotes the loss of the tumor suppressor. Significance: Together these results demonstrate new roles of USF1 in orchestring in vivo the UV- response. These results are critical to further understand how UV-irradiation direct the development of skin cancers. Publication: This work is published in 2 articles: Baron et al., Plos Genetics 2012 ; Bouafia et al., Plos Genetics: 2014. Communication: This work has been presented as invited speaker at the following international meetings (IPCC, ESP, ESPCR in 2011, 2012 and 2013). Interaction: This work has been performed in collaboration with Dr. Sharon PRINCE (PI at the University of Cape Town, South Africa). Ongoing work is performed in collaboration with Gilles SALBERT and his team (Sp@rte, IGDR). Grants: The Cancer care foundation, LNCC and the Aviesan ITMO-Cancer fund this project for 2 and 3 years respectively.

Identification of an original and new mechanism that drives melanoma aggressiveness. We have demonstrated that in melanoma cells, TYRP1 mRNA harbors sponge activity, sequestering miRNAs. The release of these miRNAs and their binding to secondary targets blocks tumor growth. Secondary targets are identified and proved to govern melanoma aggressiveness. The expression level of actors of the sponge network predicts melanoma-patient overall survival. Therapeutic tools have been designed and are currently tested in vivo. Significance: This is the first example of a sponge network governing melanoma aggressiveness, being linked to patient OS. The drug design is original and unique in cancer treatment. Publication: The sponge-network and therapeutical tools have been patented in 2013, a manuscript is in preparation. Communication: The work has been selected for oral presentations at the PrimeOncology Paris Melanoma (Paris - 2014), the Melanoma Meeting (Iceland, 2015), the ESPCR Meeting (Edinburg 2015); for poster presentation at the Jacques Monod Conference: RNA meeting (Roscoff 2013), the Cell Symposia: Regulatory RNAs (San Francisco 2014) Interaction: This work is performed in collaboration with Pr. G. GHANEM (PI at the Jules Bordet Institute, Belgium), and the melanoma network set in the PAIR Melanoma project (coordinator: M.-D. GALIBERT). Grants: The work is supported by an INCa Grant (PAIR Melanoma) and the Accelerating Technology Transfer Society (SATT), (2 years).

Pathogenesis of the ETV6/RUNX1-positive leukemia: RUNX1 function and partners: ETV6/RUNX1 fusion gene is necessary but not sufficient to promote the development of B-ALL. We have previously reported that parallel to the presence of the ETV6/RUNX1 fusion, RUNX1 is highly and specifically expressed in ETV6/RUNX1 positive B-ALL suggesting a 111

role of RUNX1 in the pathogenesis of this leukemia. To explore the role of the RUNX1 protein along with the ETV6/RUNX1 fusion one, we have determined their distribution on the genome and their respective partners by ChromatinImmunoPrecipitation coupled with Sequencing or Mass Spectrometry. Partners and targets are currently validated. Significance: Together these results will contribute to understand the pathogenesis of the ETV6/RUNX1 positive ALL and the specific role of RUNX1. Since RUNX1 is a critical actor Publication: Manuscript in preparation. Interaction: This work is performed in collaboration with Gilles SALBERT team (Sp@rte IGDR) and Jason CARROLL team (Cambridge, UK). For in vivo testing zebrafish model has been developed, in collaboration with Hatem SABAAWY (New Jersey University, USA) and the INRA platform (Rennes). Communication: This work has been presented at the ASH meeting in 2014 as a poster, and has received the Young Scientist Award from the French Physiology Society (2013). Grants: This work is funded by an European Grant (4 years), LNCC grants, and SFCE grants (2 years).

Role of CD9 in ETV6/RUNX1-positive B-ALL late relapse: Very late relapse characterize ETV6/RUNX1-positive leukemia. The underpinning mechanism and actors that support late relapse are presently poorly understood. Using in vivo an in vitro experiments, we have demonstrated that the CD9 tetraspanin directs the homing of CD9-positive blast cells to the testis, a preferential site of ALL late relapses. Significance: First evidence of how CD9-positive pre-B leukemic cells may disseminate and promote late relapse. Publication: A manuscript has been submitted to the revue Blood, minor revision. Communication: This work has been presented at ASH meeting in 2014 as a poster. Grants: This work is funded by Cancer care foundations (LNCC and Laurette Fugain).

2.2.3.2. Scientific dissemination and influence

Alain DUPUY: Member of the board of the FHU CAMIn since 2014 – Scientific Director of the Hospital/ University of Rennes since 2014 - Member of the scientific board of the French Society of Dermatology (SFD) since 2012.

Marie-Dominique GALIBERT: Co-Director of the Hospital University Federation Cancer Microenvironment and Innovation (FHU CAMIn) since 2014 - Member of the National Scientific Council LNCC from 2011 to 2015 –Scientific director of the H2P2 core facility (Histopathology) since 2010 – Member of the Scientific board of Rennes Hospital (CRBSP) since 2012 – Member of the scientific board of the European Society of Pigment Cell Research since 2009 - Member of the scientific board of the French Society of Photobiology since 2012.

Virginie GANDEMER: Member of the board of the French Society of the Child Cancer since 2010 – Chief Editor of the French revue « Oncologie Hématologie Pédiatrique », official journal of the SFCE/SHIP since 2013 – Member of the board of the Onco Bretagne network.

David GILOT: Member of the Scientific Council of the Pharmaceutical University of Rennes since 2014.

2.2.3.3. Interaction with the economic, social and cultural environment

Marie-Dominique GALIBERT: Member of the board of the cancer care LNCC CD35.

2.3. Projects, scientific strategies & perspectives (5 years)

The GEO team aims to identify the underpinning genetic and epigenetic mechanisms that drive cancer development to propose adapted therapies.

112

The landscape of somatic driver mutations is now well established. Driver mutations are causally implicated in the initiation of the tumor. They confer clonal growth advantage. Understanding how these drivers act, altering functional interactions between genes and proteins and leading to the perturbation of biological networks is thus critical to uncover the mechanisms that support the development of tumors.

Driver mutations are necessary but not sufficient. Combinations of additional alterations are required to promote drivers-dependent initiation step, and to fully induce in situ tumor development, metastasis process and relapse. Combinations of gene alterations have been identified being critical for tumor development. Yet they do not elucidate the complexity of tumor development. Recent studies have underscored the role of the dark genome as key regulatory actors of biological process, and increasing body of evidence is pointing to its implication in cancer development. Besides genome regulatory elements like enhancers and super-enhancers, non-coding RNAs (miRNA, lncRNA) are key for a fine-tuning of gene expressions and gene splicing. It is now clear that microRNA dysregulation plays an important role in cancer onset and progression where miRs can function as both tumor promoters (oncomiRs) or tumor suppressors by targeting numerous biomolecules that are important in carcinogenesis (Esquela-Kerscher and Slack, 2006). Their effects are dependent on the genome sequence (SNP, mutations) and epigenetic modifications that may alter their expression and impact on functional regulatory elements. Understanding how ncRNAs network together with genome alterations and epigenome modifications impact cell destiny according to macro and microenvironment signaling and therefore cancer biology is now necessary.

To explore these underpinning mechanisms that support tumor development the GEO team aim to investigate two cancer-types as paradigms: UV-dependent melanoma and ETV6/RUNX1 positive acute leukemia.

A. UV-dependent MELANOMA: The dark genome as passenger actors of tumor development

Melanoma is among the most aggressive and treatment-resistant human cancer. It arises from malignant transformation of melanocytes, the melanin-producing cells present at the basal membrane of the skin epidermis and responsible for pigmentation and photo-protection. Epidemiologic studies have implicated intense intermittent solar exposure and sunburn during childhood in skin cancer (Whiteman et al., 2011). UVR are responsible for the high somatic-mutation rate of sun-exposed melanoma. However the exact contribution of ultraviolet radiation (UVR) in the pathogenesis of cutaneous melanoma is still a matter of debate (few UV-dependent driver mutation). We have identified a new mechanism that drives melanoma burden that we predict to be linked to UVR. Using a combination of methods, including mRNA immunoprecipitation followed by miRNA quantification, miRNA profiling, transcriptomic studies associated to the analysis of biological readouts, we have accumulated convincing evidence demonstrating that microRNA activities are altered due to the sequestration of miRNAs on the highly expressed TYRP1 mRNA. TYRP1 mRNA acts as a micro-RNA- sponge, buffering miRNA. This microRNA-sponge activity directs human melanoma aggressiveness (manuscript in preparation & Patent: EP N° 13306524.3 – PCT/ EP2014/073961).

TYRP1 mRNA is specifically and highly expressed in the melanocytic lineage. TYRP1 is a downstream target of the lineage specific MITF transcription factor that is shown to be critical for melanocyte development and melanoma promotion. The level and activity of MITF is directly dependent on the activation of the RAS-RAF-MEK-ERK signaling pathway that is found mutated in 60% of cutaneous melanoma, NRAS-BRAF mutations being typical driver mutations. TYRP1 is also a downstream target of the UV-activated transcription factor USF1 being regulated by the p38 signaling pathways according to environmental cues. This suggests that the level of TYRP1 mRNA and its sponge activity may be modulated in response to environmental cues including UVR and growth-dependent signaling pathways. Determining the respective weight of the Mitf and USF1 transcription factors and their regulation is critical.

Another level of complexity in understanding how microRNA sponge may be regulated is linked to determine the actors that promote the stability of the sponge (mRNA-binding proteins and co-factors, Lnc- RNA…) and resolve how are regulated the miRNA within the network. An important element regarding miRNA is the amount of available miRNA. This notion is poorly documented since presently only absolute level of miRNA can be technically quantified, which does not reflect the active fraction of miRNA per cell. Quantifying actors of the microRNA sponge network is thus required.

We have demonstrated that miRNA-sponge activity of TYRP1 mRNA is correlated to overall survival of patients suffering of metastatic melanoma (n=192). The quantification of only three members of the TYRP1 network is significantly predictive of patient OS. This has prompt us to evaluate the TYRP1 network 113

as potential therapeutical targets and proposed a new concept in resetting endogeneous anti-tumor mechanisms. Doing so, we have demonstrated that sponge network are promising therapeutical targets and made the proof-of-concept for using Target Site Blockers (TSBs). It remains however critical to better characterize their mechanism and monitor the use of these TSBs to propose adapted treatment for precision medicine.

Our objectives for the following five years are recapitulated in the above Figure and aim to: (1) Evaluate the impact of UV on the TYRP1 sponge-network and the weight of Mitf and USF1 transcription factors (2) Determine the role of the dark-genome in the regulation of the TYRP1 sponge-network (3) Explore new therapeutical approaches

We anticipate pinpointing new processes in melanoma onset, by exploring the role and mechanism of UV-dependent RNA-sponge network. With the identification of major breackthroughts: (i) Give new insights in the role of UVR in melanoma development (ii) Investigate the role of miRNA-sponge (miRNA and lncRNA) in the biology of melanoma (iii) Propose new therapies for precision medicine using TSB

This work will deepen our knowledge in the biology of melanoma by deciphering important and new pathways. More importantly, since miRNA-sponges are part of the general machinery that orchestrate gene expression and drive cells biology, our findings should help to identify new miRNA-sponge network in normal contaxt and disorders.

B. ETV6/RUNX1 dependent LEUKEMIA: how transcription factor network direct leukemia development and relapse

Chromosomal translocation involving transcription factors can result in cancer. The ETV6/RUNX1 acute lymphoblastic leukemia is such paradigm being the most represented hematological malignancy found in children. The 12/21 translocation lead to the fusion of the two transcription factors: the repressor ETV6 and the activator or repressor RUNX1 according to its cofactors. The ETV6/RUNX1 fusion gene is necessary but not sufficient for leukemia development. Among the secondary alteration that may support tumor development we have reported the overexpression of the normal copy of RUNX1 (Gandemer et al., 2007). Understanding how the RUNX1 factor and the ETV6/RUNX1 fusion protein that share common features and yet have distinct roles do interplay and interact with cofactors to promote leucemogenesis is critical. Identifying these mechanisms may help to understand late relapses observed in ETV6/RUNX1 leukemic patients. Together, this will permit to establish a hierarchy of signaling programs required for leukemia development, from the onset to the relapse, and will allow us to propose targeted therapeutic targets.

For the following five years, our research interest will focus on decoding the mechanisms of action of the key transcription factor RUNX1 involved in hematopoiesis and the leukemia-initiating transcription factor ETV6/RUNX1. We thus aim to (1): - characterize RUNX1-related transcription factor complexes by identifying the transcriptional partners of RUNX1 and ETV6/RUNX1, - determine regulation activity of the complexes using histone-mark immunoprecipitation,

114

- define genome-wide the subset of genes directly modulated by those transcription factor complexes using chromatin immunoprecipitation followed by sequencing and global gene expression approaches, - determine DNA binding motifs for those transcription factor complexes, - explore the role of the transcriptonal complexes in hematopoieis using zebrafish hematopoieisis as a readout, - define the role of the complexes in the onset and progression of leukemia using leukemic cells xenografts model in mouse, - and ultimately propose those complexes as specific therapeutic target for ETV6/RUNX1 leukemia.

Our research interest will also focus on exploring molecular mechanisms responsible for relapses of leukemic patients. We have shown that the tetraspanin CD9 was part of the ETV6/RUNX1 gene expression signature. We could further demonstrate that CD9 interfere with CXCL12/CXCR4 signaling by enhancing RAC1 activation via its C-terminal sequence. We demonstrated that RAC1 activation enhanced actin rearrangement and the formation of cytoplasmic extensions, promoting chemotactic migration to towards CXCL12 in the testis, a recognized site of late relapses for leukemia. This highlight the key role of CD9 in leukemia relapse. We now aim to (2): - better characterize the interactions between RAC1 and CD9 and study their impact on cytoskeleton, - explore specificities and tumorigenic abilities of the lymphoblasts that have disseminated in testis. To achieve this, we will perform serial xenografts in immunodeficient mice and analyze their phenotypic properties according to CD9 expression, - understand the transcriptional regulation of CD9 by exploring two mechanisms: (a) the regulation of CD9 by miRNAs through an indirect and direct manner using CD9 RNA-immonuprecipitation and analysis of miRNA in patients blasts, and (b) the role of hypoxia mediated by HIF.

Together this work will deepen our knowledge and propose new prospects in understanding late relapse that we anticipate to be critical for clinical practice.

2.4. Collaboration

2.4.1. Within the IGDR

Catherine ANDRE (Genetics of melanoma) - Jean MOSSER (High throughtput technologies) - Gilles SALBERT (Genome plasticity) - Reynald GILLET (p-bodies and RNA location) - Luc PAILLARD (RNA stability) – Olivier DELALANDE (Modelisation).

2.4.2. Other collaborations

Rennes Brice FELDEN, Inserm UMR-S 835 – UR1 (miRNA/RNA) - Joel BOUSTIE, CNRS UMR6226 –UR1 (Chemist group : synthetic and naturel molecules) - Karin TARTE, Inserm UMR-S917 (lymphoproliferatif syndrome) - Biosit core facilities.

France Brigitte BRESSAC, IGR-Paris (Genetic of human melanoma) - Eliette TOUATI, Pasteur Institute-Paris (USF1 regulation and cancer development) - Graca RAPOSO, Curie Institute-Paris (Melanosome, trafficking) - Lionel LARUE, Curie Institute-Orsay (Melanoma, mice model) - Sophie TARTARE-DECKERT, C3M-Nice (Melanoma, invasion 3D models) - Bernard MARI, IPMC, Nice (RNA-seq, miRNA) - Brigitte DRENO, CRCNA- Inserm U892-Nantes (Melanoma clinical network, Biorderm) - Tristan MONTIER, Inserm UMR1078, Université de Brest (Xenograft model) - Vincent PRALORAN, CNRS-UMR5164 Université de Bordeaux (ALL-B 115

and CD9).

Europe Ghanem GHANEM & Fabrice JOURNE, Jules Bordet Institute Belgium (Melanoma) - Jean Christophe MARINE, VIB Univeristy of Leuven, Belgium (PDTX, therapeutical approaches) - Colin GODING Ludwig Institute, Oxford, UK (USF1 and the UV response) - Jason Carroll, Cancer Research Institute, Cambridge, UK (ChIP-MS analysis).

USA Hatem SABAAWY, New Jersey University (ALL-B ETV6-RUNX1 zebrafish model) - Micheal SIKES, University of North Carolina (USF1 & cancer).

Canada Guy POIRIER, CHUL, Quebec (proteomics).

South Africa Sharon PRINCE, University of Cape Town (USF1 & cancer).

2.5. SWOT Analysis STRENGTHS Complementary expertise’s and background of the team members In 2012: 2 full time reserchers with tenure position Development of models and tools for in vivo and in vitro exploration Access to patients’ samples thanks to clinical networks Fruitful collaborations with teams Leader of the Hospital University Federation of Caner, Microenvironnement and Innovation : FHU CAMIn Attractive for Masters and PhD students Patents WEAKNESSES Few post-docs Few high impact publication OPPORTUNITIES Development of a new team Development of an innovative clinical strategy for medicine of precision New models: zebrafish – transgenic mice THREATS Size of the team Competition Financial support

116

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Galibert MD, Baron Y. Identification of Specific Protein/E-Box-Containing DNA Complexes: Lessons from the Ubiquitously Expressed USF Transcription Factors of the b-HLH-LZ Super Family. 2010, In: Higgins PJ (ed.) Transcription Factors: Methods and Protocols, Springer, 647:391-406. Methods Mol. Biol., Galibert MD, Corre S. In Vivo and In Vitro Tools to Identify and Study Transcriptional Regulation of USF-1 Target Genes. 2010, In: Higgins PJ (ed.) Transcription Factors: Methods and Protocols, Springer, 647:339-355. Methods Mol. Biol., Gandemer V, Aubry M, Roussel M, Rio AG, De Tayrac M, Vallée A, Mosser J, Ly-Sunnaram B, Galibert MD. CD9 expression can be used to predict childhood TEL/AML1-positive acute lymphoblastic leu kemia: Proposal for an accelerated diagnostic flowchart. Leuk. Res., 2010, 34(4):430-437. (IF 2,9) Mogha A, Fautrel A, Mouchet N, Guo N, Corre S, Adamski H, Watier E, Misery L, Galibert MD. Merkel cell polyomavirus small T antigen mRNA level is increased following in vivo UV-radiation. PLoS ONE, 2010, 5(7):e11423. (IF 4,4) Mouchet N, Adamski H, Bouvet R, Corre S, Courbebaisse Y, Watier E, Mosser J, Chesné C, Galibert MD. In vivo identification of solar radiation-responsive gene network: role of the p38 stress-dependent kinase. PLoS ONE, 2010, 5(5):e10776. (IF 4,4) Primot A, Mogha A, Corre S, Roberts K, Debbache J, Adamski H, Dreno B, Khammari A, Lesimple T, Mereau A, Goding CR, Galibert MD. ERK-regulated differential expression of the Mitf 6a/b splicing isoforms in melanoma. Pigment Cell Melanoma Res., 2010, 23(1):93-102. (IF 5,8)

2011 Amigo JD, Ming Yu, Troadec MB, Gwynn B, Cooney JD, Lambert AJ, Chi NC, Weiss MJ, Peters LL, Kaplan J, Cantor AB, Paw BH. The identification of distal cis-regulatory elements at the mouse mitoferrin loci using zebrafish transgenesis Mol Cell Biol. 2011 Apr;31(7):1344-56. Epub 2011 Jan 19. + journal cover (IF 5,28) De Tayrac M, Aubry M, Saïkali S, Etcheverry A, Surbled C, Guénot F, Galibert MD, Hamlat A, Lesimple T, Quillien V, Menei P, Mosser J. A 4-gene signature associated with clinical outcome in high-grade gliomas. Clin Cancer Res. 2011 Jan 15;17(2):317-27. (IF 8,72) Gilot D, Le Meur N, Giudicelli F, Le Vée M, Lagadic-Gossmann D, Théret N, Fardel O. RNAi-based screening identifies kinases interfering with dioxin-mediated up-regulation of CYP1A1 activity. PLoS One. 2011 Mar 29;6(3):e18261. (IF 4,4) Journe F, Id Boufker H, Van Kempen L, Galibert MD, Wiedig M, Salès F, Theunis A, Nonclercq D, Frau A, Laurent G, Awada A, Ghanem G. TYRP1 mRNA expression in melanoma metastases correlates with clinical outcome. Br J Cancer. 2011 Nov 22;105(11):1726-32. (IF 5,04) Khan J, Ezan F, Crémet JY, Fautrel A, Gilot D, Lambert M, Benaud C, Troadec MB, Prigent C. Overexpression of active Aurora-C kinase results in cell transformation and tumour formation. PLoS One. 2011;6(10):e26512. (IF 4,4) Martin-Chouly C, Morzadec C, Bonvalet M, Galibert MD, Fardel O, Vernhet L. Inorganic arsenic alters expression of immune and stress response genes in activated primary human T lymphocytes. Mol Immunol. 2011 Mar;48(6-7):956- 65. (IF 2,97) Troadec MB, Warner D, Wallace J, Thomas KR, Spangrude GJ, Phillips J, Khalimonchuk O, Paw BH, McVey Ward D, Kaplan J. Targeted deletion of the mouse mitoferrin1 gene : from anemia to porphyria. Blood. 2011 May 19;117(20):5494-502. Epub 2011 Feb 10. + journal cover (IF 10,45)

2012 Baron Y*, Corre S*, Mouchet N, Vaulont S, Prince S, Galibert MD. USF-1 is critical for maintaining genome integrity in response to UV-induced DNA photolesions. PLoS Genet. 2012 Jan;8(1):e1002470. * co-first-author (IF 8,51) A. Biondi, M. Schrappe, P. De Lorenzo, A. Castor, G. Lucchini, V. Gandemer, R. Pieters, J. Stary, G. Escherich, M. Campbell, C-K. Li, A. Vora, G. Lönnerholm, M. Aricò, J. Harbott, V. Saha, MG. Valsecchi. Efficacy and safety of Imatinib on Top of BFM-like Chemotherapy in Pediatric Patients with Ph+/BCR-ABL+ Acute Lymphoblastic Leukemia (Ph+ALL). The EsPhALL study. Lancet Oncology 2012 Sep;13(9):936-45. doi: 10.1016/S1470-2045(12)70377-7. (IF 24,72) Droitcourt C, Le Hô H, Adamski H, Le Gall F, Dupuy A. Docetaxel-induced photo-recall phenomenon. Photodermatol Photoimmunol Photomed, 2012, 28:222-223. (IF 1,26) Fenouille N, Tichet M, Dufies M, Pottier A, Mogha A, Soo JK, Rocchi S, Mallavialle A, Galibert MD, Khammari A, Lacour JP, Ballotti R, Deckert M, Tartare-Deckert S. The epithelial-mesenchymal transition (EMT) regulatory factor SLUG 117

(SNAI2) is a downstream target of SPARC and AKT in promoting melanoma cell invasion. PLoS One. 2012;7(7):e40378. (IF 3,73) V. Gandemer, S. Chevret, A. Petit, C. Vermylen, T. Leblanc, G. Michel, C. Schmitt, O. Lejars, P. Schneider, F. Demeocq, B. Bader-Meunier, F. Bernaudin, Y. Perel, MF. Auclerc, JM. Cayuela, , G. Leverger and A. Baruchel on behalf of the FRALLE group Excellent prognosis of late relapses of ETV6/RUNX1-positive childhood acute lymphoblastic leukemia: lessons from the FRALLE 93 protocol Haematologica 2012 May 11. (IF 5,86) Laurent V, Glaise D, Nübel T, Gilot D, Corlu A and Loyer P. Highly Efficient siRNA and Gene Transfer into Hepatocyte- like HepaRG Cells and Primary Human Hepatocytes: New Means for Drug Metabolism and Toxicity Studies. "Cytochrome P450 Protocols" 3rd edition, Methods in Molecular Biology Series. 2012. Editors: Ian R. Phillips, Paul Ortiz de Montellano, Elizabeth Shephard. Publishers: Humana Press. (IF 1,29) Lemasson G, Coquart N, Lebonvallet N, Boulais N, Galibert MD, Marcorelles P, Misery L. Presence of putative stem cells in Merkel cell carcinomas. J Eur Acad Dermatol Venereol. 2012 Jun;26(6):789-95.(IF 3,10) Reboutier D*, Troadec MB*, Crémet JY, Fukasawa K, Prigent C. Nucleophosmin/b23 activates aurora-a at the centrosome through phosphorylation of serine 89. J Cell Biol, 2012 Apr 2;197(1):19-26. Epub 2012 Mar 26.* co-first- author (IF 9,78)

2013 Caye A, Beldjord K, Mass-Malo K, Drunat S, Soulier J, Gandemer V, Baruchel A, Bertrand Y, Cavé H, Clappier E. Breakpoint-specific multiplex polymerase chain reaction allows the detection of IKZF1 intragenic deletions and minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia. Haematologica. 2013 Apr;98(4):597-601. doi: 10.3324/haematol.2012.073965. (IF 5,86) Chabbert C, Adamski H, Guillet G, Sassolas B, Misery L, Perrinaud A, Machet L, Quereux G, Esteve E, Solau-Gervais E, Saraux A, Polard E, Lesimple T, Le Gall F, Dreno B, Dupuy . Cutaneous melanoma in patients treated with tumour necrosis factor inhibitors: a retrospective series of 15 patients. J. Eur. Acad. Dermatol. Venereol., 2013, 28:1540- 1544. (IF 3,10) Dupuy , Heusse J-L. SPECT/CT for sentinel lymph node detection in patients with melan. JAMA, 2013, 309:232. (IF 4,6) El Hajj P, Journe F, Wiedig M, Laios I, Salès F, Galibert MD, Van Kempen LC, Spatz A, Badran B, Larsimont D, Awada A, Ghanem G. Tyrosinase-related protein 1 mRNA expression in lymph node metastases predicts overall survival in high-risk melanoma patients. Br J Cancer. 2013 Apr 30;108(8):1641-7 (IF 5,04) Lescoat A, Droitcourt C, Stock N, Le Gall F, Dupuy. Vemurafenib-induced eccrine squamous syringometaplasia. Dermatology, 2013, 226:362-364. (IF 1,57) Reboutier D*, Troadec MB*, Cremet JY, Chauvin L, Guen V, Salaun P, Prigent C. Aurora-a is involved in central spindle assembly through phosphorylation of ser 19 in p150glued. J Cell Biol 2013 Apr 1;201(1):65-79.* co-first-author (IF 9,78)

2014 Bessede A, Gargaro M, Pallotta MT, Matino D, Servillo G, Brunacci C, Bicciato S, Mazza E, Macchiarulo A, Vacca C, Iannitti R, Tissi L, Volpi C, Belladonna ML, Orabona C, Bianchi R, Lanz T, Platten M, Della Fazia MA, Piobbico D, Zelante T, Funakoshi H, Nakamura T, Gilot D, Denison MS, Guillemin GJ, DuHadaway JB, Prendergast GC, Metz R, Geffard M, Boon L, Pirro M, Iorio A, Veyret B, Romani L, Grohmann U, Fallarino F & Puccetti P. Aryl hydrocarbon receptor control of a disease tolerance defense pathway. Nature. 2014 Jul 10;511(7508):184-90. (IF 38,59) Bouafia A, Corre S, Gilot D, Mouchet N, Prince S & Galibert MD. p53 requires the stress sensor USF1 to direct appropriate cell fate decision. PLoS Genet. 2014. May 15;10(5):e1004309. (IF 8,52) Dadzie Oe, Jablonski Ng, Mahalingam M, Dupuy . Skin cancer, photoprotection, and skin of color. 2014, 71:586. Delehouzé C, Godl K, Loaëc N, Bruyère C, Desban N, Oumata N, Galons H, Roumeliotis TI, Giannopoulou EG, Grenet J, Twitchell D, Lahti J, Mouchet N, Galibert MD, Garbis SD, Meijer L. CDK/CK1 inhibitors roscovitine and CR8 downregulate amplified MYCN in neuroblastoma cells. Oncogene. 2014 Dec 11;33(50):5675-87. (IF 8,46) Gandemer V, Pochon C, Oger E, Dalle JH, Michel G, Schmitt C, de Berranger E, Galambrun C, Cavé H, Cayuela JM, Grardel N, Macintyre E, Margueritte G, Méchinaud F, Rorhlich P, Lutz P, Demeocq F, Schneider P, Plantaz D, Poirée M, Bordigoni P. Clinical value of pre-transplant minimal residual disease in childhood lymphoblastic leukaemia: the results of the French minimal residual disease-guided protocol. Br J Haematol. 2014 May;165(3):392-401. doi: 10.1111/bjh.12749. (IF 4,71) Gillard M, Cadieu E, De Brito C, Abadie J, Vergier B, Devauchelle P, Degorce F, Dréano S, Primot A, Dorso L, Lagadic M, Galibert F, Hédan B, Galibert MD, André C. Naturally occurring melanomas in dogs as models for non-UV pathways of human melanomas. Pigment Cell Melanoma Res. 2014 Jan;27(1):90-102. + journal cover (IF 5,8) Mouchet N, Coquart N, Lebonvallet N, Le Gall-Ianotto C, Mogha A, Fautrel A,Boulais N, Dréno B, Martin L, Hu W, Galibert MD, Misery L. Comparative transcriptional profiling of human Merkel cells and Merkel cell carcinoma. Exp Dermatol. 2014 Dec;23(12):928-30. (IF 3,76)

118

Øvrevik J, Låg M, Lecureur V, Gilot D, Lagadic-Gossmann D, Refsnes M, Schwarze PE, Skuland T, Becher R & Holme JA. AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells. Cell Commun Signal. 2014 Jul 24;12:48. (IF 5,5) Pochon C, Oger E, Michel G, Dalle J, Salmon A, Nelken B, Bertrand Y, Cavé H, Cayuela J, Grardel N, Macintyre E, Margueritte G, Méchinaud F, Rohrlich P, Paillard C, Demeocq F, Schneider P, Plantaz D, Poirée M, Eliaou J, Semana G, Drunat S, Jonveaux P, Bordigoni P, Gandemer V. Follow-up of post-transplant minimal residual disease and chimerism in childhood lymphoblastic leukaemia: 90 d to react. Br J Haematol. 2014 Dec 19. doi: 10.1111/bjh.13272. (IF 4,71) Tatyana Y. Doktorova, Reha Yildirimman, Liesbeth Ceelen, Mireia Vilardell, Tamara Vanhaecke, Mathieu Vinken, Gamze Ates, Anja Heymans, Hans Gmuender, Roque Bort, Raffaella Corvi, Pascal Phrakonkham, Ruoya Li, Nicolas Mouchet, Christophe Chesne, Joost van Delft, Jos Kleinjans, Jose Castell, Ralf Herwig, Vera Rogiers. Testing chemical carcino genicity by using a transcriptomics HepaRG-based model ? EXCLI Journal 2014;13:623-637 – ISSN 1611-2156 published: May 28, 2014

2015 Pracht M*, Mogha A*, Lespagnol A, Fautrel A, Mouchet N, Le Gall F, Paumier V, Lefeuvre-Plesse C, Rioux-Leclerc N, Mosser J, Oger E, Adamski H, Galibert MD*, Lesimple T*. Prognostic and predictive values of oncogenic BRAF, NRAS, c- KIT and MITF in cutaneous and mucous melanoma. J. Eur. Acad. Dermatol. Venereol., 2015, Janv.* equal contribution (IF 3,1) El Hajj P, Gilot D, Migault M, Theunis A, van Kempen LC, Salés F, Fayyad-Kazan H, Badran B, Larsimont D, Awada A, Bachelot L, Galibert MD, Ghanem G, Journe F. SNPs at miR-155 binding sites of TYRP1 explain discrepancy between mRNA and protein and refine TYRP1 prognostic value in melanoma. Br J Cancer. 2015 Jun 30;113(1):91-8. (IF 4,81) Arnaud MP, Vallée A, Robert G, Bonneau J, Leroy C, Varin-Blank N, RIO AG, Troadec MB, Galibert MD, and V Gandemer. CD9, a key actor in the dissemination of lymphoblastic leukemia, modulating CXCR4-mediated migration via RAC1 signaling, Blood (minor revision) (IF 10,45)

2. Patents (with licence)

New compounds as key modulators of the pigmentation, 18/03/2012 - WO2013156738 – Extensions US CN JP EP / ANR émergence.

Identification of a natural miRNA sponge and its use in Managing Human melanoma aggressiveness, 06/11/2013 – EP13306524. Extensions PCT/EP2014/073961 / SATT Ouest Valorisation.

3. Conferences (actual team members)

- International

2010 Marie-Dominique GALIBERT: UV, USF and DNA repair in melanoma. Melanoma Workshop : Emerging Concepts in Melanoma Biology Pave the Road to new thérapies, Nice, France, June 16-18 (Invited speaker) Marie-Dominique GALIBERT: TYRP1 function in melanoma progression. 16th Meeting of the Euroepan Society of Pigment Cell Research, Cambridge, UK, September 4-7 (Invited speaker) Marie-Dominique GALIBERT: Solar Radiation and Gene expression. 3rd Joint Photobiology Meeting of the French and Italian Society of Photobiology. Paris, France, October 25-26 (Invited speaker) Nicolas MOUCHET: In vivo identification of solar radiation-responsive gene network : rôle of the p38 stress-dependent kinase. 16th Meeting of the Euroepan Society of Pigment Cell Research, Cambridge, UK, September 4-7 (Selected speaker)

2011 Marie-Dominique GALIBERT: How does solar UV radiation initiate specific cellular responses. XXIst International Pigment Cell Conference (IPCC), Bordeaux, France, September 21-24 (Invited speaker - Chairman) Marie-Dominique GALIBERT / Sebastien CORRE : USF1 orchestrates a specific gene expression program in the DNA repair pathway in response to UV-induced DNA-damage. 41st Annual European Society of Dermatology Research (ESDR) Meeting, Barcelona, Spain, September 7-10 (Invited Speaker) Arianne MOGHA: Uncovered role of Tyrosinase-related Protein 1 (TYRP1) in melanoma cells aggressiveness. XXIst International Pigment Cell Conference (IPCC), Bordeaux, France, September 21-24 (Selected speaker)

2012 David GILOT: miRNA sponge : the ping-pong effect in melanoma. Jacques Monod Conference : ‘RNA: a key to coordination of gene expression’, Roscoff, France, November (Selected speaker)

119

Marie-Dominique GALIBERT: Characterization and comparison of canine and human melanomas : which types and which breds are good models for human melanomas. Melanocytes and Melanoma meeting : from basic science to clinical application, Malmö, Sweden, June 18-20 (Selected speaker) Marie-Dominique GALIBERT: USF1 and the UV response. 17th Meeting of the Euroepan Society of Pigment Cell Research, Geneva, Switzerland, September 11-13 (Invited speaker)

2013 Marie-Dominique GALIBERT: USF1 and the UV-response. Human Biology Departemental Seminar series, Cape Town, South Africa, April 25 (Invited speaker) Marie-Dominique GALIBERT: UV-solar radiation and skin cancers : the rôle of the USF1 transcription factor. 81 Congress of the ACFAS – Oncology meeting. Quebec, Canada, May 7–8 (Invited speaker) Marie-Dominique GALIBERT: New insights into the rôle of UV-radiation in melanoma development, How does USF1 orchestrate the UV-response. 5th European Melanoma Workshop : Basic and clinical research join forces to defeat the melanoma. Marseilles, France, June 26-28 (Invited speaker & Chairman) Marie-Dominique GALIBERT: How does USF1 orchestrate the UV-response ? 15th Congress of the Euroepan Society of Photobiology, Liège, Belgium, September 2-6 (Invited speaker) Marie-Dominique GALIBERT: How does USF1 orchestrate the UV-response ? 18th Meeting of the Euroepan Society of Pigment Cell Research, Lisbon, Portugal, September 9-12 (Selected speaker)

2014 Marie-Pierre ARNAUD: Disruption of CD9 expression affects adhesion, migration, and actin polymerization through RAC1 signalling pathway in ETV6/RUNX1 pre-B lymphocytes. 6th European Conference on Tetraspanins, Lille, France, June 18-20, (Selected speaker) Marie-Dominique GALIBERT: microRNA as a potent anti-melanoma mechanism : a new therapeutic strategy. Prime Oncology, Melanoma Conference, Paris, France, July 4-5 (Selected speaker) Marie-Dominique GALIBERT: XXIInd International Pigment Cell Conference (IPCC), Singapore, September 4-7 (Chairman) Marie-Dominique GALIBERT: USF1 transcription factor coordinates the skin response to UV-induced DNA-damage. Restoring microRNA activity to reset a potent anti-melanoma mechanism. 1rst Young Life Scientist’ Symposium Rennes, France, October 24. (Invited speaker)

2015 Marie-Dominique GALIBERT: Restoring microRNA activity to reset a potent anti-melanoma mechanism : a new therapeutic strategy. Melanoma meeting : from basic science to clinical application, Reykjavik, Iceland, June 24-26 (Selected speaker) Marie-Dominique GALIBERT: Restoring microRNA activity to reset a potent anti-melanoma mechanism. European Society of Pigment Cell Research (ESPCR), 19th meeting Edinburgh, Scotland, September 15-18. (Chairman & Selected speaker)

- National

2010 Marie-Dominique GALIBERT: Roles du facteur de transcription USF1 dans la réponse aux irradiations UV. Association Nationale des Professeurs et Maitres de Conférences Biochimistes des Facultés de Médecine, Lille, 9-10 Septembre (Invited speaker)

2011 Marie-Dominique GALIBERT: Mélanome et mutations en pratique ! Réunion du Groupe Ouest Melanome (GROUM), Concarneau, 14-15 Octobre (Invited speaker) Marie-Dominique GALIBERT: USF and the NER régulation in response to DNA-Damage, Séminaire Institut Curie Orsay, 4 Mars (Invited speaker)

2012 Marie-Dominique GALIBERT: La recherche des mutations BRAF. Réunion du Groupe Ouest Melanome (GROUM), Lorient, 4-5 Octobre (Invited speaker)

2013 Marie-Dominique GALIBERT: l’apport des nouveaux traitements dans la prise en charge du mélanome. Réunion du Groupe Ouest Melanome (GROUM), Saint-Quay Portrieux, 10-11 Octobre (Invited speaker)

120

Marie-Dominique GALIBERT: Les modèles animaux en cancérologie. Recherche en Santé les rencontres du Grand Ouest, Cancéropole GO, Nantes, 3 Décembre (Invited speaker) Marie-Bérengère TROADEC: 4ème Séminaire du réseau EFOR, Réseau d’Etudes Fonctionnelles chez les organismes modèles, Paris, 14 Février : The use of zebrafish to understand human leukemia (Invited speaker) Marie-Bérengère TROADEC: 8ème Congrès de Physiologie, Pharmacologie et Thérapeutique, Angers 24 Avril : Deciphering onset and relapse of childhood leukemia using zebrafish and mouse (Invited speaker)

2014 Marie-Pierre ARNAUD: 21ème congrès de la Société Française d'Hématologie, Le Club Hématopoïèse et Oncogenèse (CHO), Grasse, 28 Septembre- 1 Octobre : Disruption of CD9 expression affects adhesion, migration, and actin polymerization through RAC1 signalling pathway in ETV6/RUNX1 pre-B lymphocytes. (Invited speaker) Marie-Bérengère TROADEC: 5ème Séminaire du réseau EFOR, Réseau d’Etudes Fonctionnelles chez les organismes modèles, Paris, 13 Février : Zebrafish and cancer : the role of runx1 in childhood leukemia (Invited speaker)

2015 Marie-Dominique GALIBERT: Les aspects mécanistiques de la photoprotection : USF1 et p53 deux acteurs clés. Journées de la Société Francaise de Photobiologie, Paris 4-5 Juin (Invited speaker)

4. Funding

2007-2010 Région Bretagne – ARED 88,5 k€ 2008-2010 Université de Rennes 1 5,5 k€ 2010 FP7-ERG N°230985 2010-2012 (S. CORRE) 45 k€ 2010 FRM Post-Doc (Marie-Bérengère TROADEC) - 24 mois 88 k€ 2010 ARED LNCC/Region Bretagne (Amine BOUAFIA) - 42mois 105 k€ 2010 LNCC 35 (M.D. GALIBERT) 28 k€ 2010 LNCC 35 (V. GANDEMER) 25 k€ 2011 Institut Fédératif de Recherche 140 - Génétique fonctionnelle, Agronomie et Santé 6,7 k€ 2011 ARC 15 K€ 2011 Bourse de mobilité entrante (S. WANSLEBEN, South Africa) 4 k€ 2011 Collaboration de Recherche (S. PRINCE, South Africa) 5,4 k€ 2011 ANR Emergence (2011-2013 /M.D. GALIBERT) 184 k€ 2011 LNCC 35 (M.D. GALIBERT) 35 k€ 2011 Institut Mère Enfant (V. GANDEMER) 9 k€ 2011 UR1- Action Incitative 1,5 k€ 2011 Contrat Société Clarins (M.D. GALIBERT) 65 k€ 2011-2014 Région Bretagne et Ligue Nationale Contre le Cancer 90 K* 2012 SFD (2012-2013 /M.D. GALIBERT) 30 k€ 2012 LNCC 35 (M.D. GALIBERT) 35 k€ 2012 LNCC 35 (V. GANDEMER) 22,5 k€ 2012 Contrat Société Auriga (30 mois M.D. GALIBERT) 110 k€ 2012 AVIESAN ITMO Cancer (2012-2015 M.D. GALIBERT/C. ANDRÉ) 85 k€ 2012 UR1- Action Incitative 10 k€ 2012 FP7-People (2012-2016/ M.B. TROADEC) 100 k€ 2012 PHC Tournesol Mobilité France/ Belgique 1 k€ 2012 ARED LNCC/Region Bretagne (Marie-Pierre ARNAUD) - 42 mois 105 k€ 2012-2013 Ligue Nationale Contre le Cancer 15 K€ 2013 Laurette Fugain (2013-2015 / V. GANDEMER) 40 k€ 2013 LNCC 35 (M.B. TROADEC) 23 k€ 2013 LNCC 35 (S. CORRE) 30 k€ 2013 Equipment Rennes metropole (M.B. TROADEC) 40 k€ 2013 BIOSIT (M.B. TROADEC) 12 k€ 2013 AVIESAN M.D. GALIBERT Coordinator 360 k€ 2013 INCa PAIR Melanome Coordinator 350 k€ 235 k€ 2013 Université de Rennes 1 1 k€ 2013 LNCC PhD program (Emmanuelle DONNOU) - 36 mois 90 k€ 2013 Bourse Ministère (Kévin TUTORÉ) - 24 mois 60 k€ 2014 SFCE (2014-2016 M.B. TROADEC) 25 k€ 2014 Ligue Nationale Contre le Cancer 23 K€ 2014 LNCC 35 (M.B. TROADEC) 25 k€ 2014 SATT (18 mois M.D. GALIBERT) 224 k€ 2014 LNCC 35 (D. GILOT) 25 k€ 2014 ARED LNCC/Region Bretagne (Lydie DEBAIZE) - 36 mois 90 k€ 2014-2015 Ligue Nationale Contre le Cancer 15 K€ 2015 Cancéropole GO (MD. GALIBERT) 61 k€ 2015 LNCC 35 (S. CORRE) 30 k€ 2015 BIOSIT 4 k€ 121

2015 UR1 Mobility (D. GILOT) 4 k€ 2015 ARED LNCC/Region Bretagne (Arthur GAUTRON) - 36 mois 90 k€ 2015 ARED CAMIn/Region Bretagne (Jérémie GAUDICHON) - 36 mois 135 k€ 2015 Bourse Ministère (Hélène JACOBCZYK) - 36 mois 90 k€

5. Training Teaching Marie-Dominique GALIBERT organize the Master2 "Fondamental Oncology" courses. Faculté de Pharmacie 2013 - Laura FERRAND, 4e année de pharmacie UR1 (1 month) - Marion QUINIOU, 4e année de pharmacie UR1 (1 month) - Fabien LINDENBERG, 5e année de pharmacie UR1 (1,5 month) Master 2010 - Murielle GREGOIRE, M2 UR1 (6 months) - Na GUO, M2 UR1 (5 months) 2011 - Jena Kanha CHUN, M2 UR1 (5 months) - Na GUO, M2 UR1 (5 months) 2012 - Mélodie MIGAULT, M1 UR1 (4 months) - Catherine GUEZENNEC & Guillaume ROBERT, M1 UE11 UR1 (2 weeks) - Ronan ULVE, M2 UR1 (5 months) 2013 - Pauline SARAROLS, M1 UR1 (2 months) - Meryl ROBERT, M1 UR1 (3,5 months) - Lucie LEFEUVRE & Marion PINSARD, M1 UE11 UR1 (2 weeks) - Charlotte DEGORRE, M1 biologie santé UR1 (1 month) - Guillaume ROBERT, M2 UR1 (5 months) - Kévin TUTORE, M2 UR1 (5 months) - Morgane LENROUILLY, 3e année Ingénieur Agronome (5 months) 2014 - Naila BENKALFATE & Marie GRABAS, M1 UE11 UR1 (2 weeks) - Allan JINQ, M2 UR1 (5 months) 2015 - Erwan BODIOU, M2 UR1 (5 months) - Arthur GAUTRON, M2 UR1 (5 months) - Marie GRABAS, M2 UR1 (9 months) - Hélène JAKOBCZYK, M2 UR1 (5 months) Students Internships from foreign Universities 2012 - Sabina WANSLEBEN, PhD student from the University of Cape Town, South Africa (6 months) 2013 - Jagjeet KAUR SONDH, third year of University of , UK (8 months) 2015 - Sohely SIKDAR, PhD student from the University of Brussels, Belgium (2 months) PhD 10/02/2010: Nicolas MOUCHET (CIFFRE Proclaim)- Dir. Thèse M.D. GALIBERT, he is now a post-doctoral fellow in the team 12/07/2010: Ariane MOGHA (Ministère & team)- Dir. Thèse M.D. GALIBERT, she is now working as a technical & application specialist in the start-up CellProthera (tenure position) 28/06/2011: Audrey VALLEE (Région & ARC)- Dir. Thèse M.D. GALIBERT, she is now working at the genomic platform of Nantes Hospital, as an Engineer (Tenure position) 27/03/2014: Amine BOUAFIA (Région & Ligue) - Dir. Thèse M.D. GALIBERT & S. CORRE, he is now a post-doctoral fellow in the Niessen lab, Department of Dermatology and CECAD Cologne, University of Cologne 30/03/2015: Marie-Pierre ARNAUD (Région & Ligue) - Dir. Thèse V. GANDEMER, she has been accepted to enter the 3rd year of medical school Since 10/2012: Emmanuel DONNOU (LNCC) - Dir. Thèse M.D. GALIBERT & D. GILOT Since 12/2012: Mélodie MIGAULT (team contrat) –- Dir. Thèse D. GILOT & M.D. GALIBERT 10/2013-04/2015: Kévin TUTORE (Ministère) - Dir. Thèse M.D. GALIBERT & S. CORRE, stop thesis Since 10/2014: Lydie DEBAIZE (Région & Ligue) – Dir. Thèse M.B. TROADEC Starting 10/2015: - Arthur GAUTRON (Région & Ligue) – Dir. Thèse D. GILOT & M.D. GALIBERT - Hélène JACOBCZYK (Ministère) - Dir. Thèse M.B. TROADEC - Jérémie GAUDICHON (Région) - Dir. Thèse V. GANDEMER Post-doctorants 2008-2012 : Sébastien CORRE (CNRS & team contrat), post-doctoral fellow 3 years & 11 months – Now Researcher CR1 INSERM. 2010-2012 : Marie-Bérengère TROADEC (FRM & team contrat), post-doctoral fellow 2 years & 1 month – Now Researcher CR1 CNRS. 2010-2011 : Ariane MOGHA (team contrat), post-doctoral fellow 11,5 months. 2011-2016 : Nicolas MOUCHET (team contrat, ANR & Aviesan Itmo Cancer), post-doctoral fellow 5 years & 2 months. CDD 2012-2016: Laura BACHELOT - Engineer assistant UR1 and SATT OV, contracts 1 year & 8,5 months and 1 year & 6 months

122

Team 7 "Cytoskeleton and cell proliferation" Leader: Régis GIET

123

2.1. Team presentation

The mitotic spindle is a microtubule-based structure used to segregate the two lots of duplicated chromosomes in the two daughter cells. Therefore it is crucial to maintain euploidy. The mitotic spindle is also used during stem cell asymmetric division to differentially segregate cell fate determinants (anchored at the apical or basal cell cortex) in the daughter cells. This asymmetric cell division allows the two daughters to adopt different cell fates: one maintains a stem cell identity and continues to proliferate while the other daughter is subjected to differentiation. Defects in spindle orientation lead to abnormal delivery of cell fate determinants. As a consequence both daughter cells may acquire the proliferative stem cell fate, leading to tumour formation. Spindle orientation and assembly are therefore two highly important features that maintain tissue homeostasis and euploidy. We are interested by the role of microtubules and their regulation by Microtubule Associated Proteins (MAPs) during cell division in different cell types. We aim to analyse the consequences of MT misregulation on a multicellular traceable organism, Drosophila melanogaster, that can be easily imaged and manipulated genetically.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

GIET Régis DR2 CNRS PASCAL Aude TCE UR1 RICHARD-PARPAILLON Laurent MCU UR1

2.2.2.2. Temporary staff

CAOUS Renaud PhD student (2012/2016) METIVIER Mathieu PhD student (2015/2018)

2.2.3. Achievements

2.2.3.1. Scientific achievements

1) We showed that Drosophila Aurora A, a master kinase required for cell division is required to moderate dynein/dynactin affinity for spindle microtubules (MTs) following p150glued phosphorylation. This moderator function of Aurora A is crucial for spindle morphogenesis (Romé et al., JCB, 2010).

2) We established that CDK11p58 is required to target Plk4 on mitotic centrioles. This targeting is essential for subsequent centriole duplication during the following interphase (Franck et al., PLoS ONE 2012).

3) Our most important contribution is the study/ analysis of the MT interactome in Drosophila during syncytial development (M phase) and during the early steps of embryo morphogenesis (interphase/differentiation). We described (by RNAi screening) several genes required for cell division and development of the Drosophila larval brain (Gallaud et al., JCB, 2014).

124

4) The detailed study of the Drosophila Ensconsin (isolated in this screen) showed a dual role for this MAP for centrosome separation (interphase, in cooperation with its binding partner Kinesin 1) and in the control of Mt dynamics during M phase, a crucial step needed to promote the maintenance of a correct spindle length (Gallaud et al., JCB, 2014).

5) Contribution of mitotic timing to tumor suppression. We show that Drosophila aurA NBs are delayed in mitosis, and perform accurate chromosome segregation in a SAC independent manner maintaining the ability of aurA NB-like cells to proliferate and promote tumor formation, unlike other centrosomal mutants (Caous et al., Ms in preparation).

2.2.3.2. Scientific dissemination and influence

Dr RICHARD-PARPAILLON is in charge in designing, coordinating and teaching for the teaching unit of: "Cell communication", BSc, and "Molecular and Cellular Developmental Biology", Master 1. He teaches: "Integrated cell biology", BSc, and" Dynamic equilibrium in cell communication", Master 2, University of Rennes 1.

2.2.3.3. Interaction with the economic, social and cultural environment

Popularisation of science: Several PhD student have been deeply involved in the short movie science festival “Science en court” (2010: Pierre ROMÉ, Prize of the college student, 2012 Emmanuel GALLAUD et Renaud CAOUS, Special Jury prize).

2.3. Projects, scientific strategies & perspectives (5 years)

Introduction Microtubules are highly dynamic proteic fibres involved in the delivery of cellular cargoes and also in the segregation of chromosomes during cell division. Our work seeks to identify proteins that regulate the stability of the microtubules during mitosis (Microtubule Associated Proteins; MAPs) and how this impacts cell division, cell proliferation and the morphogenesis and development of tissues. For these studies we utilise the fruit fly Drosophila melanogaster, and specifically the Central Nervous System in which a small number of stem cells (Neuroblasts; NBs) give rise to most differentiated cells. This system shows a high homology to vertebrates while lacking much of its gene redundancy. Importantly, Drosophila offers a tractable system for examining protein functions throughout development, something not possible with vertebrate tissue culture cells and which is costly and time consuming using mammalian models such as the mouse. The originality of our work is to combine a biochemical approach to identify putative new candidate genes required for cell division and cell proliferation with the use of a genetic system to determine which of the corresponding genes are required for these two processes. Our group has identified and characterised Ensconsin/MAP7 as a crucial regulator of both centrosome distribution and chromosome segregation and we will continue to decipher the detailed function of this MAP. In addition, we believe that TBCE (an other gene identified by us as a putative “mitotic gene”) deserve to be characterised in details. Confirming that TBCE is involved during mitosis and disrupt mitotic spindle assembly (as suggested by our preliminary results), would represent a breakthrough in our understanding of Mt assembly during cell division. In addition to the molecular characterisation of Ensconsin/MAP7 and TBCE, a large aspect of our research will be to develop strategies to modify MT dynamics, MT bundling during cell division to modify spindle shape, length and assay how these changes impact Neural Stem Cell division (spindle orientation, asymmetric cell division, chromosome segregation) and ultimately tissue proliferation.

Separating ensconsin functions during stem cell division Our previous results showed that Ensconsin is required to promote (1)Mt polymerisation (to control spindle length during M phase) and (2) kinesin 1 activation (to stimulate centrosome separation, that occurs during interphase interphase in fly NBs) (Gallaud, E. et al., J Cell Biol 2014;204, 1111-1121). This is though to be mediated by 2 distinct domains that displays separate functions. These domains are located at the N and C-terminus domain respectively. However, the results from different papers are controversial. While one group showed that the N-terminal (MT binding domain) is dispensable for ensconsin function in cultured cells and that only the Kinesin 1 binding domain is sufficient to fulfil all ensconsin functions, an other group showed that the only function of ensconsin is to target Kinesin 1 to

125

the MT network (Metzger, T. et al., Nature 2012;484, 120-124) (Sung, H.H. et al., Dev Cell 2008;15, 866- 876) (Barlan, K. et al., Curr Biol 2013; 23(4):317-22). The fly model represents an ideal system to challenge each of these hypotheses in different tissues. We are currently expressing ensconsin truncation mutants in an ensc null mutant in an attempt to separate (and rescue) each of these 2 functions and to investigate their respective role during fly brain development but also during the development of the whole fly.

Ensconsin is required for male and female meiotic spindle assembly My team have obtained Preliminary exciting observations suggesting that meiotic spindle assembly is strongly impaired during female meiosis. We therefore started the detailed study of ensc mutant phenotype on a collaboration basis with Pr OHKURA (). In addition, Ensconsin also seems to play a key role during spermatocyte division. By contrast to ensc fly brain NBs, cytokinesis frequently fails during meiosis 1 and 2 suggesting that Ensconsin contribution to cell division varies between different cell types (Collaboration with Dr SAVOIAN, University Massey, NZ). In agreement with this hypothesis, Ensconsin association with spindle MT is down regulated during early (but not late) meiosis. We propose that this specific regulation is correlated with the presence of high tubulin levels (needed to build the flagellum) during spermatid elongation (Lattao, R. et al, J Cell Sci 2012;125, 584-588).

Do changes in spindle "architecture" interfere with NB asymmetric cell division? Ensc null flies have lower Mt velocity leading to short spindle formation in brain neuroblasts but without effect on cell division and mitotic spindle alignment (Gallaud, E. et al., J Cell Biol 2014;204, 1111-1121). This mutant is therefore a great tool to investigate if NBs harbouring short spindles interferes with the robust size asymmetry normally observed following NB division. In parallel, we also plan to examine more generally the consequences of modifying spindle size/architecture for cell size asymmetry and cell division. Spindle size but also spindle shape and morphogenesis can easily be modified as many conserved MAPs have already been identified (Goshima, G. et al., Science 2007;316, 417-421) (Goshima, G. et al., Curr Biol 2005;15, 1979-1988. This can be achieved by modifying the levels of key known MT motors and MAPs in the central nervous system by various genetic means including RNAi, overexpression or mutations. We will therefore challenge in live cells (in an intact tissues) how these modifications of the shape and length of the mitotic spindle interfere with asymmetric cell division as well as with NB fate and central brain development (Tumor formation or impaired development).

"Chaperoning" the mitotic spindle My team also aims to decipher the exact function of a protein identified in our previous "MAP screen" and named TBCE (Tubulin Co Factor E). This protein was described for decades as a Tubulin Chaperone but several results obtained recently by my team suggests it could play a more specific role during chromosome segregation and mitotic spindle assembly. We plan to use the Drosophila model to manipulate TBCE levels in space and time in the fly to investigate a possible role of TBCE during mitosis. We will use RNAi, available mutants and fast gene inactivation (DeGRAD GFP) to decipher the function of this poorly studied gene. Interestingly, in humans mutations were found in the TBCE gene. Affected individuals suffer of mental retardation, hypo parathyroidism and facial dysmorphism (Parvari, R. et al, Horm Res 2007; 67, 12-21) (Parvari, R. et al., Nat Genet 2002; 32, 448-452). However, evidences in these patients showed that tubulin levels are not affected, unlike microtubule networks. This is in agreement with a prominent role of TBCE in regulating Mt arrays rather than in regulating tubulin folding (an improper folding would lead to tubulin degradation) in the cell. These results in humans are corroborated by studies in Drosophila; flies lacking DmTBCE display normal tubulin levels but affected Mt network formation (Jin, S. et al.; Development 2009;136, 1571-1581). Confirming that TBCE is involved during mitosis and disrupt mitotic spindle assembly (as suggested by our preliminary results), would represent a breakthrough in our understanding of Mt assembly during cell division. Furthermore, we believe this could open the way for the possible design of new anti-cancer therapies. These pioneered experiments should be really helpful to decipher the primary role of TBCE and the possibility that this “tubulin chaperone” may become a unique and promising target to develop cell division inhibitors.

Investigating the role of the Deubiquitinating enzymes (DUBs) in Drosophila (Collaboration with the Group of Peter Deak, , Hungary) Protein ubiquitination is a central mechanism regulating the progression through the eukaryotic cell cycle and other intracellular processes. Similarly to other covalent modifications, such as

126

phosphorylation or methylation, ubiquitination is reversible (D'Arcy, P. & Linder, S., The international journal of biochemistry & cell biology 2012; 44, 1729-1738) (Eletr, Z.M. & Wilkinson, K.D., Biochim Biophys Acta 2014; 1843, 114-128). The project identified approximately 90 so called deubiquitinating enzymes or DUBs, which cleave ubiquitin off substrates and terminate ubiquitin-mediated signalling. DUBs have only lately been recognized as essential and specific components of diverse cellular pathways. Despite growing understanding of DUB enzymology and biochemistry, little is known about their physiological importance. In this project, we plan to examine the physiological roles of DUBs in vivo in a genetically well tractable model organism, Drosophila melanogaster. This goal will be achieved first through systematic RNAi screens of the 46 DUB genes in the fly central nervous system. The results of the RNAi screen will then be confirmed genetically through mutations in the corresponding genes. Mutants with interesting phenotypes will be subjected to detailed genetic, cytological and molecular biological analyses and the distribution of the DUBs protein will be examined by a combination of live microscopy.

2.4. Collaboration

2.4.1. Within the IGDR

Our team has set up national collaborations with several IGDR teams: Véronique DAVID (Mounden et al., 2015) - Christian JAULIN (Rakkaa et al., 2013) - Gilles SALBERT/Sébastien HUET (Gallaud et al., 2014) - Denis CHRETIEN (Gallaud et al., 2014).

2.4.2. Other collaborations

National Antoine GUICHET, PI at the Jacques Monod Institute (joined ARC libre application) - Roger KARESS, for the study of centrosome and SAC mutants to mitotic timing aneuploidy and tumor formation (Caous et al., ms in preparation).

International Our team has established International Collaboration with the group of: H. OHKURA (University of Edimburgh, UK to study female meiosis) - M. SAVOIAN (University Massey, NZ to study male meiosis) - P. DEAK (University of Szeged, Hun, for the fly DUB project) - G. POIRIER (Univeristy Laval, Quebec, CA, for all proteomic studies, Gallaud et al., 2014) - R. LÜHRMANN (Max Planck Institute, Germany, Schneider et al., 2010).

2.5. SWOT Analysis STRENGTHS Focused projects based on the passed expertise of the team (Project 32 and 36 are planned to be done on a collaboration basis). Good publication in the field of cell division in solid Cell Biology journals Constant national and local funding based of regular publication The team project is a niche, as most competitors in the field (although working on the same system) are looking at other aspects of stem cell biology. Existence of a Microtubule “hot spot” in Rennes (CHRETIEN and PECREAUX’s Labs) Presence of microscopy facilities CCP’s PI becomes regularly invited for national and international PhD committees (increased team standing). WEAKNESSES Lack of the top grants (ANR, ERC…) that are required to hire post-docs and increase team size to develop more ambitious projects. Team size (The PI is the only full time permanent research staff), the number of people in the group is sub-optimal. This makes problematic to combine student supervision, bureaucracy work, meeting participation…etc. The team reputation/standing (Invitation to give seminars, meetings…) has to be improved. This is to some extend caused by the youth of the team (the team was created in 2012) and the lack of top papers in the best journal in the field (Nature, Science, Cell…)

127

The number of International collaborations should be ameliorated to combine the efforts of the group with those of other peoples to address questions related to cell division, stem cell biology in other systems. The team strength is mostly dependent upon recruitment of PhD students, which is variable and tends to decrease over the years. OPPORTUNITIES Our group has potentially good papers in preparation that should secure continuous funding and (hopefully) top research grants. Our team has set up national collaborations with several IGDR teams (with the groups of V. DAVID, C. JAULIN, G. SALBERT/ S. HUET, D. CHRETIEN…) as well as with the group of A. GUICHET (joined ARC application, PI at the Jacques Monod Institute) and R. KARESS. Our team has estabished International Collaboration with the group of H. OHKURA (University of Edimburgh), the group of M. SAVOIAN (University Massey, NZ) and the group of G. POIRIER (University Laval, Quebec, CA). Future possible interaction/collaboration with the group of G. Rabut (IGDR) for the DUB project. This group that has a solid expertise in the field of protein ubiquitylation. THREATS

128

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

Within the team Cell Cycle (Claude PRIGENT) until Jan 1st 2012

2010 Schneider M, Hsiao HH, Will CL, Giet R, Urlaub H & Lührmann R. Human Prp4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation (2010). Nature Structural Molecular Biology, 17(2):216-221. (IF 11.6) Romé, P., Prigent., and Giet, R. (2010) Centrosome, microtubule and cancer. Medecine Sciences. 26(4):377-383. (IF 0.5) Romé, P., Montembault, E., Franck, N., Pascal, A., Glover, D. M., and Giet, R. (2010). Aurora A participates to dynactin phoshorylation and function during mitosis. Journal of Cell Biology, 189(4):651-9. (IF 9.7)

2011 Franck, N., Montembault, E., Romé, P., Pascal, A., Cremet, J. Y. and Giet, R. (2011). CDK11 is required for centriole duplication and Plk4 targeting to the mitotic centrosome. PLoS ONE, 6(1):e14600. (IF 3.5)

Team Cytoskeleton and Cell Proliferation (Regis GIET) created Jan 1st 2012

2014 Rannou Y, Salaun P, Benaud C, Khan J, Dutertre S, Giet R and Prigent C. (2012). MNK1 kinase activity is required for abscission. Journal of Cell Science, 15(125):2844-42. (IF 5.3) Rakkaa T, Escudé C, Giet R, Magnaghi-Jaulin L, Jaulin C. CDK11p58 kinase activity is required to protect sister chromatid cohesion at centromeres in mitosis. (2014). Chromosome Research, 22(3) :267-76. (IF 2.478) Gallaud E, Caous R, Pascal A, Bazile F, Gagné J-P, Poirier GG, Chrétien D, Richard-Parpaillon L and Giet R An ensconsin/MAP7-dependent prophase aster positioning and spindle assembly pathway. (2014). Journal of Cell Biology 204(7):2011-21. (IF 9.7)

2015 Mouden C, de Tayrac M, Dubourg C, Rose S, Carré W, Hamdi-Rozé H, Babron M-C, Akloul L, Héron-Longe B, Odent S, Dupé V, Giet R and David V. Homozygous STIL mutation causes holoprosencephaly and microcephaly in two siblings. PLoS ONE, 10(2):e0117418. (IF 3.5)

2. Patents (with licence)

N/A

3. Conferences (actual team members)

- International

2010 Conference Jacques Monod in Roscoff (France). Spindle Assembly in Space and time.

2012 Conference Jacques Monod in Roscoff (France). Cell division. EMBO meeting in EMBL-Microtubule Meeting. Heidelberg (Germany).

2013 EMBO Cell division in Drosophila meeting in Totnes (Devon UK).

2014 Conference Jacques Monod à Roscoff (France). Cell division meeting. EMBO meeting in EMBL-Microtubule Meeting. Heidelberg (Germany).

2015 EMBO Cell Cycle meeting Budapest (Hungary). Europeen Drosophila meeting in Heidelberg (Germany)

129

- National

2013 French Microtubule Network in Marseille (France)

2015 French Microtubule Network in Grenoble (France)

4. Funding

Before the creation of the team

2007-2010 ANR Programme Jeunes chercheuses et jeunes chercheurs 125,5 K€ 2010 ARED Region Bretagne (Emmanuel GALLAUD) 90 k€ 2010 ARC libre 144 k€ 2010 UR1 défits émergents 25 k€ 2011 BIOSIT microscopy 13 k€ 2011 LNCC 25 k€

After the création of the team Jan 1st 2012

2012 PhD fellowship Bourse Ministère (Renaud CAOUS) 90 k€ 2013 LNCC 25 k€ 2014 Projet Fondation ARC 25 k€ 2014 LNCC 4e année (Emmanuel GALLAUD) 15 k€ 2015 ARED LNCC/Region Bretagne (Mathieu METIVIER) 90 k€ 2015 Rennes Métropole (Visiting student Agota NAGY) 3.2 k€

5. Training Member of HDR Committees 2010: Referee for the HDR of Isabelle ARNAL (University of Rennes1, Dr D. CHRETIEN’s laboratory) 2012: Referee for the HDR of Stéphane BRUNET (University of Paris 6, Dr M.H. VERLHAC’s laboratory) 2015: Referee for the HDR of Anne ROYOU (). Member of thesis committees Jury member of theses 2010: Manuel BREUER (University of Paris 6, Dr M-H. VERLHAC’s laboratory). 2013: - Algassimou DIALLO (University Of Rennes 1, Dr C. PRIGENT’s laboratory). - Audrey GUESDON (University Of Rennes 1, Dr D CHRETIEN’s laboratory). - Lenaig DEFACHECHELLES (Université Diderot-ParisVII, Dr R. KARESS’s laboratory) - Vincent GUEN (University of Rennes 1, Dr P. COLAS, Roscoff) 2014: Elvira NIKALAYEVITCH (University of Edinburgh, Pr H.OHKURA’s Lab). 2015: Thomas JUNGAS (University of Toulouse, Dr A. DAVY’s Laboratory). Master 2012 Renaud CAOUS, M2 University of Rennes 1 (6 months) 2014 Clémentine VILLENEUVE, M1 University of Rennes 1 (2 months) 2015 Mathieu METHIVIER, M2 University of Rennes 1 (6 months) PhD 28/10/2011: Pierre ROME, University of Rennes 1 23/04/2014: Emmanuel GALLAUD, University of Rennes 1 Since 10/2012: Renaud CAOUS, University of Rennes 1 Starting 10/2015: Mathieu METHIVIER, University of Rennes 1 Post-doctorants 2008-2010: Nathalie FRANCK, post-doctoral fellow 2 years & 5 months

130

Team 8 "Translation and folding" (TAF) Leader: Reynald GILLET

131

2.1. Team presentation

The team “Translation and Folding” is dedicated to the study of the macromolecular interactions involved in the translation and folding of proteins. They include the study of the ribosome, of the molecular chaperones that act upon the nascent polypeptides and of some misfolded protein conformations that aggregate and trigger conformational diseases. For the new contract the team will mainly switch to the investigation of “structures and functions of the RIBOSOME under stress conditions”.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

DECAUX Olivier PU-PH2 UR1-CHU (10%) GIUDICE Emmanuel MCU UR1 DELAMARCHE Christian PU1 UR1 THOMAS Daniel DREM CNRS GARNIER Cyrille MCU UR1 CHAT Sophie IE2 UR1 GILLET Reynald PU2 UR1

2.2.2.2. Temporary staff

GUYOMAR Charlotte PhD Student (2015-2018) MACE Kévin PhD Student (2013-2016)

2.2.3. Achievements

2.2.3.1. Scientific achievements

Our team has a strong expertise and is internationally known in EM methods and image processing. We conducted the first cryo-electron microscopy studies of the bacterial ribosome during the quality control of protein synthesis by trans-translation. After setting up the team "CNRS UMR 6290, Translation and Folding", we combined these studies with a collaborative project with Dr. V. RAMAKRISHNAN (MRC- LMB, Cambridge, UK) on the crystal structures of similar ribosomal complexes at atomic resolution (Neubauer, Gillet et al., Science 2012). Thanks to the amazing recent advances of cryo-EM, we are now

132

capable to determine our ribosome molecular structures at high resolution (<4Å). Several of these structures, including yet unknown steps of trans-translation or unknown precursors of bacterial ribosomes are currently under study in the lab and will be released soon (see an example below).

Bacterial ribosome ongoing trans-translation (Giudice and Gillet, unpublished)

We also obtained by electron tomography the first structural data on Processing bodies (P-bodies) in situ and their intricate links with active polysomes (Cougot et al., J Mol Biol 2012; RNA Biol 2013). We demonstrated the links between P-bodies disassembly and cancer progression in human HER2- positive (HER2+) breast cancer cells overexpressing MLN51 (J Cell Science 2014).

For the new contract the team will be reorganized and joined by a group of 4 microbiologists whose main topic is dedicated to stress response in bacteria. Such an expertise will strongly complement our current studies on trans-translation. Indeed, this system is particularly attractive for the development of new broad-spectrum antibiotics. Thanks to this strong input in microbiology we already started to synthesize new anti-trans-translation molecules and screen the molecules specifically targeting the trans- translational activity of bacterial cells. For this we have developed a double system, with a single recombinant plasmid carrying a green fluorescent and a red mCherry reporter. This is system is currently patented in partnership with "Ouest-Valorisation".

The PI recently obtained a junior Chair at the "Institut Universitaire de France" (IUF). This prestigious and honorific distinction is awarded to very few (< 2%) professors, and aims to recognize and promote high-quality and interdisciplinary research done by university professors at the international level. We obtained during the recent period (2010-2014) several grants from ANR (x4); Ligue National contre le Cancer (x3); IUF for a total amount of ~1000k€. We also obtained allocations for computational time on national super calculators (DARI), for a total amount of 1 770 000 “CPU hours” over the last 4 years (estimated cost: ~48675 Euros/year).

Peer-reviewed publications (2010-2015): 38 accepted; 4 under review; 4 front covers:

2.2.3.2. Scientific dissemination and influence

Reynald GILLET was nominated in 2015 as a permanent Associate Editor on the editorial board of Frontiers in Microbiology (IF 3.9).

Reynald GILLET Guest Editor of the Special Issue of BIOCHIMIE (IF 3.55) devoted to "Quality Control in Protein Synthesis". BIOCHIMIE is an International Journal of Biochemistry and Molecular Biology published under the auspices of the Société Française de Biochimie et Biologie Moléculaire. Issue to be published in July 2015 (17 articles, ~150 pages).

Reynald GILLET is an expert reviewer for several funding agencies that include the french ANR, CNRS; ville de Paris, région Aquitaine, FNRS (Belgium), NSERC (Canada).

Reynald GILLET and colleagues are regular reviewers for several international peer-reviewed journals including Nucleic Acids Res; J Biol Chem; RNA; Biochimie; Frontiers in Microbiology, etc.

2.2.3.3. Interaction with the economic, social and cultural environment

Reynald GILLET: supervisor of the Department ‘Biology and Expression of Genomes’: 9 teams, >120 persons from the Institute of Genetics and Development of Rennes (IGDR) - will be nominated as a 133

Director of the Institute for the next contract (2017-2022) - creator of a "Sciences Café" within the Institute in 2012 - elected member of the Scientific Board of the University of Rennes 1 since 2012 and member of its strategic committee (comité de pilotage) since 2015 - an elected member of the board of the French Society of Biochemistry and Molecular Biology (SFBBM) 2010-2014 - a co-organizer of the national meeting of the "Institut Universitaire de France", Rennes, France (june 2016).

Daniel THOMAS was designated in 2011 as an honorary member of the French Society of Microscopies (SFµ).

Emmanuel GIUDICE has been an expert for Go Capital (https://www.gocapital.fr/).

The team participated to the exhibition "Sciences and Arts" 10 april-15 June 2014 (campus de Beaulieu, Rennes) for the international year of crystallography.

2.3. Projects, scientific strategies & perspectives (5 years)

Our research program will consist of three lines of investigation whose paths cross one another constantly into the general theme of "structures and functions of the RIBOSOME under stress conditions". The ribosome is the fundamental ribonucleoprotein complex that mediates protein synthesis in all living cells. At a first level we will study ribosome biogenesis in bacteria. The precursors to the small and large ribosomal subunits accumulate under severe heat stress. We will use these conditions to purify these assembly intermediates and identify their structures and composition. At a second level, we will study quality control events during trans-translation, the main system ensuring the recycling of stalled translating ribosomes and the degradation of incomplete nascent proteins when incomplete messenger RNAs (mRNAs) are translated under several stress conditions. This should lead to the presentation of a near-complete mechanistic model of trans-translation at the molecular and cellular levels. Strikingly, trans-translation is essential to the survival of many pathogenic bacteria. At a third level, we will screen molecules specifically targeting the trans-translational activity of bacterial cells. We will validate the molecular and cellular effectiveness of the molecules in vitro and in vivo on a large panel of pathogenic bacteria, including multi-resistant hospital strains which pose a major public health problem.

Innovative essence of the project The innovative essence of the project is its multidisciplinary and multiscale approach which aims to unravel the still unexplored details of bacterial translation during translational stresses. The amount of molecular and structural information collected will permit an in-depth understanding of how bacterial stress adaptation enables bacteria to synthesize functional ribosomes and proteins. The complementarity between our varied approaches will improve our capacities for achieving quite significant progress in the discovery of new structures of ribosome intermediates, ribosomes ongoing trans-translation, in vitro and in vivo. This is a prerequisite for developing new antibiotics targeting trans-translation in pathogenic bacteria, a worldwide threat to public health.

Towards this aim the team will be reorganized by the recruitment of seven permanent and two temporary people, according to the following new flowchart:

Permanent staff (people who arrive in the team in Jan. 2017 are in red):

BLANCO Carlos PR1 UR1 THOMAS Daniel DREM CNRS BOUJARD Daniel PRHC UR1 TRAUTWETTER Annie MCU UR1 ERMEL Gwennola PR2 UR1 CHAT Sophie IE2 UR1 GILLET Reynald PU2 UR1 GEORGEAULT Sylvie TCN UR1 GIUDICE Emmanuel MCU UR1 UGUET Marie ADT UR1 (35%) GOUDE Ronan MCU UR1

134

Temporary staff:

GUYOMAR Charlotte PhD Student (2015-2018) THOMET Manon PhD Student (2015-2018)

Research plan for the next 5 years

Line 1: Ribosome biogenesis

Work package 1 (WP1): structure; identification of authentic ribosomal precursors Coordinator: Annie TRAUTWETTER The ribosome is an essential ribonucleoprotein enzyme responsible for protein synthesis, and its biogenesis is a fundamental process in all living cells. Though, the details of the fine-tuned assembly process still remain unknown. Investigations will focus on elucidating the cellular processes that facilitate biogenesis of the bacterial ribosomal subunits in vivo. Towards this aim, authentic precursors, (not degraded or dead-end particles) 32S and 45S particles (precursors to large 50S subunits) and 21S particles (precursors to small 30S subunits) will be overproduced under severe heat stress in Escherichia coli that is reminiscent of that observed at lower temperatures in E. coli mutants lacking the molecular chaperones dnaK or dnaJ genes. Using genetic screens, single particle analysis from cryo-EM images and proteomic data we will describe the structures and composition of the assembly intermediates.

Work package 2 (WP2): post-transcriptional and post-translational modifications Coordinator: Gwennola ERMEL The bacterial ribosome is made of three distinct ribosomal RNAs (16S, 23S and 5S rRNAs) and about 50 different proteins. The ribosome in Escherichia coli contains 36 modified nucleotides such as base or ribose methylations and pseudouridines. These modifications may impact assembly and activity of the ribosome. Moreover, ribosomal proteins encounter posttranslational modifications including acetylation and methylation. All the genetic determinants are known but the functions of these modifications are poorly understood. We plan (i) to analyse the influence of stress on ribosomal modifications and (ii) to study the impact of these modifications on both efficiency and specialisation of ribosomes for selective translation of stress mRNAs.

Line 2: Structural aspects of ribosome rescue

Work package 1 (WP1): Structural analysis of the ribosome during the trans-translation Coordinator: Emmanuel GIUDICE We will conduct the studies of ribosomal complexes ongoing trans-translation by cryo-electron microscopy: cryo-EM, imaging and single particle reconstruction. We will focus specifically on the late

135

steps of trans-translation, as well as on the interactions of tmRNA.SmpB with RNase R and the ribosome. We have access to both local and remote state-of-the art cryo-EM facilities as well as the Dutch NeCEN platform (www.necen.nl), an open access facility for advanced cryo-transmission electron microscopes, making it possible to get trans-translational ribosomal image reconstructions at a resolution of <4Å. Our expertise in biomolecular modeling and "in-house" computational facility will allow for the reconstruction process of the entire structure inside the electron density map.

Work package (WP2): To obtain a deeper understanding of polysomes stalling and rescue Coordinator: Renan GOUDE Messenger RNAs (mRNAs) are recognized and read by numerous ribosomes that are clustered into polysomes, held together by the mRNA, and move along the mRNA as the polypeptide chains elongate. While the spatial organization of bacterial polysomes has been well studied in vitro, little is known about how they cluster when cellular conditions are highly constrained. We will focus this WP on the three dimensional organization of prokaryotic native polysomes under stress conditions, when the rescue systems are absent or overwhelmed. High-pressure freezing of cells over-producing polysomes and cryo- electron tomography of vitreous sections (TOVIS) will be used to identify the topology of polysomes required to maintain optimal translation efficiency and cell survival.

Line 3: A new class of antibiotics inhibiting bacterial trans-translation

Work package 1 (WP1): screening and characterization of new inhibitors of bacterial trans- translation Coordinator: Reynald GILLET From a broad screening of over 600,000 compounds, less than 50 compounds were recently selected for inhibiting trans-translation and having antibiotic activity (Ramadoss et al., 2013). Of these, we will conduct a medicinal chemistry program based on the structure–activity relationship (SAR) of the compounds. To screen the molecules specifically targeting the trans-translational activity of bacterial cells, we have developed a double system, with a single recombinant plasmid carrying a green fluorescent and a red mCherry reporter. When trans-translation is ineffective, the GFP protein is no longer removed and the cells become fluorescent green. When the compound targets the proteases that degrade trans- translated proteins, the cells become yellow (green + red).

Work package 2 (WP2): Activity and resistance of the compounds in vivo Coordinator: Carlos BLANCO Following this initial analysis, we will test the toxicity and then the effect of different molecules against bacteria harvested from clinical isolates. In collaboration with the Hospital of Caen that has access to a large collection of pathogens, and is the National Reference Centre (CNR) for enterococci drug resistance, such as enterobacteriaceae highly resistant to beta-lactamases (ESBL), methicillin-resistant staphylococci (MRSA) or vancomycin-resistant enterococci (VRE). In addition, synergy studies with antibiotics typically used in therapy will be conducted. This project will be funded by a new ANR grant running until 2018. Global expected achievements Ribosomal translation of genetic code into proteins is the foundation of life for all cell types. However ribosomes regularly stall on mRNAs, jeopardizing cell survival, and the main bacterial rescue mechanism for addressing this is trans-translation. Trans–translation, which does not exist in eukaryote cells, is essential for pathogen virulence. The system is driven by the unconventional transfer-messenger RNA (tmRNA, an RNA having both tRNA and mRNA capabilities) and the protein SmpB. Our team will use a multidisciplinary and integrated structural biology approach to analyse several elusive steps of trans- translation. This should lead to a near-complete mechanistic model of trans-translation. We will use the same strategy to solve yet unknown intermediates of ribosome biogenesis in bacteria. Each of these intermediates is a very attractive target for the development of new broad-spectrum antibiotics, and the ability to solve related structures in atomic detail is a prerequisite for their development.

Our team has a strong background and is internationally-known in cryo-EM studies of the bacterial ribosome. The team has permanent access to Rennes’ electron microscopy facility (IBISA), which provides academic or private research teams with transmission electron microscopy techniques. However, to improve resolution, the most promising samples (as estimated by the presence of tmRNA and partners in the 3D maps of the ribosomes) will be processed on the NeCEN (Netherlands Center for Electron Nanoscopy) open access facility. The NeCEN, with whom we have worked for three years, offers access to two FEI Titan Krios microscopes equipped with very sensitive Falcon direct electron detectors 136

(http://www.necen.nl/).

We have also two in-house computing clusters, permitting the use of advanced methods of image processing and parallel computation for investigation of complex large macromolecular structures using single particle analysis.

Towards this aim we recently obtained two important contracts dedicated to this ambitious project: ANR ASTRID recherche duale civile et militaire "La trans-traduction comme cible pour une nouvelle classe d’antibiotiques" R. GILLET, 300 k€ (2014-2017) and "Homme & Animal : Une Seule Santé" SATT Idfinnov, Pulsalys et Ouest Valorisation E. GIUDICE, 200 k€. We are also expecting the results of the recent ANR blanche "transBact" (R. GILLET coordinator). "Trans-translation rescues stalled ribosomes in bacteria: a multiscale structural analysis" 511 k€ (ranked 14th over hundreds of applications during the first round).

Running this ambitious project implies combining complementary expertise. The transdisciplinarity of our team’s permanent staff as key members is a must for the success of the project, as they have a strong background and expertise in biochemistry, microbiology, cryo-EM studies of macromolecular complexes, and molecular modelling.

2.4. Collaboration

2.4.1. Within the IGDR

Team Jean-François HUBERT (molecular modeling ; cryo-EM ; SAXS on ribosomes and dystrophin) - Team Luc PAILLARD (RNAseq).

2.4.2. Other collaborations

Rennes Pierre VAN de WEGHE, ISCR UMR CNRS 6226, Rennes (antibiotics).

National Jean-Christophe GIARD, Unité de Recherche Risques Microbiens (U2RM), Caen (antibiotics) - Bruno SARGUEIL, Laboratoire de Cristallographie et RMN Biologiques, CNRS, Paris (ribosome structures) -Sylvie NONIN, Laboratoire de Cristallographie et RMN Biologiques, CNRS Paris (trans-translation) - Patrice VACHETTE, Institut de Biologie Intégrative de la Cellule, UMR 9198, Gif sur Yvette (SAXS) - Guy SHOEN, EMBL, Grenoble (cryo-EM).

International Alan WARREN, MRC LMB, UK (ribosome structures) - Venki RAMAKRISHNAN, MRC-LMB, UK (ribosome structures) - Isabella MÖLL, Max perutz laboratory, Vienna, Austria (specialized ribosomes).

2.5. SWOT Analysis STRENGTHS Up-to-date expertise in EM methods Grants (total amount ∼ 600 k€) A lab. with a multidisciplinary approach Implication in higher education Implication in the Institute Track record Manpower WEAKNESSES Dispersion (too many topics) Low level of collaborative projects Implication in higher education

137

OPPORTUNITIES Amazing advances in cryo-EM ("resolution revolution") In house EM platform Partnership with french Army THREATS High level of competition Microbiology in an "eukaryotic" institute Ongrowing dependence on international EM facilities (need of a last generation microscope equipped with direct electron detector) Computational facilities

138

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Moules V, Ferraris O, Terrier O, Giudice E, Yver M, Rolland JP, Bouscambert Duchamp M, Bergeron C, Ottmann M, Fournier E, Traversier A, Boule C, Rivoire A, Yipu L, Hay A, Valette M, Marquet R, Rosa-Calatrava M, Naffakh N, Schoehn G, Thomas D, Lina B. (2010) In vitro characterization of naturally occurring influenza H3NA- viruses lacking the NA gene segment: toward a new mechanism of viral resistance? Virology. 404:215-24 (IF 3.3) L. Moullintraffort, M. Bruneaux, D. Allegro, A. Nazabal, P. Barbier, D. Thomas, C. Garnier. The molecular chaperone Hsp90's oligomers: when the bird makes its nest (2010). FEBS J 277(1) 292. (IF 3.1) Moullintraffort L, Bruneaux M, Nazabal A, Allegro D, Giudice E, Zal F, Peyrot V, Barbier P, Thomas D, Garnier C. (2010) Biochemical and biophysical characterization of the Mg2+-induced 90-kDa heat shock protein oligomers. J Biol Chem. 285 :15100-10 (IF 5.3) Pouget E, Fay N, Dujardin E, Jamin N, Perrin L, Pandit A, Rose T, Valéry C, Thomas D, Paternostre M, Artzner F. (2010). Elucidation of the self-assembly pathway of Lanreotide octapeptide into beta-sheet nanotubes: role of two stable intermediates. Journal of the American Chemical Society. 132:4230-4241 (IF 9) Thomas D. and Cavalier A. (2010) Observation of membrane proteins in situ: AQPcic, the insect aquaporin example. In Membrane protein structure determination, J.-J. Lacapere editor, Methods in Molecular Biology, Humana press. 654:171-85. S. Valleix, P. Derreumaux, C. Garnier, F. Briki, M. Boimard, J. Doucet, N. Rioux-Leclercq, L. Martin, G. Grateau, M. Delpech, P. Le Pogamp. The VLITL aggregation-prone motif might trigger amyloid fibril formation of fibrinogen A alpha-chain frameshift variants in vivo (2010) Amyloid 17(1): 96-97. (IF 1.4) Weis F, Bron P, Rolland JP, Thomas D, Felden B, Gillet R. (2010) Accommodation of tmRNA-SmpB into stalled ribosomes: A cryo-EM study. RNA. 16:299-306 (IF 6) Weis F, Bron P, Giudice E, Rolland JP, Thomas D, Felden B, Gillet R. tmRNA-SmpB: a journey to the centre of the bacterial ribosome. EMBO J. 2010 Nov 17;29(22):3810-8. (IF 10.7) This paper was highlighted in EMBO J (EMBO J. 2010 Nov 17;29(22):3747-9) and also featured in Faculty of 1000 Biology (http://f1000.com.gate1.inist.fr/prime/7143956) Weis F, Moullintraffort L, Heichette C, Chrétien D, Garnier C. (2010). The 90-kDa heat shock protein HSP90 protects tubulin against thermal denaturation. J Biol Chem. 285:9525-34. (IF 5.3)

2011 Felden B., Gillet R. SmpB as the handyman of tmRNA during trans-translation. RNA biology, 8:440-9, 2011. (IF 5,6) Legrand B, Giudice E, Nicolas A, Delalande O, Le Rumeur E. Computational study of the human dystrophin repeats: interaction properties and molecular dynamics. PLoS One. 2011;6(8):e23819. (IF 4) Moulès V, Terrier O, Yver M, Riteau B, Moriscot C, Ferraris O, Julien T, Giudice E, Roland JP, Erny A, Bouscambert- Duchamp M, Frobert E, Rosa-Calatrava M, Yi Pu L, Hay A, Thomas D, Schoehn G, Lina B. (2011) Importance of viral genomic composition in modulating glycoprotein content on the surface of influenza virus particles. Virology 414:51- 62 (IF 3.2) Tarabout C, Roux S, Gobeaux F, Fay N, Pouget E, Meriadec C, Ligeti M, Thomas D, IJsselstijn M, Besselievre F, Buisson DA, Verbavatz JM, Petitjean M, Valéry C, Perrin L, Rousseau B, Franck Artzner F, Paternostre M, Cintrat JC. (2011) Control of peptide nanotube diameter by chemical modifications of an aromatic residue involved in a single close contact. Proc. Natl. Acad. Sci. U.S.A. 108:7679-7684. (IF 9.8)

2012 Cougot N, Cavalier A, Thomas D, Gillet R. The dual organization of P-bodies revealed by immunoelectron microscopy and electron tomography. J. Mol. Biol., 2012, 420 :17-28. (IF 4) Fournier E, Moules V, Essere B, Paillart JC, Sirbat JD, Isel C, Cavalier A, Rolland JP, Thomas D, Lina B, and Marquet R. A Supramolecular Assembly Formed by Influenza A Virus Genomic RNA Segments. Nucleic Acids Res., 2012, 40:2197-2209 (IF 8.8) Fournier, E., Moules, V., Essere, B., Paillart, J.-C., Sirbat, J.-D., Cavalier, A., Rolland, J.-P., Thomas, D., Lina, B., Isel, C. Marquet, R. Interaction network linking the human H3N2 influenza A virus genomic RNA segments. Vaccine, 2012, 30: 7359-7367 (IF 3) Gavazzi, C., Isel, C., Fournier, E., Moules, V., Cavalier, A., Thomas, D., Lina, B., Marquet, R. An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus. Nucleic Acid Res., 2012, 40: 2197-2209 (IF 8.8)

139

Gillet R. Origine, évolution et métamorphose du ribosome. À la recherche des vestiges d’un ancien «Monde ARN » in Variations, évolutions, métamorphoses, B. Pouderon et J. Casas éds, Presses Universitaires de Saint-Etienne, octobre 2012, p233-246 Neubauer C., Gillet R., Kelley AC., Ramakrishnan V. Decoding in the absence of a codon by tmRNA and SmpB in the ribosome. Science, 335:1366-9, 2012 (IF 31,364) This paper was highlighted in Nature Reviews Molecular Cell Biology 13, 280 (May 2012) and also featured in Faculty of 1000 Biology. (http://f1000.com.gate1.inist.fr/prime/14252956) Renard D, Garnier C, Lapp A, Schmitt C, Sanchez C. Structure of arabinogalactan-protein from Acacia gum: from porous ellipsoids to supramolecular architectures. Carbohydr Polym. 2012 Sep 1;90 (1):322-32 (IF 3.6)

2013 Cougot N., Giudice E., Gillet R., Thomas D. Analyse in situ d’interactions macromoléculaires par Tomographie Electronique : l’exemple des « Processing Bodies ». Rev. Fr. Histotechnol., 2013, 26, 1 :13 -20 Cougot, N., Molza, AE., Giudice, E., Cavalier, A., Thomas, D., Gillet, R. Structural organization of the polysomes adjacent to mammalian processing bodies (P-bodies). RNA Biol. 2013, 10: 314-20. (IF 4.8) Emily M., Talvas A. and Delamarche C. (2013) MetAmyl: a METa-predictor for AMYLoid proteins. PLoS One, 8, e79722. (IF 3.7) Giudice E., Gillet R. The task force that rescues stalled ribosomes in bacteria. Trends Biochem Sci. 38:403-11, 2013 (IF 10,847) Giudice E, Molza AE, Laurin Y, Nicolas A, Le Rumeur E, Delalande O. Molecular clues to the dystrophin-nNOS interaction: a theoretical approach. Biochemistry. 2013 Nov 5;52(44):7777-84. (IF 3.3)

2014 Cougot N, Daguenet E., Baguet A., Cavalier A., Thomas D., Bellaud P., Fautrel A., Godey F., Bertrand E., Tomasetto C., Gillet R. Overexpression of MLN51 triggers P-body disassembly and formation of a new type of RNA granules. J Cell Sci. 2014, 127: 4692-701. (IF 5.9) Cougot N., Molza AE., Delesques J., Giudice E., Cavalier A., Rolland JP., Ermel G., Blanco C., Thomas D., Gillet R. Visualizing compaction of polysomes in bacteria. J Mol Biol. 2014, 426: 377-88. (IF 4) Giudice E and Gillet R. Bacterial trans-Translation: From Functions to Applications, IN "Encyclopedia of Molecular Cell Biology and Molecular Medicine", Wiley-VCH Verlag, Weinheim, Germany, 2014, RNA Regulation, pp 1-33. Giudice E, Macé K, Gillet R. Trans-translation exposed: understanding the structures and functions of tmRNA-SmpB. Front Microbiol. 2014 Mar 21;5:113. (IF 3.9) Lepvrier E, Doigneaux C, Moullintraffort L, Nazabal A, Garnier C. Optimized Protocol for Protein Macrocomplexes Stabilization Using the EDC, 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide, Zero-Length Cross-Linker. Anal Chem. 2014 Nov 4;86(21):10524-30. (IF 5.7) Nicolas A, Raguénès-Nicol C, Ben Yaou R, Ameziane-Le Hir S, Chéron A, Vié V, Claustres M, Leturcq F, Delalande O, Hubert JF, Tuffery-Giraud S, Giudice E, Le Rumeur E; the French Network of Clinical Reference Centres for Neuromuscular Diseases (CORNEMUS). Becker muscular dystrophy severity is linked to the structure of dystrophin. Hum Mol Genet. 2014 Oct 27 pii: ddu537. (IF 7.7) Renard D, Lepvrier E, Garnier C, Roblin P, Nigen M, Sanchez C. Structure of glycoproteins from Acacia gum: an assembly of ring-like glycoproteins modules. Carbohydr Polym. 2014 99:736-47. (IF 3.5)

2015 De Sa Peixoto P, Bouchoux A, Huet S, Madec MN, Thomas D, Floury J, Gesan-Guiziou G. Diffusion and partitioning of macromolecules in casein microgels: evidence for size-dependent attractive interactions in a dense protein system. Langmuir 2015, in press (IF 4.3) De Sa Peixoto P, Roiland C, Thomas D, Briard-Bion V, Le Guellec R, Parayre S, Deutsch SM, Jan G, Guyomarc’h F. Recrystallized S-Layer protein of a probiotic propionibacterium: structural and nanomechanical changes upon temperature or pH shifts probed by solid-state NMR and AFM. Langmuir 2015, 31 :199-208. (IF 4.3) Gillet R. Quality control in protein synthesis. Biochimie. 2015 (in press) (Apr 29. pii: S0300-9084(15)00122-4) (IF 3.1) E. Lepvrier, L. Moullintraffort, M. Nigen, R. Goude, D. Allegro, P. Barbier, V. Peyrot, D. Thomas, A. Nazabal and C. Garnier. Hsp90 Oligomers Interact with the Aha1 Co-Chaperone: A new Outlook for the Hsp90 Chaperone Machineries. Anal Chem 2015, in press (IF 5.7) E. Lepvrier, L. Moullintraffort, M. Nigen, D. Allegro, P. Barbier, D. Thomas, A. Nazabal and C. Garnier. Hsp90 Oligomerization Process: How Can p23 Drive the Chaperone Machineries, Biochim. Biophys. Acta Proteins and Proteomics 2015, in press. (IF 2.747) Macé K, Giudice E, Gillet R. La synthèse des protéines par le ribosome : un chemin semé d’embûches. Médecine Sciences. 2015 31(3):282-90. (IF 0.52)

140

Félix Weis, Emmanuel Giudice, Mark Churcher, Li Jin, Christine Hilcenko, Chi C. Wong, David Traynor, Robert R. Kay, Alan J. Warren. Preparing the 60S ribosomal subunit for translation: structural basis of eIF6 release, Nature Structural and Molecular Biology 2015, in press (IF 11.633)

Articles peer reviewed and published in the proceedings of international bioinformatics meetings Altamiranda J, Aguilar J, and Delamarche C. (2011) Similarity of Amyloid Protein Motif using an Hybrid Intelligent System, IEEE Latin America transactions, ISSN: 1548-0992, 9, 700-710. Altamiranda J., Aguilar J. and Delamarche, C. (2011) Similitud de Motivos de Proteínas Amiloideas utilizando un Sistema Hibrido Inteligente, Revista IEEE Latinoamerica Transactions, 9, 1-11. Altamiranda J., Aguilar J. and Delamarche C. Comparacion y fusion de Motivos de Proteina β-amiloidea utilizando Computacion Inteligente. Revista de Estudios Transdiciplinarios RET, 3. Altamiranda J., Aguilar J., Torres R. and Delamarche C. (2012) Algorithm based on the ant colony optimization for DNA motif fusion, Mathematical models and methods in modern science, 3, 177-182. Altamiranda J., Aguilar J. and Delamarche C. (2013) Comparison and Fusion Model in Protein Motifs, Proceedings of the 2013 39th Latin American Computing Conference, CLEI. IEEE Xplore, 2, 229-240.

2. Patents (with licence)

Patent on the software "Metamyl »A METa-Predictor for AMYLoid Proteins" DELAMARCHE C., EMILY M. (AgroCampus), TALVAS A (IRISA) (Ongoing with Ouest Valorisation).

3. Conferences (actual team members)

- International

2013 Université Libre de Bruxelles. Institut de biologie et de médecine moléculaires (IBMM) Charleroi 20 septembre 2013. Quality control of protein synthesis under the eye of the electron microscope. R GILLET* (Invited speaker)

2014 Ribosome Alumni Meeting Cambridge UK 9th July 2014; Trans-translation exposed: understanding the structures and functions of tmRNA-SmpB; R GILLET* (Invited speaker) Embo Workshop Recoding: Reprogramming genetic decoding; Killarney, Ireland, 13 – 18 May 2014 Switching templates in the course of synthesizing one single protein R GILLET* (Invited speaker)

- National

2013 Marseille, AFMB. Contrôle qualité de la synthèse protéique sous l'œil du microscope électronique : le ribosome dans tous ses états ! 18 Mars 2013. R GILLET* (Invited speaker)

2014 6th Bordeaux RNA Club Workshop June 26-27, 2014. Trans-translation exposed: understanding the structures and functions of tmRNA-SmpB. R GILLET* (Invited speaker)

4. Funding

2009-2011 ANR Programme Jeunes chercheuses et jeunes chercheurs 209 K€ 2009-2012 ANR programme microbiologie, R. GILLET - partners 40 k€ 2010-2014 Computational time on national super calculators (DARI), E. GIUDICE 48,6 k€/year (total amount of 1 770 000 “CPU hours” over the last 4 years) 2011-2012 Ligue Nationale contre le cancer 131,5 K€ 2012 Prix Association Française contre l'Amylose, E. LE MOUEL (interne CHU), C. DELAMARCHE 20 k€ 2012 Biosit, R. GILLET 7 k€ 2011-2016 Institut Universitaire de France, R. GILLET 100 k€ 2011 Mobilité des doctorants J. ALTAMIRANDA (U. Murcia Vénézuéla), C. DELAMARCHE - co-tuteur 4,8 k€ 2011 Ligue Nationale contre le cancer, C. GARNIER 36 k€ 2012-2013 Région Bretagne – SAD 50 K€ 2011-2015 ANR JCJC, C. GARNIER 200 k€ 2012-2015 ANR programme blanc, R. GILLET & D. THOMAS - partners 40 k€ 2013 Défis scientifiques émergents UR1, C. DELAMARCHE (Coll. F. LE BIHAN, IETR Rennes) 1,20 k€ 141

2013 Ligue Nationale contre le cancer, R. GILLET 35 k€ 2014 ANR "ASTRID", R. GILLET 300 k€ 2015 "Homme & Animal : Une Seule Santé" SATT Idfinnov, E. GIUDICE 200 k€

5. Training Teaching Permanent people in the team include two assistant professors (E. GIUDICE and C. GARNIER) and two full professors (C. DELAMARCHE and R. GILLET). According to the French system, each Professor has to teach at least 192 hours per year: - Christian DELAMARCHE, Professor, Université Rennes 1: Fundamental Biochemistry and Bioinformatics - Reynald GILLET, Professor, Université Rennes 1: Molecular and Structural Biology - Cyrille GARNIER, Assistant Professor, Université Rennes 1: Biochemistry and Structural Biology - Emmanuel GIUDICE, Assistant Professor, Université Rennes 1: Bioinformatics Noticeable involvement in training Christian DELAMARCHE is in charge of the Continuing Education Center at the University of Rennes 1. Emmanuel GIUDICE and Christian DELAMARCHE are in charge of the master degree in "Bioinformatics & Genomics", University of Rennes 1. Emmanuel GIUDICE is a member of the educational board of the "Ecole Doctorale" VAS, in charge of the PhD students in biology in Rennes. Reynald GILLET is in charge of: Masters 1 and 2 Biochemistry from 2012, Faculty of Sciences, University Rennes 1. Reynald GILLET is in charge of the courses in "Molecular Biology" Master International "Molecular and Medicinal Chemistry" Hanoi, Viet-Nam. Reynald GILLET is a board member of the committee from the French society of biochemistry and molecular biology (SFBBM) dedicated to "Teaching Biochemistry". Emmanuel GIUDICE is in charge of the educational team of Bioinformatics within the Biology department of the University of Rennes 1. He represents the Biology department at the Informatics committee of the University. Licence 2012 - Manon FRADIN, L3 UR1 (6 weeks) 2013 - Cyrielle DOIGNEAUX, L2 UR1 (6 weeks) - Clémence BLOQUET, Licence Pro IUT de Carcassonne (4 months) 2014 - Cyrielle DOIGNEAUX, L3 UR1 (2 months) Master 2012 - Yoann LAURIN, M1 UR1 (2 months) - Sarah LEVIN-LAÏCHE, M2 UR1 (5 months) 2013 - Steven FRIED, M1 UR1 (3 months) - Kévin MACE, M2 UR1 (6 months) 2014 - Sylvain OYER, M1 UR1 (2 months) - Nadia SADOK, M1 UR1 (2 months) - Edwige LE MOUEL, M2 UR1 (9 months) 2015 - Marie GIEU & Ondine MASSANR, M1 UE11 UR1 (2 weeks) - Nathan ALARY, M1 UR1 (2 months) - Damien TOULLEC, M1 UR1 (2 months) - Marital BONAREK, M2 UR1 (5 months) - Caroline POCHLAUER, Erasmus UR1 (8 weeks) PhD 22/03/2012: Laura MOULLINTRAFFORT – Dir. Thèse Daniel THOMAS 04/11/2010: Félix WEIS – Dir. Thèse Reynald GILLET 15/12/2014: Claire SHIRMER – Dir. Thèse Christian DELAMARCHE & Cyrille GARNIER 10/03/2015: Fabrice GORREC (validation d’acquis d’expérience) – Dir. Thèse Reynald GILLET Since 10/2013: Kévin MACE – Dir. Thèse Reynald GILLET Post-doctorants 2011-2013: Nicolas COUGOT, post-doctoral fellow 2 years & 9 months 2012-2013: Jérémy DELESQUES, post-doctoral fellow 1 year & 6 months 2012-2013: Aurélie NICOLAS, post-doctoral fellow 1 year CDD 2011-2012: Anne-Elisbeth MOLZA –Engineer UR1, contract 4 months 2011-2014: Eléonord LEPVRIER –Engineer CNRS, contract 2 years & 9 months 2013: Audrey BROSSARD –Engineer assistant UR1, contract 2 months

142

Team 9 "Oocyte dynamics and implantation in mammals" Leader: Guillaume HALET

143

2.1. Team presentation

The team was created in 2009 by G. HALET, after the award of the ATIP fellowship from the CNRS. Research in the team is primarily focused on the signalling mechanisms that regulate oocyte meiosis and preimplantation embryo development, using the mouse as a model. The aim is to understand how oocytes/embryos develop in order to be competent for fertilization and implantation, and to highlight the key mechanisms involved, and thus possibly defective in human infertilities. As the main theme, we are studying the role of Rho GTPases in oocyte polarization and asymmetric divisions (emission of the polar bodies). We have also a strong interest in phosphoinositide signalling, especially PI3-kinase activation to produce the lipid PIP3 in oocytes and embryos. Our favorite approach is to record the dynamics of signalling intermediates involved in the signalling cascades elicited by Rho GTPase activation, or PI3- kinase activation, using imaging techniques. We are currently focusing on actin and myosin II dynamics in oocytes. We wish to expand our research interests towards follicular activation (role of PI3-kinase signalling) and embryo implantation (markers of uterine receptivity).

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

GUERRIER Daniel CR1 INSERM MORCEL Karine PH CHU Gynaecologist HALET Guillaume CR1 CNRS BOURDAIS Anne AI CNRS

2.2.2.2. Temporary staff

VIARD Patricia AHU UR1-CHU

2.2.3. Achievements

2.2.3.1. Scientific achievements

Identification of the GTPase Cdc42 as a key regulator of polarity establishment and maintenance, and polar body formation, in mouse oocytes. Polarization of the mouse oocyte cortex was first described over 30 years ago, yet its molecular mechanisms are still uncompletely understood. In regards of previous findings in other systems, we investigated a role for the small GTPase Cdc42 in oocyte polarization. Using imaging techniques in live oocytes, we have shown that Cdc42 is activated (GTP- 144

loaded) in a polarized fashion, in the cortical area facing the meiotic spindle. Cdc42 activation requires the generation of a gradient of active Ran GTPase, emanating from meiotic chromosomes, in a microtubule-independent manner (in contrast to Cdc42 activation in Xenopus oocytes). One role for Cdc42 in the polarized cortex is to recruit and activate its effector N-WASP, which promotes the formation of a polarized actin cap (via the activation of the Arp2/3 nucleator). Our data suggest that Cdc42-dependent actin filaments promote the membrane deformation necessary for polar body protrusion, especially in meiosis II. Inhibition of Cdc42 (using a dominant-negative mutant) results in N-WASP detachment from the cortex, the disappearance of the polarized actin cap, and a failure of polar body protrusion. Cdc42 thus appears as a key intermediate in the signalling cascade triggered by meiotic chromosomes to establish and sustain oocyte polarity, and to execute asymmetric divisions leading to polar body formation. Related publication: Dehapiot et al., Dev. Biol. 2013.

Identification of Ran GTPase activation as a trigger for the polarized inactivation of Ezrin/Radixin/Moesin (ERM), in oocyte polarization. Another feature of oocyte polarization is the loss of membrane microvilli in the polarized cortex overlying the meiotic spindle. This aspect of oocyte polarization has been little studied, though it defines the boundaries of the sperm-binding region on the oocyte surface. One likely mechanism for microvilli disappearance is the inactivation (dephosphorylation) of proteins of the ERM family, as revealed by the loss of phospho-ERM immuno-staining. We have shown that, alike actin cap formation, polarized ERM inactivation was under the control of the chromatin- centered Ran-GTP gradient. However, it does not require polarized Cdc42 activation. We conclude that Ran activates at least two distinct signalling cascades to polarize the oocyte cortex. Related publication : Dehapiot and Halet, Cell Cycle 2013.

Identification of the role of PI3-kinase (PI3K) beta in preimplantation embryo viability. In a previous study, we have reported for the first time the dynamics of PIP3 –the lipid product of class I PI3K- in mouse preimplantation embryos. Unexpectedly, PIP3 synthesis appeared to be constitutive, from the zygote to blastocyst stage. Pharmacological inhibition of PI3K resulted in developmental arrest at an early developmental stage, and massive apoptosis in blastomeres. To get further insights into the role and regulation of PI3K in early embryos, we have collaborated with the group of Prof. Bart VANHAESEBROECK (Institute of Cancer, London) who has generated a variety of mouse lines with constitutive (Knock In) inactivation of the PI3K catalytic subunit p110 (alpha, beta or delta isoforms). We have shown that homozygous inactivation of p110beta resulted in female subfertility, due to developmental arrest during preimplantation development of the embryos. The results suggest the involvement of maternal p110beta provided by the oocyte cytoplasm. Related publication : Guillermet-Guibert et al. PloS Genetics, in press

Identification of ITIH5 as a functional marker of uterine status (Work of D. GUERRIER and K. MORCEL, initiated before their joining the team in january 2015). Since 2010, D. GUERRIER, within the GPLD team (Head V. DAVID) at IGDR, conducted a research program focused on the characterization of the genetic mechanisms responsible for the congenital utero-vaginal aplasia syndrome (MRKH syndrome). This research field, where very few new data have appeared internationally, has led him to lean on a particular gene, ITIH5, which appeared to be rearranged in a patient, and therefore could be involved in the syndrome. After a very long break for health reasons, he has now joined the team, and is developping a translational research program based on ITIH5 protein, with promising developments for human reproductive health. The ITIH5 gene (Inter-α-Trypsin Inhibitor 5) encodes a secreted protein and component of the extracellular matrix, alike other members of this family (ITIH-1 to -4). In a seminal study in mice (where the coding parts of the gene share 81% homology with human and 88% at the protein level), we identified two isoforms of this protein, an embryonic form and an adult one. In the same animal model, the embryonic expression of ITIH5 specifically increases with female genital tract differentiation. In the adult uterus, its expression is found at low levels in the absence of gestation, whatever the stage of the estrous cycle. Conversely it greatly increases (∼ 50-fold), in the uterus during gestation with a first peak of expression corresponding to the implantation window, and a second one while the pregnant uterus increases in size. These results suggested that ITIH5 could be a functional marker of female genital tract differentiation, and of the uterine status in adults, especially at the critical time of implantation. This first led us to evaluate whether the ITIH5 protein could be measured in sera from 150 women presenting in the Emergency Department of Gynaecology (Rennes Univ. Hospital). Indeed, being an excreted molecule, assessing ITIH5 levels in a serum assay seemed feasible. Furthermore, it would represent the first uterus-specific marker assayable without invasive practice unlike other potential markers previously described. This first series of experiments, funded by the local research federation (SFR BIOSIT Rennes - "Funding for innovative/at risk scientific projects"), showed that ITIH5 levels could vary up 100-fold 145

according to the patients and thus their pathophysiological state. These first results allowed us, through a partnership with the technology transfer society SATT ("Société d'Accélération de Transfert de Technologies"), to conduct a second "maturation" phase, now nearing completion (June 2015). The latter, supported by the European Regional Development Fund (FEDER), aims at providing proofs of concept in humans, and was subject to a patent extended to Europe and the USA (No. WO2013135836 priority date 14/03/2012). It also allowed us to develop the first immunological tools (monoclonal antibodies) dedicated to the identification of the adult isoform of ITIH5, and its quantification by ELISA. Related publications : Morcel et al., 2011 ; Morcel et al., 2012.

2.2.3.2. Scientific dissemination and influence

Daniel GUERRIER: Coordinator of a clinical research network on Mullerian aplasia (Programme de Recherches sur les Aplasies Mullériennes/PRAM) ;

Daniel GUERRIER: member of the INSERM Specialized Scientific Committee 2 “Genetics, Epigenetics, Cancerology”;

Daniel GUERRIER: member of the AERES evaluation committee to evaluate INSERM unit 958 (Genetics of Diabetes, Head C. Julier) in 2013;

Guillaume HALET: 3 invited conferences since 2010 (see below);

Guillaume HALET: F1000 Biology Associate Faculty Member (since 2010);

Guillaume HALET (2013): honoured with the “Top 10% Best Reviewer” by the editor of Molecular Human Reproduction ;

Guillaume HALET: Scientific director of the animal facility of University Rennes 1 (since January 2015);

2.2.3.3. Interaction with the economic, social and cultural environment

Guillaume HALET: interviewed in 2015 by the "Sciences Ouest" magazine (in association with the Science Center "Espace des Sciences" of the city of Rennes) for a special issue dedicated to the current research developments in reproductive biology, and destined to a lay audience. Also, participation to the "Science café" that followed (podcast available at http://www.espace-sciences.org).

2.3. Projects, scientific strategies & perspectives (5 years)

• In the continuity of our past research activity, we will pursue our efforts into the identification of key signalling intermediates promoting chromatin-induced oocyte polarization and asymmetric division, with a focus on Rho GTPase signalling, and actin and myosin II dynamics;

• We will expand our research interests by exploring two major "black boxes" of reproductive biology: follicular activation, and embryo implantation/uterine receptivity.

We aim to recruit a new PhD student and post-doc (funding permitting) for the oocyte project; a dedicated engineer will be recruited to implement the industrial transfer of the concepts validated in the ITIH5 project.

Mechanism of oocyte polarization and asymmetric division

We aim to identify the molecular mechanisms by which Ran GTPase, activated in the vicinity of meiotic chromosomes, promotes oocyte polarization and polar body formation, in order to achieve correct haploidization of the maternal genome. Using imaging techniques (mainly confocal on live/fixed specimen), molecular tools and genetically-modified mouse strains, we will address the following points:

- Myosin II is activated in a polarized fashion in Metaphase II (MII) oocytes, and its role is still debated. We have identified a new, Cdc42-dependent mechanism for Myosin II activation at the oocyte cortex; we will describe a new model for asymmetric division of activated MII oocytes, that challenges the current view on MII oocyte asymmetric division. Our model will be based on the activation of two differentially-regulated pools of Myosin II with distinct functions;

- We will investigate whether Ran-induced oocyte polarization proceeds via the regulation of - 146

cargo complexes, and we will attempt to identify such cargos via a candidate approach. In particular, we will examine the involvement of protein phosphatases of the PP1/PP2A family in Ran-induced ERM phosphoregulation. We will assess a possible crosstalk between ERM dephosphorylation and Cdc42 activation at the cortex;

- We have identified Cdc42 as an upstream regulator of Par6beta polarization in the cortical region overlying the meiotic spindle. However, polarized Par6beta is not associated with atypical PKC, suggesting a non canonical role for oocyte Par6beta. We will investigate this role using the Cre/Lox strategy to knock out Par6beta specifically in oocytes (using the ZP3-Cre transgenic strain);

- The mechanisms regulating actin filament dynamics in oocytes are still poorly understood. We have recently characterized the LIM-kinase/cofilin pathway in mouse oocyte meiosis, and our data suggest a role for cofilin activation in the normal process of oocyte maturation, as well as during the MII arrest. Our data suggest a hierarchical actin monomer usage by competing actin filament nucleation machineries, such that the balance between cortical actin polymerization vs cytoplasmic actin meshwork maintenance, is altered in oocytes lacking functional cofilin. We will investigate the upstream signalling pathways regulating LIM-kinase activity in oocytes, and the phosphatase(s) involved in cofilin dephosphorylation. We will examine the functional consequences of cofilin activation (dephosphorylation) on cortical vs cytoplasmic actin dynamics, spindle positioning and asymmetric division. We will investigate the relative contributions of diverse actin filament nucleators, e.g. formins and the Arp2/3 complex, to cortical F-actin dynamics;

- Upon fertilization of the mouse oocyte, emission of the second polar body proceeds via a remarkable symmetry-breaking “spindle rotation” phenomenon, the mechanism of which has remained unexplained. We will examine whether symmetry is broken by a subtle imbalance in the Ran-induced signalling cascade (Cdc42/N-WASP/F-actin/Myosin II), based on the relative distance between the chromatin clusters at the poles of the anaphase spindle, and the overlying oocyte cortex. We will attempt to manipulate the stength of this signalling cascade at one pole (eg laser ablation) to check if the orientation of spindle rotation can be reversed, or whether it is predetermined. We will also examine the relative contribution of the two Myosin II pools (Cdc42-dependent vs Rho-dependent) in the symmetry breaking, using dominant-negative mutants and small molecule inhibitors;

- PI3-kinase activation was reported to regulate spindle migration to the oocyte cortex, possibly via the regulation of Formin-2 and the cytoplasmic actin meshwork. We have observed for the first time, constitutive, as well as ligand-induced PIP3 synthesis at the oocyte plasma membrane, but not on cytoplasmic structures as reported by others. To solve this contradiction, we will re-examine the role of PIP3 using improved PI3K inhibitors, PTEN overexpression and PI3K-inactivated oocytes (see below). We will describe the signalling pathways leading to PIP3 synthesis and we will examine the effects of the signalling lipid on cortical and cytoplasmic F-actin dynamics, spindle positioning and asymmetric division. PIP3 effectors involved in this process will be investigated.

PI3-kinase (PI3K) signalling in developing ovarian follicles

Follicular activation is a major enigma in reproductive biology. The PI3K/Akt pathway has recently received a lot of attention in this field, as an increasing number of studies point to oocyte PI3K activation being the trigger for follicular activation. This discovery holds hope for treatment of infertilities, based on assisted follicular activation protocols. However, the data presented so far are based on indirect evidence for oocyte PI3K activation (e.g. Foxo3A nuclear exclusion), which role in promoting follicule activation has been questioned.

We wish to examine directly whether follicular activation is associated with PIP3 synthesis in oocytes and whether PI3K inactivation prevents follicular activation. We will test various strategies (eg live imaging, tissue sections, immuno-EM, using PIP3-specific pleckstrin homology domains and anti-PIP3 antibodies) to detect PIP3 in live or fixed oocytes. In addition, through our existing collaboration with the group of Bart VANHAESEBROECK (Institute of Cancer, London), we will perform isoform-specific PI3K invalidation in oocytes (using the Cre/Lox strategy or PI3K Knock-In mutant mice) to demonstrate unambiguously that oocyte PI3K activation is required for follicular activation and/or development and survival of growing follicles. The PI3K isoform(s) involved will be investigated.

ITIH5 in uterine receptivity

Embryo implantation is a dynamic process observed during a limited period of time called implantation window, relying on both the receptivity of the endometrium, and the quality of the embryo 147

(blastocyst). In clinical practice, in vitro fertilization techniques provide informations -based on morphological and kinetic criteria- on the quality of the preimplantation embryo, but still do not allow the assessment of endometrial receptivity, the ultrasonographic measurement of endometrial thickness remaining insufficient to predict a favorable uterine environment for implantation. In this context, the pregnancy rate is currently 25 to 30% following embryo transfer.

In the next five years, the "implantation" project will primarily focus on the development and validation of a prognostic and diagnostic tool for implantation failure. This will take place in the framework of a close partnership between our research institute, the university hospital, and the local biological resources center. Moreover, the eligibility of the project for funding through the "One Health" call (which aim is to consolidate proof-of-concept research programs in order to validate new tools for improving Human and animal health, and to promote their translation to industry), offers the possibility to finalize and prepare it for industrial transfer by the end of 2015.

The next phase of the project will consist in a pre-clinical retrospective study aiming at correlating the levels of ITIH5 and other relevant markers (annexin A4, HOXA10, progesterone receptor and prolactin), to a successful implantation. Indeed it is well established that embryos cannot implant in a poorly matured endometrium, the development of which being related to the expression of various effectors including ITIH5. This should provide additional evidence for the role of ITIH5 in the establishment and in the quality of the uterine wall. This project has already been endorsed by the French Agence de la Biomédecine and funded. It also represents the starting point of a fundamental research project aiming at studying the regulation and the mode of action of ITIH5 in relation to implantation. This study, using the mouse as a model, will aim at analyzing the process of interaction between the embryo and the uterus, of which currently mostly the embryonic side is described. We will investigate the still controversial mechanisms of accession of the embryo to the endometrium and next, those of the trophoblast invasion with respect to endometrial receptivity. A precise follow-up of the above-mentioned effectors during the early phases of implantation will be conducted using immuno-histological approaches. This will be achieved through a direct comparison of the implantation site with the adjacent unmodified endometrial tissue.

2.4. Collaboration

2.4.1. Within the IGDR

Dr Sébastien Huet, Gilles SALBERT team (for the designing of image analysis tools to quantify dynamic events in mouse oocytes).

2.4.2. Other collaborations

National Dr Julie GUILLERMET-GUIBERT, Cancer Research Institute in Toulouse, France ( for the study of PI3K isoform specific signalling in ovarian follicles).

International Prof. John CARROLL, Monash University, Australia (for the study of Akt activation in oocytes, and polarization mechanisms) - Prof. Bart VANHAESEBROECK, Institute of Cancer, London UK (for the study of PI3K isoform-specific signalling in oocytes and early embryos).

2.5. SWOT Analysis STRENGTHS Range of complementary skills in the team International visibility regarding our oocyte research; rare expertise (France) ITIH5 project: no competition, originality, potential industrial development WEAKNESSES Small size of the team Delay in the progress/publication of our data, due to the reason above 148

Need for multi-year funding for the oocyte project Strategy not to publish the current results on ITIH5 until completion of the project OPPORTUNITIES We have recently made strong links with the local IVF unit (Laboratory for reproductive biology, Rennes Univ. Hospital) and we aim at setting up scientific collaborations to translate our findings and/or technical expertise into improved human reproductive medicine Partnership with hospital practitioners Participation to the creation of a start-up company THREATS Little opportunities of research funding targeted to reproductive biology Increasing worldwide competition in the field of oocyte biology Lengthy process for the recruitment of cohorts of patients Difficulties with the animal facility, which has reached saturation, slowing down the progress of our research

149

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2012 Adhikari D, Zheng W, Shen Y, Gorre N, Ning Y, Halet G, Kaldis P and Liu K (2012). Cdk1, but not Cdk2, is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes. Hum. Mol. Genet. 21:2476-2484. [IF: 6.7]

2013 Dehapiot B, Carrière V, Carroll J, Halet G (2013). Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes. Dev. Biol. 377:202-212. [IF: 3.6] Dehapiot B, Halet G (2013). Ran promotes oocyte polarization by regulating ERM (Ezrin/Radixin/Moesin) inactivation. Cell Cycle 12:1672-1678. [IF: 5.0]

2014 Rougier JS, Albesa M, Syam N, Halet G, Abriel H, Viard P (2014). Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote alpha2delta1-induced downregulation of Cav1.2 channels. Pflugers Arch. In press [IF: 3.1]

2015 Guillermet-Guibert J, Smith LB, Halet G, Whitehead MA, Pearce W, Rebourcet D, León K, Crépieux P, Nock G, Strömstedt M, Enerback M, Chelala C, Graupera M, Carroll J, Cosulich S, Saunders PTK, Huhtaniemi I and Vanhaesebroeck B (2015). Nover role for p110β PI 3-kinase in male fertility through regulation of androgen receptor activity in Sertoli cells. PLoS Genetics In press [IF: 7.5]

Other publications: relevant publications by D. GUERRIER and K. MORCEL, before their arrival in the team Morcel K, Watrin T, Jaffre F, Deschamps S, Omilli F, Pellerin I, Levêque J and Guerrier D (2012). Involvement of ITIH5, a candidate gene for congenital uterovaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome), in female genital tract development. Gene Expr. 15:207-214. [IF: 1.7] Morcel K, Watrin T, Pasquier L, Rochard L, Le Caignec C, Dubourg C, Loget P, Paniel BJ, Odent S, David V, Pellerin I, Bendavid C and Guerrier D (2011). Utero-vaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome) associated with deletions in known DiGeorge or DiGeorge-like loci. Orphanet J. Rare Dis. 6:9 [IF: 3.9]

2. Patents (with licence)

Patents by D. GUERRIER and K. MORCEL, before their arrival in the team 2012 : "ITIH5 as a diagnostic marker of uterine development and functional defects". European Patent # 13709136.9- 1405. North America # USP201261610590. Patent holders: D. GUERRIER and K. MORCEL.

3. Conferences (actual team members)

- International

2012 HALET Guillaume: Actin dynamics and Cdc42 signalling during asymmetric division in the mouse oocyte. Eggs in the UK- University College London, London, UK, 13 July 2012.

2013 HALET Guillaume: Cortical actin dynamics in the polarized mouse oocyte. EMBO Workshop Oocyte maturation and fertilization: lessons from canonical and emerging models. Banyuls-sur-mer, France, 12-15 June 2013.

- National

2014 HALET Guillaume: Regulation of oocyte maturation and polar body emission by Rho GTPases. “Ovocyte et Aptitude au Développement” Ecole Nationale Vétérinaire d’Alfort, 9 July 2014, France.

150

4. Funding

2009-2010 Communauté d'agglomération Rennes Métropole 75 K€ 2009-2010 Institut Fédératif de Recherche 140 - Génétique fonctionnelle, Agronomie et Santé 12 K€ 2009-2011 ATIP (CNRS grant) : 270 k€ 2010 Région Bretagne 62.5 k€ 2010 Partenariat Hubert Curien "PLATON" 6.5 k€ 2012 Université de Rennes 1 10 k€ 2013 Society for Reproduction and Fertility 3.5 k€ 2013 Structure Fédérative BIOSIT 4,7 k€ 2013 Agence de la Biomédecine 20 k€ 2013 Structure Fédérative BIOSIT 4.7 k€ 2013-2014 Fondation pour la Recherche Médicale 15,6 k€ 2014 Ligue Nationale Contre le Cancer 24.8 k€ 2014 Structure Fédérative BIOSIT 6 k€

Other relevant contracts obtained by D. GUERRIER or K. MORCEL, before their arrival in the team 2012 FEDER (D. GUERRIER) 100 k€ 2013 Structure Fédérative BIOSIT (D. GUERRIER) 6 k€ 2014 Agence de la Biomédecine (K. MORCEL) 15 k€

5. Training Licence 2010 - Pamy YA, L3 UR1 (6 weeks) 2012 - Glenn PANAGET, L2 UR1 (3 weeks) Master 2010 - Guillaume BERNAS, M1 UR1 (4 months) 2012 - Fabienne LE CANN, M1 UR1 (4 months) 2013 - Clotilde RIVES-LANGE, M1 UE11 UR1 (2 weeks) 2015 - Emilie LE BLÉVEC, M2 UBO Brest (6 months) PhD 27/05/2014: Benoît DEHAPIOT – Dir. Thèse Guillaume HALET Post-doctorants 2010-2013: Virginie CARRIÈRE, post-doctoral fellow 3 years & 3 months 2010-2014: Patricia VIARD post-doctoral fellow 2 years & 9 months

Other training by D. GUERRIER or K. MORCEL, before their arrival in the team Faculté de Médecine 2011 - Solène DUROS – DES Gyn. Ostr. UR1 (2 weeks) - Lydia FLAUX, 5e année de médecine de Rennes (2 weeks) 2014 - Kévin MARCHE, 1e année de médecine de Rennes (2 weeks) Licence 2010 - Gaëlle COLLET, L3 (6 weeks) 2012 - Pauline SARAROLS, L3 UR1 (6 weeks) 2014 - Laure LE CALVEZ, L3 UR1 (6 weeks) Master 2010 - Ali MTEYREK, M2 UR1 (5 months) 2011 - Nicolas FOLLEA - M1 UR1 (2 months) - Marie-Catherine VOLTZENLOGEL – M2 UR1 (5 months) 2012 - François-Galaad SCHORP - M1 UR1 (2 months) - Gaëlle COLLET, M2 (8,5 months) 2013 - Pauline BERNEAU - M1 UE11 UR1 (2 weeks) - Florent RABAULT, M1 UR1 (2 months) - Julien SALLAIS, M1 UR1 (2 months) 2014 - Solène DUROS, M2 UR1 (6 months)

151

Team 10 "Structure and molecular interactions" Leader: Jean-François HUBERT

153

2.1. Team presentation

How muscle cell membranes resit to mechanical stress due to contraction and relaxation is a challenging question. A network of subsarcolemal filamentous proteins interacts with the membrane bilayer through transmembrane proteins that are themselves associates to components of the extracellular matrix. Among this network, a key element is the protein dystrophin. Lack of dystrophin leds to the Duchenne Muscular Dystrophy (DMD), a severe genetic disease that affects 1/3800 male newborns. The less severe Becker Muscular Dystrophy (BMD) is caused by the expression in patients of truncated forms of dystrophin. The current development of gene therapies aim to restore the expression of short but efficient forms of dystrophin. However, very little is known about the structure of native as well as pathologic dystrophins. The expertise inside the team is structural biology. Our research project aims at better understanding the molecular basis for muscle membrane scaffolding in normal and DMD or BMD cells. Moreover, understanding the variability of BMD is to date challenging. Aiming to provide molecular explanations for this question, we develop specific bioinformatics tools. The team combines its in vitro and in silico structure to function approaches with data from our network of cell biologists and clinicians collaborators.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

DELALANDE Olivier MCU UR1 RAGUENES-NICOL Céline MCU UR1 HUBERT Jean-François PU1 UR1 ALLAIRE Agnès ADT UR1 (20%) LE RUMEUR Elisabeth MCU-PH1 UR1-CHU CHERON Angélique AI UR1

2.2.2.2. Temporary staff

CHENUEL Thomas AI MIAS-LUCQUIN Dominique PhD student DOS SANTOS MORAIS Raphaël PhD student (2015/2018) (2014-2017) MOLZA Anne-Elisabeth PhD student (2012-2015)

2.2.2.3. Permanent staff who left the team during the contract BONDON Arnaud DR2 CNRS until 09/2013 MOURET Liza MCU UR1 until 11/2014 POTTIER Sandrine AI UR1 until 02/2015 154

2.2.3. Achievements

2.2.3.1. Scientific achievements

J Biol Chem 2011, Sarkis et al. and the FASEB J. 2013 Sarkis et al. We demonstrated that the Actin Binding Domain 2 of dystrophin is able to create in vitro a functional link between a biomimetic membrane and filamentous cytoskeletal actin. This work was done by micro-rhelology methods in close interaction with the Physics Institute of Rennes. The shear stress properties of membrane were significantly modified from a viscous to a viscoelastic system when the actin binding domain 2 of human dystrophin was present in the sub phase together with filamentous actin. These results are from Joe SARKIS PhD work.

Orphanet J, 2012, Nicolas et al. We developed a database devoted to in frame mutations of dystrophin. This "edystrophin" tool allows to correlate dystrophin structure and function with clinical outcomes in Duchenne (DMD) and Becker (BMD) Muscular Dystrophy patients. This database allows to connect people from clinics to structural biology that aim to understand how Becker muscular dystrophy could be linked to specific protein domains following a given single or poly exon deletion.

J. Biol. Chem 2012 Acsadi et al., Hum. Mol. Genet. 2014, Nicolas et al., Annals of Neurology 2014, Findlay et al. We highlighted links between genotype - protein structure - phenotype of the disease in Becker Muscular Dystrophy (BMD) in collaboration with a network of American and French clinicians. All together, these results raised a point of blocking concerning the understanding of the severity of the phenotype of Becker Muscular Dystrophy. The primary to tertiary structure analysis by molecular modeling approaches allowed to simulate with a high degree of confidence the behavior of a given truncated dystrophin. This represents a highly valuable new tool for the community of scientists that aim to design or optimize strategies for muscular dystrophy.

Biochemistry 2013, Guidice et al., Molza & Mangat et al., submitted. We investigated at the atomic level the association mode of dystrophin with the neuronal nitroxyde synthase nNOS In collaboration with Dr. N. Menhart in Chicago. The proper targeting of nNOS to sarcolemma is a key step for muscular cell function. This targeting depends on the binding of the enzyme to repeats 16-17 of dystrophin. This part of dystrophin is located in a highly strategic an area corresponding to the hot spot of mutations leading to muscular dystrophies. Our in silico work together with in vitro work from Dr. Menhart allow to map at a residue level the interacting mode of both partners. Additionally, this allows to predict what the behavior of the interaction is when part of dystrophin is lacking in some BMDs.

Faraday discuss 2014, Molza et al., Delalande et al. submitted. We obtained the structure of the central domain of dystrophin. These structures cannot be solved through conventional X-ray of NMR methods. Therefore, we combined experimental (mainly Small Angle X-ray Scattering) and computational methods. We show that specifically placed kink angles within dystrophin result in a tortuous filamentous structure that is profoundly modified by the most frequent in-phase mutation found in Becker muscular dystrophy, partially explaining its severe phenotype. Both native and mutated dystrophin structures provide a guide for the future design of curative therapies.

Indeed, no structure of the protein was yet available. This represented an obstacle for further understanding of dystrophin structure to function relationship. Our findings allowed to overcome this difficulty and open the way for our ongoing studies about binding and dynamic properties of the protein, in native as well as in pathologic conditions.

2.2.3.2. Scientific dissemination and influence

Thanks to our findings from the 5 past years, the team is recognized as a major expert in structure-function studies of dystrophin in vitro and in silico.

Invitation Elisabteh LE RUMEUR to Alpbach symposium on coiled coil proteins (2013), Institute of Myology seminars (2014-2015), Olivier DELALANDE to Faraday Discussions (2014).

Olivier DELALANDE has been Workshop organiser at the IEEE VR international symposium (2015).

Jean-François HUBERT, Elisabteh LE RUMEUR and Olivier DELALANDE created the collaborative network with French and US clinicians (G. ACSADI, R. BEN YAOU, J. CHELLY, F. LETURCQ, S. TUFFERY- GIRAUD, K. FLANIGAN). The aim is to collect clinical data about BMD patients, in order to correlate them with the biophysical and bioinformatical data from the lab. The general goal is to understand the 155

molecular bases of the heterogeneity of BMD phenotypes. The network is constituted by clinicians from Rennes, the Hospital Cochin, The Institut de Myologie (Paris), the Faculté de medecine de Montpellier, with the help of the French network of clinical reference centres for neuromuscular diseases (CORNEMUS).

Jean-François HUBERT and Olivier DELALANDE initiated a collaborative work with the Soleil Synchrotron and Neutron line of LLB CEA. Our previous findings concerning the development of specific molecular modelling tools allowed us to obtain the three dimensional structures of most of the parts of the central domain of dystrophin under experimental SAXS constraints. On the basis of the potency of the approach, with Dr. S. COMBET at the CEA Saclay and Laboratoire Leon Brilloin (France) I developed a project that was selected for a PhD grant. Then, Raphaël DOS SANTOS MORAIS from the University of Nancy has been starting his PhD work in October 2014 in our team. The project was selected on the basis of the complementarity of the experimental and theoretical approaches that we proposed for the study of the binding of the actin binding domain 2 of dystrophin to membrane lipid models.

2.2.3.3. Interaction with the economic, social and cultural environment

The team each year contributes to the "fête de la science" in Rennes for exhibitions concerning our work and muscle linked genetic diseases. We were co-organisers of the local committee for all events of "the year of crystallography 2014". Members of the team regularly are involved in science discovering research sessions in high schools.

Olivier DELALANDE was invited to give a talk to a TEDx session in Rennes in 2014.

We have been asked by the US "Cure Duchenne Foundation" to provide scientific information to patient’s families. Our e-dystrophin database tool has proven particularly efficient for that purpose. We regularly are able to explain to patients families what exactly are the consequences of the mutation their boys are suffering from. This partnership led "Cure Duchenne" to fund a post-doc for one of our research projects in 2013.

Some clinicians for any parts of the world also come to us for molecular explanations of the protein phenotype of their patients.

We presented in 2014 our results to the Association Française contre les Myopathies (AFM).

2.3. Projects, scientific strategies & perspectives (5 years)

The name of the team moves from “structure and molecular interactions” to "Dystrophin Structure and Function in myopathies". Following the retirement of E. LE RUMEUR and the transfer of C. RAGUENES-NICOL to another research unit for scientific reasons, the work of the team will be focused on the molecular bases of dystrophin structure and function.

State of the art

Resistance of cell membranes to mechanical stress that occur during muscle contraction and relaxation is ensured by multiproteic complexes tightly associated with sarcolemma, the muscular cell membrane. One of the key components of these complexes is dystrophin, a large filamentous protein of 427 kDa, encoded by the longest human gene with 79 exons (DMD). This protein is organised in, a N- terminal calponin-like actin binding domain, a long filamentous central domain constituted by 24 spectrin- like repeats, and a C-terminal domain involved in binding to sarcolemma. Dystrophin is sub sarcolemmal and interacts with membrane lipids; it also bears binding sites for transmembrane proteins (beta- dystroglycan) and cytosolic proteins such as neuronal nitroxide synthase (nNOS), as well as binding sites for cytoskeletal proteins: F-actin, tubulin, intermediary filaments and plectin. The overall organisation and dynamics of these protein-lipid complexes are largely unknown.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are rare genetic diseases characterised by sarcolemma ruptures that led to muscular degeneration. The diseases are caused by mutations in the DMD gene. The protein is either completely lacking (DMD) or partially expressed as truncated forms (BMD). All attempts aiming to cure patient suffering from DMD and BMD failed until now. Indeed, due to the length of the wild type protein, no expression of the full length dystrophin can be expected. From mouse and dog animal models lacking dystrophin, therapeutic strategies have been proposed 1) exon skipping strategies aiming to restore the expression of a truncated but as functional as possible dystrophin (“mini-dystrophins”) 2) injection of mini genes. The challenge is 156

to design and express the best shortened form of dystrophin that would mimic the mildest phenotypes of BMD patients. In addition to the difficulties of obtaining the tools for exogenous protein expression in vivo, one major drawback to improve gene therapies is the lack of knowledge of the structural bases of native dystrophin function. Due to its size and its fibrous repetitive coiled-coil structure, no conventional X-ray crystallography nor NMR methods can be used for structure analysis of dystrophin. No full length structure of the protein is available. However, our work on dystrophin fragments of various size recently allowed to significantly increase knowledge by obtaining atomic structures covering the whole central domain of human native dystrophin (Delalande et al., submitted).

The aim of the present project is to continue the analysis of structure-function relationship of dystrophin. Based on the native structures that we recently obtained, our goal is to determine the structure of multiple BMD shortened forms of dystrophin, and therefore to better understand the link between structure-partnership-severity of the diseases. This will bring knowledge that will be helpful for future therapies improvements.

The project focuses in the field of structural biology, which is the actual expertise inside the team. To answer fundamental questions, collaborations with cell biologists and clinicians will be pursued. It should aim 1) to bring fundamental knowledge about the role of dystrophin in membrane scaffolding by interacting with sub-sarcolemmal cytoskeleton, and 2) to provide data useful for the design of mini- dystrophins for the cure of muscular dystrophies (DMD and BMD). Discovering the interacting modes of either native or pathologic dystrophin will be at the heart of our concerns.

Main parts of the project

Links between severity of Becker Muscular dystrophy and "proteic phenotype" Characterisation of the truncated dystrophins found in BMD patients Mutations found in the DMD gene are either frameshift (exon deletions or duplications, single mutations) or in frame exon deletions. The later are characterised by the expression in muscle of truncated dystrophins that ensure part of normal functions of the protein. Hundreds of such mutations have been identified in humans. These dystrophins are shortened in their central domain and are associated with BMDs. However the degree of severity of the BMD disease is not correlated to the length of the central domain. Moreover various phenotypes may be associated for a single kind of exon deletion. Our recent data from primary and secondary structure analysis, biochemistry experiments and molecular modelling, showed that some BMD-related structure alterations partly explain the clinical phenotype. Following our contribution to the predictive comprehension of the link between BMD mutations and severity of the disease, researchers more and more frequently contact us to correlate their experimental findings on cellular or animal models to our molecular modelling or biochemical data. As well, we are consulted by clinicians about the putative consequences of some mutations found in the family of BMD patients. We will express and purify selected forms of truncated dystrophins. Their structure will be obtained by the combination of SAXS and molecular modelling data, as we recently did for wild type fragments (Delalande et al. submitted). These three dimensional models will led to a fine mapping of interacting surfaces and transconformational potency. These parameters will be compared to those responsible of the behaviour of the wild type protein. This should allow us to establish the links with the corresponding phenotypes. These goals will be achieved in collaboration with our network of clinicians. The truncated forms that will be analysed first will be the most frequently found in the “hot spot” around exon 45. This part of the project is already funded by AFM grant obtained from January 2015 and for 2-3 years. Expected results: a better comprehension of the Becker Muscular Dystrophy.

Analysis of polymorphisms As exposed earlier, for a given exon deletion, severity of the resulting BMD is extremely variable. We would like to explore the hypothesis that the polymorphisms of dystrophin and its protein partners could be involved this large range in BMD phenotypes severity. We will investigate the nucleotidic polymorphism for targeted genes (DMD, nNOS) of healthy people by data mining in banks such as “the 1000 genome project”. This should allow to identify protein areas that would be critical for normal function of dystrophin or its partners: high-frequence polymorphism regions should provide an inversed mapping of strongly conserved regions. In parallel, in collaboration with the Myology Institute and France Génomique, we will look for polymorphisms in cohorts of BMD patients bearing each a single kind of exon deletion. The number of patients bearing deletions in the so called “hot-spot” is sufficient to expect doing these studies with a reasonable statistical 157

confidence. Depending on the results of this bio-informatics approach, the links between primary structure, 3D structure, flexibility and binding affinities will be experimentally studied and correlated to clinical data. Funding application for this project is in progress.

Dystrophin-lipid interactions: analysis by small angle diffraction (neutrons and X rays) and molecular modelling. Interactions between cytoskeletal actin, dystrophin and sarcolemma are a key point of cell membrane resistance to muscle mechanical stress. Dystrophin bears two actin binding domains. Actin binding site 1 (ABD1) is located at the N-terminal part and the second site (ABD2) is constituted by the repeats 11 to 15 from the central domain of dystrophin. ABD2 is also a lipid binding domain. Actin as well as lipids can simultaneously bind dystrophin and induce changes of the rheologic properties of lipid layers (Sarkis et al.; FASEB J 2013). It therefore ensures a fundamental function in lipid bilayers scaffolding but little is known at the molecular level for the lipid-protein interaction while transconformations have been found to occur (LeGardinier et al. J Mol Biol 2009) Low resolution analysis by SAXS and SANS will be undertaken on dystrophin-membranomimetic systems complexes and will provide molecular shapes. Thereafter, molecular modelling under experimental constraints will provide high resolution structures of the protein interacting with liposomes or bicelles, amenable to dynamical studies. In addition, the methodological tools that we will develop will be usable for analysing lipid binding properties for other fragments of dystrophin. In this field, analysis of some BMD forms of dystrophin appear highly promising for understanding some bases of the disease. Feasibility of this project is high because the Synchrotron SOLEIL, the Laboratoire L. Brillouin (Saclay- France) and the Conseil Régional de Bretagne specifically funded a PhD for it. In addition, Drs. S. Combet and J. Perez, the local beamlines experts in SANS and SAXS methods, take part in the scientific management of the PhD. Both will contribute to the development of specific tools, particularly for extinction of the signal due to lipids in the SANS studies. This staff will be actively involved in the shuttle between experimental data obtained in from the beamlines and theoretical data obtained by our team. Expected results: at an atomic level, the conformational changes exhibited by the central domain of dystrophin, native or BMD like, upon binding to membrane bilayers.

Structural analysis of complexes of dystrophin and protein partners Interactions between dystrophin and actin have been well described for ABD1. However little is known for ADB2 interacting with F-actin, while this interaction together with the one with lipids may be crucial for muscle membrane scaffolding. We will perform electron microscopy studies to obtain low resolution images of F-actin: ABD2 complexes. The cryoEM experiments and data analysis will be performed in close collaboration with the IGDR experts (R. Gillet and D. Chretien). From the experimentally obtained shapes, molecular modelling and dynamics added to interactive flexible fitting (Molza et al., Faraday Discussion F169 2014) will provide high resolution structures of the complexes. In addition to the provided basic knowledge, this approach will allow to understand the behaviour of some BMD forms of dystrophin, in regard to actin binding and therefore membrane scaffolding. In addition, we will consider the nNOS binding region, the repeats 16-17 of dystrophin, which is very close to the ABD2 domain (repeats 11 to 15). We recently built a map of interactions between dystrophin and nNOS based on complementary biophysical and theoretical data (Molza et al, submitted). We will next build an accurate 3D model of the three protein complex: dystrophin, actin, nNOS based on newly acquired experimental data. In the second half of the contract, the goal will be to use the same tools to analyse the complexes of dystrophin with other known partners, namely PAR1B and tubulin. A phage display screening in progress in our lab likely will evidence other yet unknown partners for which specific interacting modes will be studied by biochemical, biophysical and theoretical approaches. Expected results: 3D structure of dystrophin complexes, links with function.

2.4. Collaboration

2.4.1. Within the IGDR

Denis CHRETIEN (actin- dystrophin complexes- electron microscopy) - Yannick ARLOT-BONNEMAINS (protein complexes modelling) - Emmanuel GUIDICE, Reynald GILLET (molecular modelling).

158

2.4.2. Other collaborations

Rennes Véronique VIE, Institut de physique de Rennes (lipid protein-interactions).

National Sylvie TUFFERY-GIRAUD, U Montpellier (DMD & BMD clinics) - Marc BAADEN, Curie Institute Paris (molecular modelling) - Rabah BEN YAOU, France Leturcq, Cochin Hospital Paris (DMD & BMD clinics) - Denis FURLING, Myology Institute Paris (molecular modeling of pathological dystrophin) - Mirjam CZCZEK, CNRS Roscoff (SAXS studies) - PEREZ Javier, Synchrotron soleil Saclay (SAXS Studies) - Sophie COMBET, CEA Saclay (SANS studies).

International Steeve WINDER, University of Sheffield UK (dystroglycan complex) - Nick MENHART, Illinois tech Institute USA (nNOS-dystrophin interactions) - Gyulia ACSADI, Michigan Children Hospital, USA ((DMD & BMD clinics) - Kevin FLANIGAN, Ohio State University USA (DMD & BMD clinics).

2.5. SWOT Analysis STRENGTHS The complementarity of experimental and theoretical approaches ; in both domains the expertise is present in the team The fiability of molecular modelling approaches under experimental constraints is now very good A network of clinicians easily provides BMD patient’s data Our team is considered as a reference by the scientific community for the impact of our molecular view of dystrophin and BMDs (Institut de Myologie, AFM, Nationwide Children’s Hospital, Columbus USA, Hôpital Cochin, …); the team is regularly answered by clinicians for expertise about the characteristics of BMD mutations Our SAXS and SANS projects are well inserted into the synchrotron and LLB programs which fund a PhD thesis and provide beam time. We are able to regularly obtain calculation facilities through national French programs (DARI) WEAKNESSES The team is relatively small (1 professor, 1 assistant professor, 1 ingeneer, 1 PhD). Teaching duties are fulfilled and time consuming. Technical help in the fields of biochemistry and molecular modelling is needed. OPPORTUNITIES The scientists developing gene therapies aiming to cure muscular dystrophy more and more take in account the necessity to deal with the functional quality of the truncated dystrophins potentially expressed in treated DMD patients. Our team is a precursor in the field, we are able to predict the structure and therefore the behavior of the molecules. Our work will led to the emergence of new methodologies. These will be usable for studying other fibrous proteins or other kinds of lipid-protein interactions. THREATS Some dystrophin deletion mutants may be difficult or impossible to obtain in vitro. This may lead to a significant lack of knowledge for some series of deletions. There is a very high level of applications from french and international teams to obtain beam time for X rays and neutrons. The polymorphism analysis is quite at risk; no money for this project is yet obtained.

159

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Daval, S., Rocher, C., Cherel, Y., and Le Rumeur, E. Several dystrophin-glycoprotein complex members are present in crude surface membranes but they are sodium dodecyl sulphate invisible in KCl-washed microsomes from mdx mouse muscle. Cell Mol Biol Lett 15, 134-152 (IF = 1.45) Delalande O., Desvaux, H., Godat, E., Valleix, A., Junot, C., Labarre, J., Boulard, Y., Cadmium-glutathione solution structures provide new insights into heavy metal detoxification, FEBS J., 277(24), 5086-96 (IF = 4.01) Delalande O., Férey, N., Laurent, B., Gueroult, M., Hartmann, B., Baaden, M. Multi-resolution approach for interactively locating functionally linked ion binding sites by steering small molecules into electrostatic potential maps using a haptic device. Pacific Symposium on Biocomputing (PSB 2010), 205-215 (IF = no) Le Rumeur E, Winder SJ, Hubert J-F. Dystrophin, more than just the sum of its parts. Biochim Biophys Acta 1804: 1713-1722 (IF = 4.66) Saladin, A., Amourda, C., Poulain, P., Férey, N., Baaden, M., Zacharias, M., Delalande, O., Prévost, C., Modeling the early stage of DNA sequence recognition within RecA nucleoprotein filaments, Nucleic Acids Res., 38, 6313-23 (IF = 7.83) Vie V., Legardinier S., Chieze L., Le Bihan O., Qin Y., Sarkis J., Hubert J-F. Renault A., Desbat B., Le Rumeur E. Specific behaviour of two distinct dystrophin rod sub-domains interacting with phospholipid Langmuir films studied by atomic force microscopy and PM-IRRAS. Biochim Biophys Acta. 1798:1503-1511 (IF = 4.66)

2011 Legrand B, Giudice E, Nicolas A, Delalande O, Le Rumeur E. Computational Study of the Human Dystrophin Repeats: Interaction Properties and Molecular Dynamics. Plos One 6: e23819. (IF = 3.55) Delalande O, Sacquin-Mora, S., & Baaden, M., Conformational substates of Mycobacterium Tuberculosis guanylate kinase reveal structural fluctuations linked to enzymatic function, Biophys. J., 101, 1440-1449 (IF = 4.21) Legrand, B., Laurencin M., Sarkis J., Duval E., L. Mouret, Hubert J-F., Collen M., Vie V, Zatylny-Gaudin C., Henry J., Baudy-Floc'h M., Bondon A. Structure and mechanism of action of a de novo antimicrobial detergent-like peptide. Biochim Biophys Acta 1808:106-116. (IF = 4.66) Le Guiner, C., Jouvion, G., Delorme, B.,Lieubeau, B., Carlus, M., Fornasari, B., Theret, M., Orlando, P., Ledevin, M., Zuber, C., Leroux, I., Deleau, S., Guigand, L., Testault, I., Le Rumeur, E., Fiszman, M., and Cherel, Y. Systemic delivery of allogenic muscle stem cells induces long-term muscle repair and clinical efficacy in Duchenne muscular dystrophy dogs. Am J Pathol , 2501-2518. (IF=4.89) Sarkis J., Hubert J-F., Legrand B., Robert E., Chéron A., Jardin J., Hitti E., Le Rumeur E., Vié V. Spectrin-like repeats 11-15 of human dystrophin show adaptations to a lipidic environment. J Biol Chem. 286(35):30481-91. (IF= 4.65)

2012 Acsadi G, Moore S A., Cheron A, Delalande O, Bennett L, Kupsky W, El-Baba M, Le Rumeur E And Hubert J-F. Novel mutation in spectrin-like repeat 1 of dystrophin central domain causes protein misfolding and mild Becker muscular dystrophy. J Biol Chem 2012; 287:18153-62. (IF= 4.65) Garcin, P., Delalande, O., Zhang, J.-Y., Cassier-Chauvat, C., Chauvat, F., & Boulard, Y., A transcriptional-switch model for Slr1738-controlled gene expression in the cyanobacterium Synechocystis, BMC Struct Biol; 2:12. (IF = 2.22) Ismed F, Lohézic-Le Dévéhat F, Delalande O, Sinbandhit S, Bakhtiar A, Boustie J. Lobarin from the Sumatran lichen, Stereocaulon halei. Fitoterapia. 2012; 83:1693-8. (IF = 2.20) Le Rumeur E, Hubert J-F, Winder S.J. A new twist to coiled coil. FEBS Lett 2012; 586: 2717-22. (IF = 3.35) Nicolas A, Lucchetti-Miganeh C, Yaou Rb, Kaplan Jc, Chelly J, Leturcq F, Barloy-Hubler F, Le Rumeur E. Assessment of the structural and functional impact of in-frame mutations of the DMD gene, using the tools included in the eDystrophin online database. Orphanet J Rare Dis 2012; 7:45. (IF = 3.95) Sahni N, Mangat K, Le Rumeur E, Menhart N. Exon edited dystrophin rods in the hinge 3 region. Biochim Biophys Acta. Biomembranes; 1824:1080-9. (IF = 3.45)

2013 Da Costa G, Bondon A, Delalande O, Mouret L, Monti Jp. Elucidation by NMR solution of neurotensin in small unilamellar vesicle environment: molecular surveys for neurotensin receptor recognition. J Biomol Struct Dyn 2013; 31:809-17. (IF = 3.00)

160

Giudice E, Molza Ae, Laurin Y, Nicolas A, Le Rumeur E, Delalande O. Molecular clues about the dystrophin-neuronal nitric oxide synthase interaction: a theoretical approach. Biochemistry 2013; 44:7777-84. (IF = 3.20) Sarkis J, Vié V, Winder Sj, Renault A, Le Rumeur E, Hubert J-F. Resisting sarcolemmal rupture: dystrophin repeats increase membrane-actin stiffness. FASEB J 2013; 27:359-67. (IF = 5.50) Simon M, Metzinger-Le Meuth V, Chevance S, Delalande O, Bondon A.Versatility of non-native forms of human cytochrome c: pH and micellar concentration dependence. J Biol Inorg Chem 2013; 18:27-38. (IF = 3.20)

2014 Ameziane-Le Hir S, Raguénès-Nicol C, Paboeuf G, Nicolas A, Le Rumeur E, Vié V. Cholesterol favors the anchorage of human dystrophin repeats 16 to 21 in membrane at physiological surface pressure. Biochim Biophys Acta. Biomembranes; 1838:1266-1273. (IF = 3.45) Molza Ae, Férey N, Czjzek M, Le Rumeur E, Hubert J-F, Tek A, Laurent B, Baaden M, Delalande O. FD169: Innovative interactive flexible docking method for multi-scale reconstruction elucidates dystrophin molecular assembly. Faraday Discuss 2014; 169:45-62. (IF = 4.20) Nicolas A, Delalande O, Hubert J-F, Le Rumeur E. The spectrin family of proteins: a unique coiled-coil fold for various molecular surface properties. J Struct Biol 2014; 186:392-401. (IF = 3.40) Paquet F, Delalande O, Goffinont S, Culard F, Loth K, Asseline U, Castaing B, Landon C. Model of a DNA-Protein Complex of the Architectural Monomeric Protein MC1 from Euryarchaea. PLoS One 2014; 9:e88809. (IF = 3.55)

2015 Findlay A, Wein N, Kaminoh Y, Taylor L, Dunn D, Mendell J, King W, Pestronk A, Florence J, Mathews K, Finkel R, Swoboda K, Howard M, Day J, Mcdonald C, Nicolas A, Le Rumeur E, Weiss R, Flanigan K. Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45. Annals Neurol 2015; 77:668-74. (IF = 11.90) Nicolas A, Raguénès-Nicol C, Ben Yaou R, Ameziane-Le Hir S, Chéron A, Vié V, Claustres M, Leturcq F, Delalande O, Hubert J-F, Tuffery-Giraud S, Giudice E, Le Rumeur E; the French Network of Clinical Reference Centers for Neuromuscular Diseases (CORNEMUS). Becker muscular dystrophy severity is linked to the structure of dystrophin. Hum Mol Genet 2015; 24:1267-1279. (IF = 6.70) Rau F, Lainé J, Ramanoudjame L, Ferry A, Arandel L, Delalande O, Jollet A, Dingli F, Lee K-Y, Peccate C, Lorain S, Kabashi E, Athanasopoulos T, Koo T, Loew D, Swanson Ms, Le Rumeur E, Dickson G, Allamand V, Marie J Furling D. Abnormal splicing switch of DMD’s penultimate exon compromises muscle fiber maintenance in Myotonic Dystrophy. Nature Comm 2015, in press. (IF = 10.75)

2. Patents (with licence)

N/A

3. Conferences (actual team members)

- International

2010 Joe SARKIS: Human Dystrophin Rod 11-15 sub-domain: A membrane interacting zone modulated by Lipid Packing 55th annual biophysical meeting Baltimore, USA

2013 Elisabeth LE RUMEUR: Overview: Spectrin-like repeats and Dystrophin. Alpbach int meeting fibrous proteins. Olivier DELALANDE: Investigating the structure of the dystrophin central domain - a theoretical approach. MDA conference, Washington, USA.

2014 Olivier DELALANDE: Exploring the ternary complex involving F-Actin, Dystrophin central domain and nNOS. Faraday discussions, Nothingam, UK. Elisabeth LE RUMEUR: Becker muscular dystrophy severity is linked to dystrophin structure. ICNMD, Nice.

- National

2011 Jean-François HUBERT: Interaction of the actin binding domain 2 of dystrophin with lipid membrane is modulated by lipid packing. Soc Myologie, Lille.

161

Emeline POLLET: Coarse-grained Simulations as a Bridge to Solve Atomic Structures of Dystrophin Essential Fragments from SAXS Envelopes. JOBIM, Rennes.

2012 Jean-François HUBERT: Resisting sarcolemma rupture: dystrophin repeats increase membrane-actin stiffness. GEIMM, Paris.

2013 Jean-François HUBERT: Structure et fonction de la dystrophine: modifications dans les myopathies de Becker. Soc. Myologie, Montpellier. Olivier DELALANDE: Molecular dynamics on truncated dystrophin in Becker Muscular Dystrophies. GGMM, Paris. Olivier DELALANDE: Experimental and theoretical characterization of macromolecular assemblies at low resolution. CFCAM Discussion Meeting.

4. Funding

2011 Institut Fédératif de Recherche 140 - Génétique fonctionnelle, Agronomie et Santé 6,7 k€ 2011 RTR Biologie-Santé de l'Université Européenne de Bretagne (UEB) 10 k€ 2011 Rennes Métropole AIS 40 k€ 2012 AFM – Thèse (Anne-Elisabeth MOLZA) 90 k€ 2013 AFM- fonctionnement 40 k€ 2012 Cure Duchenne Fundation Post doc (Asma FREDJ) 45 k€ 2013 Rennes Metropole accueil doctorant (Khushdeep MANGAT) 2.5 k€ 2013 Univiversité Rennes 1 projet émergent 12 k€ 2013 Biosit interdisciplinary project 10 k€ 2014 AFM-Téléthon – fonctionnement 10 k€ 2015 AFM-Téléthon – fonctionnement 30 k€ 2015 AFM Téléthon Salary Technician 29 k€ 2015 Genethon micro-dystrophines fonctionnement 8 k€ 2015 Généthon micro-dystrophines salary 6 month ingeneer 20 k€

2012 Ressources informatiques – GENCI – DARI 300 000 hours 2013 Ressources informatiques – GENCI – DARI 500 000 hours 2014 Ressources informatiques – GENCI – DARI 200 000 hours 2015 Ressources informatiques – GENCI – DARI 520 000 hours

5. Training Licence 2013 - Pauline SCOTTO di VETTIMO, L3 BCGMP UR1 (6 weeks) - Charazed SMIR, L3 Chimie UR1 (6 weeks) BTS 2014 - Elise FLEJOU ESSEIVA, BTS ABM lycée Bréquigny Rennes (6 weeks) - Medéric FONTAINE, BTS ABM lycée Bréquigny Rennes (6 weeks) 2015 - Quentin le GALLUDEC, BTS ABM lycée Bréquigny Rennes (6 weeks) Master 2010 - Elisa MARIVIN, M1 SCMV UR1 (2 months) - Claire SCHIRMER, M1 SCMV UR1 (2 months) 2011 - Hillel JEAN-BAPTISTE ADOLPHE, M1 MSB UR1 (2 months) - Sarah LEVIN, M1 SCMV UR1 (2 months) 2012 - Yoann LAURIN, M1 BIG UR1 (2 months) - Maria MATARD, M1 SCMV UR1 (2 months) - Aurélie NGUEA, M1 SCMV UR1 (2 months) - Emeline POLLET, M2 BIG UR1 (6 months) 2013 - Emeric BANKOLE, M1 BIG UR1 (2 months) - Maelys BOUTHEMY, M1 SCMV UR1 (2 months) - Guillaume MASSON, M1 SCMV UR1 (2 months) - Philippe MOUA, M1 SCMV UR1 (2 months) - Xavier WARNET, M1 SCMV UR1 (2 months) - Awa DIALLO, M2 BIG UR1 (6 months) 2014 - Nalhuarati MOHAMED, M1 SCMV UR1 (2 months) 2015 - Sidwell RIGADE, M1 BIG UR1 (2 months) - Dominique MIAS-LUCQUIN, M2 BIG UR1 (6 months) PhD 04/11/2011: Joe SARKIS – Dir. Thèse Jean-François HUBERT 162

23/10/2012: Aurelie NICOLAS – Dir. Thèse Elisabeth LE RUMEUR & Frédérique HUBLER 14/11/2012: Sarah AMEZIANE – Dir. Thèse Céline RAGUENES-NICOL & Véronique VIE 29/11/2012: Matthieu SIMON – Dir. Thèse Arnaud BONDON Since 10/2012: Anne Elisabeth MOLZA – Dir. Thèse Elisabeth LE RUMEUR &Olivier DELALANDE Since 10/2014: Raphaël DOS SANTOS MORAIS – Dir. Thèse Jean-François HUBERT et Sophie COMBET Starting 10/2015: Dominique MIAS-LUCQUIN – Dir. Thèse Jean-François HUBERT Post-doctorants 2013-2014: Asma FREDJ, post-doctoral fellow 1 year 2013-2014: Aurélie NICOLAS, post-doctoral fellow 7 months Welcome of foreign student 2014: Khushdeep MANGAT, doctoral fellow 3 months CDD 2015-2016: Thomas CHENUEL - Engineer UR1, contract 1 year

163

Team 11 "Epigenetics and cancer" Leader: Christian JAULIN

165

2.1. Team presentation

The team "epigenetics and cancer" was created in 2004 in Montpellier and moved to Rennes in 2008. Since its creation, the team has been interested in the role of histone modifications in mitotic processes. Since centromeres orchestrate many mitotic processes, our research activity is focused on histone modifications at centromeres and, more specifically, our efforts are directed towards identifying new histone modifications at centromeres and discovering their function(s). We are interested in both centromere epigenetic propagation and centromere mitotic functions such as maintenance of sister chromatid cohesion, kinetochore building, chromosome dynamics or mitotic checkpoint regulation. Our main aim is to understand centromere function(s) at the molecular level but we keep in mind that centromeres may represent a novel therapeutic target in oncology. Indeed, mitosis is a prime target for treatment against cancer but the only mitotic target which is currently the subject of therapeutic protocols is the mitotic spindle. We believe that other mitotic functions such as the spindle assembly checkpoint or sister chromatid cohesion may also represent functional compartments that can be targeted as part of an antitumor strategy.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

EOT-HOULLIER Grégory CR2 CNRS MAGNAGHI-JAULIN Laura CR1 CNRS JAULIN Christian DR2 INSERM MONIER-TSCHOPE Solange CR1 INSERM

2.2.2.2. Temporary staff

N/A

2.2.2.3. Permanent staff who left the team during the contract

MIRONOV Svetlana AJT2 CNRS until 10/2013

2.2.3. Achievements

166

2.2.3.1. Scientific achievements

Published work:

- Calpain 2 is required for sister chromatid cohesion (Magnaghi-Jaulin et al, Chromosoma 2010; 119(3): 267-274): We reported that depletion of calpain 2 leads to loss of sister chromatid cohesion and defects in Aurora B recruitment at centromeres.

- CDK11p58 kinase activity is required to protect sister chromatid cohesion at centromeres in mitosis (Rakkaa et al, Chromosome Res. 2014 Sep;22(3):267-76): We found that the kinase activity of CDK11p58 is necessary to protect sister chromatid cohesion and we reported that CDK11p58 acts upstream of Sgo1 and Bub1 in cohesion protection.

Collaborative work:

- Role of HDAC8 in cohesin deacetylation and Cornelia de Lange syndrome (Deardorff et al, Nature 2012; 489(7415): 313-317). Our part of this work has focused on the role of the histone deacetylase HDAC8 in the cohesin acetylation cycle. The identification of HDAC8 mutations in CdL syndrome was performed by our Japanese collaborators.

- Bloom's syndrome and PICH helicases cooperate with topoisomerase IIα in centromere disjunction before anaphase (Rouzeau et al, PLoS One 2012;7(4): e33905). We show that the BLM helicase, known to be involved in the repair of collapsed replication forks, is present at centromeres during mitosis and helps to decatenate DNA at mitosis onset.

Projects completed but not yet published:

- Aurora A-dependent CENP-A phosphorylation at centromeres is required for protection of sister chromatid cohesion during mitosis (Fulcrand et al. in preparation): During mitosis, the centromere- specific histone variant CENP-A is sequentially phosphorylated on serine 7 by Aurora kinases A and B. We report the detection of Aurora at centromeres in mitosis and we show that replacement of endogenous CENP-A with a non-phosphorylatable version weakens the protection of sister chromatid cohesion at centromeres. We show that, while Aurora A and shugoshin do not require each other for their respective targeting to centromeres, they both require the mitotic kinase Bub1 for their recruitment at centromeres. We propose the existence of a signaling pathway involving a cross-talk between centromere chromatin and factors involved in the protection of sister chromatid cohesion in mitosis.

- NSD3 histone methyltransferase is required for cohesin loading (Gallais et al. in preparation): In a previous study, we reported that a specific histone methylation at centromere could be involved in sister chromatid cohesion (Eot-Houllier et al, Genes Dev 2008; 22(19): 2639-2644). Using a screen aimed at identifying histone methyltransferases involved in cohesion, we have identified a histone methyltransferase, NSD3, whose depletion leads to early separation of sister chromatids. This premature separation is not accompanied by a recruitment defect of the factors involved in the protection of centromere cohesion (Shugoshine, H2AT120 phosphorylation, dimethylation of H3K4). We therefore determined the cell cycle stage at which the loss of cohesion takes place and showed that cohesin loading is defective in G1. We also show that the recruitment of the cohesin loading factors SCC2 and SCC4 depends on the presence of NSD3.

- CENP-A is not restricted to the centromere outer side (Magnaghi-Jaulin et al. in preparation): Previous published data suggested that the centromere-specific histone variant CENP-A is only present on the outer side of mitotic chromosomes, H3-containing nucleosomes being located on the inner side (Blower et al, Dev Cell, 2002; 2(3): 319-330). We show that this is due to an experimental artifact: the fraction of CENP-A that is phosphorylated on serine 7 during mitosis is detectable on inner centromeres but is not bound by the anti-CENP-A antibody used in previous studies.

2.2.3.2. Scientific dissemination and influence

The team leader (Christian JAULIN) is deputy director of the IGDR and is, or was, a member of the following bodies: National Committee for Scientific Research (CoNRS, Section 26) 2009-2012 - ANR, National Expertise Committee Projects "Blue Sky" SVSE2, 2011-2012 - National League Against Cancer, National Commission of Expertise No. 1, 2013-2017.

167

2.2.3.3. Interaction with the economic, social and cultural environment

N/A

2.3. Projects, scientific strategies & perspectives (5 years)

Histone post-translational modifications (acetylation, methylation, phosphorylation, etc.) play an important role in all chromatin-based processes. Specific combinations of histone modifications can specify various functions downstream. This chromatin "language" is known as the "histone code". Studies on the structure and function of this code have been focused on its role in regulating gene expression. However, several experimental data (including ours) show that post-translational modifications of histones play an important role in major centromeric functions such as sister chromatid cohesion or chromosome segregation.

We have undertaken an ambitious project, funded by an ANR grant, aimed at purifying centromere chromatin, isolating individual histones and characterizing centromere-specific histone modifications. Centromeric chromatin is characterized by the presence of a centromere specific variant of histone H3, CENP-A. Previous studies have shown that centromeres are made up of blocks of nucleosomes containing CENP-A alternating with blocks of nucleosomes containing histone H3. We take advantage of the physical link between the CENP-A and H3 nucleosomes to purify centromeric chromatin. We immunoprecipitated CENP-A in conditions of controlled chromatin digestion in order to copurify adjacent centromeric H3 nucleosomes. Individual histones have been purified and their post-translational modifications profiles are currently being established using FT-MS (Fourier-Transform Mass Spectrometry), a mass spectrometry technique that allows to identify multiple modifications on a given histone polypeptide. It took us almost three years to obtain purified fractions of centromeric chromatin suitable (in terms of purity and amounts) for FT-MS. The main reason for this delay is that centromere chromatin biochemical properties differ widely from those of "regular" chromatin and we had to set up new protocols and adapt them to large scale preparations (10 liters of cultured cells in suspension). The search for the functions of the modifications that we are currently identifying should represent the main activity (if not all) of the team for the next 5 years.

Several approaches are possible, depending on the identified modifications:

If a modification is found on CENP-A, it is possible to construct the corresponding mutants and analyze their phenotype as we did in our study on Aurora A-mediated CENP-AS7 phosphorylation. For example, we have identified two previously unreported histone modifications on CENP-A. Their location suggest that they are involved in CENP-A loading and/or maintenance at centromeres. The corresponding positions are being mutated and reintroduced in a CENP-A depleted background to assess the function of the corresponding modifications in centromere function(s) and/or CENP-A loading and maintenance (project undertaken by R. VILLOT under the supervision of G. EOT-HOULLIER). The endogenous CENP-A will be depleted by RNA interference and the localization of the mutated CENP-A will be followed by quantitative fluorescence microscopy along several cell generations. We do not restrict the search for phenotypes to the stability and/or maintenance of CENP-A. For each mutant, we will look at the following functional mitotic compartments: - Kinetochore attachment checkpoint (also known as "spindle attachment checkpoint"). Most known factors involved in this checkpoint need to be recruited at centromeres to fulfil their function. We will look whether, upon mutant expression, cells are still able to activate this checkpoint. We will assess if microtubules depolymerization upon nocodazole treatment is still able to block cells in prometaphase. If a defect is found, we will look for the presence of known factors (Bub1, BubR1, Mad1, Mad2, Mps1, etc.) at centromeres to determine how a mutation of CENP-A affects the spindle attachment checkpoint. - Kinetochore construction. CENP-A is considered as the first brick on which the kinetochore is being built. A defect in kinetochore building may not lead to an obvious cellular phenotype and we will look, by fluorescence microscopy, whether the most proximal (CENP-C) and most distal (Hec1) components of the kinetochore are properly recruited to centromeres. - Protection of sister chromatid cohesion at centromeres. In vertebrates, the dissociation of sister chromatids occurs in two steps: during prophase, cohesins are removed from chromosome arms but remain protected at centromeres until the metaphase to anaphase transition. At anaphase onset, the remaining centromeric cohesins are cleaved by the proteolytic enzyme separase. Protection of 168

cohesin at centromeres during the early mitotic stages relies on several proteins and histone marks, including shugoshin, Bub1, haspin, H3K4diMe, H3T3P, H2AT120P, etc. We will look at sister chromatid cohesion upon mutant expression (by examining chromosome spreads) and, if a defect is found, we will check for the presence of these factors at centromeres and look for their possible binding to CENP-A.

However, the strategy described above is not suitable for modifications found in core centromere histones (H2A, H2B, H3, H4) since the replacement of such a core histone by a mutated version is predicted to have severe pleiotropic effects unrelated to centromere function. Three independent alternative approaches are thus conceivable and can be used in combinations (Project undertaken by L. MAGNAGHI-JAULIN) : - Screening for the histone modifying enzyme involved in the modification of interest, as we did in previous studies (Eot-Houllier et al, Genes Dev, 2008; Deardorff et al, Nature, 2012). We already have a collection of siRNA for histone deacetylases and histone methyltransferases. We will screen, by microscopy, for the enzyme involved in the deposition of the centromere histone mark of interest and look for the mitotic defects associated with its depletion as described above. - Identifying binding partners by peptide pull down. We will synthesize streptavidin-coupled peptides with the modification of interest (and their unmodified counterpart) and compare, by mass spectrometry, the profiles of proteins bound to these peptides. Protein extracts will be obtained from both unsynchronized cells and cells blocked in mitosis to ensure identification of factors that would "prime" a mitotic function but be absent from mitotic extracts. - Expressing a mutant in yeast and screening for mitotic defects. For most cell cycle and chromosome dynamics related studies, S. pombe has proved to be a model better related to metazoans than S. cerevisiae. For example, most mammalian cell cycle control mechanisms are better conserved in S. pombe than in S. cerevisiae. We plan to construct S. pombe strains with conditional expression of a given histone by deleting the endogenous gene(s) (between one and three copies of the gene depending on the histone) and expressing the same histone under the control of an inducible promoter. Mutant histones will be introduced in these strains and the expression of their wild-type counterpart will be switched off. Mitotic defects and chromosome mis-segregation will be assessed using specific strains containing selectable artificial chromosomes and/or strains containing chromosomes that can be specifically labelled at centromeres or on chromosome arms.

This latter approaches requires skills in S. pombe genetics and the principle of a collaboration with Dr Pei-Yun Jenny WU's laboratory, within the IGDR, has already been acknowledged.

An interesting spinoff of our efforts to purify centromere chromatin is that we are able, by modulating immunoprecipitation stringency, to co-purify intact kinetochores (Project undertaken by S. MONIER-TSCHOPE). We have obtained centromere chromatin fractions that contain both proximal and distal kinetochore components and we are thus confident that the intermediate components are also present. The composition of centromere-bound proteins has been described for interphase cells (Foltz et al. Nat Cell Biol, 2006) but little is known about the dynamics of these components during the cell cycle and, more specifically, during mitosis. Cultured cells will be synchronized using various methods (thymidine block, nocodazole, serum deprivation, cdk inhibitors...) and released for a short time. Centromeres will be purified at different times after release and the centromere-bound proteins will be identified by mass spectrometry. This should allow to investigate the dynamics of centromere bound complexes during the cell cycle. However, classical mass spectrometry is notoriously poorly quantitative and we expect to have to refine our results using more quantitative (but more expensive and time- consuming) approaches such as SILAC.

All experiments (except for the S. pombe approach) will be performed in the epithelial aneuploid cancerous HeLa cell line and duplicated in the retina immortalized (by telomerase expression) diploid RPE cell line in order to ensure that the results obtained are not a consequence of carcinogenesis. If a difference of phenotype between HeLa and RPE cells is found, the origin of this difference will be investigated.

2.4. Collaboration

2.4.1. Within the IGDR

Pei-Yun Jenny WU (analysis of core histone modifications at centromeres in S. pombe) - Erwan WATRIN 169

(Function of HDAC8 in the cohesin acetylation cycle) - Gwenaël RABUT (Ubiquitination of CENP-A) - Régis GIET (Role of cdk11 in sister chromatid cohesion).

2.4.2. Other collaborations

National Christophe ESCUDE, CNRS/MNHN Paris (Role of cdk11 in sister chromatid cohesion) - Christophe THIRIET, CNRS/Université de Nantes (Centromere chromatin biochemistry).

International Yoshinori WATANABE, University of Tokyo, Japan (Function of centromere histone modifications) - Jonathan HIGGINS, Harvard Medical School, Boston, USA (haspin kinase in sister chromatid cohesion).

2.5. SWOT Analysis STRENGTHS Expertise in chromosome dynamics and centromere epigenetics. "Hot" research theme. Excellent atmosphere in the team. The laboratory has been well funded and well equipped. WEAKNESSES The Pi has been involved in too many administrative tasks. Lack of major publications since 2008. Only 2 years financial visibility. Not enough post-docs. OPPORTUNITIES Numerous collaborations opportunities within the institute. Variety of experimental models available at the IGDR. Support from Région Bretagne. THREATS Lack of technical help. Bureaucracy. Time-consuming laboratory renovation during 2013-2014. Strong competition.

170

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Eot-Houllier, G., M. Venoux, S. Vidal-Eychenie, M. T. Hoang, D. Giorgi and S. Rouquier (2010). "Plk1 regulates both ASAP localization and its role in spindle pole integrity." J Biol Chem 285(38): 29556-29568. (IF : 5) Magnaghi-Jaulin, L., A. Marcilhac, M. Rossel, C. Jaulin, Y. Benyamin and F. Raynaud (2010). "Calpain 2 is required for sister chromatid cohesion." Chromosoma 119(3): 267-274. (IF : 3)

2012 Deardorff, M. A., M. Bando, R. Nakato, E. Watrin, T. Itoh, M. Minamino, K. Saitoh, M. Komata, Y. Katou, D. Clark, K. E. Cole, E. De Baere, C. Decroos, N. Di Donato, S. Ernst, L. J. Francey, Y. Gyftodimou, K. Hirashima, M. Hullings, Y. Ishikawa, C. Jaulin, M. Kaur, T. Kiyono, P. M. Lombardi, L. Magnaghi-Jaulin, G. R. Mortier, N. Nozaki, M. B. Petersen, H. Seimiya, V. M. Siu, Y. Suzuki, K. Takagaki, J. J. Wilde, P. J. Willems, C. Prigent, G. Gillessen-Kaesbach, D. W. Christianson, F. J. Kaiser, L. G. Jackson, T. Hirota, I. D. Krantz and K. Shirahige (2012). "HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle." Nature 489(7415): 313-317. (IF : 42) Rouzeau, S., F. P. Cordelieres, G. Buhagiar-Labarchede, I. Hurbain, R. Onclercq-Delic, S. Gemble, L. Magnaghi-Jaulin, C. Jaulin and M. Amor-Gueret (2012). "Bloom's syndrome and PICH helicases cooperate with topoisomerase IIalpha in centromere disjunction before anaphase." PLoS One 7(4): e33905. (IF : 4)

2014 Rakkaa, T., C. Escude, R. Giet, L. Magnaghi-Jaulin and C. Jaulin (2014). "CDK11 kinase activity is required to protect sister chromatid cohesion at centromeres in mitosis." Chromosome Res 22(3):267-76. (IF : 3)

2. Patents (with licence)

N/A

3. Conferences (actual team members)

- International

2010 Christian JAULIN: EMBO workshop “chromosome segregation and aneuploidy” June 2010, Edinburgh (poster presentation) Laura MAGNAGHI-JAULIN: XXème colloque de l'Association des Cytogénéticiens de Langue Française, September 20- 21, 2010, Aix-en-Provence (poster presentation)

2011 Christian JAULIN: 1st Annual World Congress of Molecular & Cell Biology (BMCC-2011) August 6-9, 2011, Beijing, China (guest speaker)

2012 Christian JAULIN: 4th Meeting of the Asian Forum of Chromatin and Chromosome Biology on 'Epigenetic Mechanisms in Development and Disease "22-24 November 2012. Hyderabad, India (guest speaker).

2013 Tarik RAKKAA: EMBO workshop on Chromosome Segregation and Aneuploidy, June 22-26, 2013 Breukelen, The Netherlands (poster presentation)

- National

2011 Christian JAULIN: INRA, Nouzilly, April 19, 2011 (guest speaker, invited by Yves COMBARNOUS) Christian JAULIN: Meeting of the Société de Biologie Cellulaire de France “Cell Cycle, Cancer and Development", May 2011, Saint Malo (poster presentation) Christian JAULIN: Institut Curie, Paris, July 8, 2011 (guest speaker, invited by Geneviève ALMOUZNI)

171

Christian JAULIN: ENS Lyon, September 14, 2011 (guest speaker, invited by Benjamin AUDIT) Christian JAULIN: 7th SFBBM Meeting - Figeac - "nucleic acid and protein interactions: the key to decipher the Rosetta Stone of the genome?" 17-21 September 2011 (guest speaker).

2013 Christian JAULIN: Unité "Epigénétique et destin cellulaire", Paris, February 13, 2013 (guest speaker, invited by Claire FRANCASTEL) Christian JAULIN: Muséum National d'Histoire Naturelle, Paris, March 28, 2013 (guest speaker, invited by Christophe LAVELLE)

2014 Christian JAULIN: 1st meeting RepiCGO, June 18, 2014, Les Sables d'Olonne (guest speaker)

2015 Grégory EOT-HOULLIER and Romain VILLOT: Meeting of the Société de Biologie Cellulaire de France “Cell Cycle and Cancer "April 7-10, 2015, Marseille (poster presentation)

4. Funding

2009-2010 SAD Conseil Régional de Bretagne (Coord. C. JAULIN) running costs 48 k€ + Equipment 50 k€ 2009 Ligue Contre le Cancer (Grand-Ouest) (Coord. C. JAULIN) 30 k€ 2009-2010 Association Pour la Recherche sur le Cancer (ARC) (Coord. C. JAULIN) 50 k€ 2009-2011 FRM Fondation pour la Recherche médicale 88,8 k€ 2010-2011 Cancéropôle Grand Ouest/Région Bretagne (Coord. C. JAULIN) 99.6 k€ 2010-2011 Ligue Contre le Cancer 25 k€ 2010-2013 Région Bretagne et Ligue Nationale Contre le Cancer 89 k€ 2011-2014 ANR "Programme Blanc" (Coord. C. JAULIN - partner C. THIRIET, UMR 3204 Nantes) 421.6 k€ 2015 Région Bretagne 15 k€

5. Training Licence 2015 - Alice URIEN - L3 (6 weeks) BTS 2013 - Tiffany BERGOT – BTS 2e année (8 weeks) 2014 - Alexis DETAIN – BTS 1e année (7 weeks) Master 2013 - Gwendoline BELLAUD, M2 (8 months) 2015 - Romain VILLOT, M2 UBO Brest (6 months) PhD 18/12/2013: Tarik RAKKAA – Dir. Thèse Christian JAULIN Post-doctorant 2009-2013: Rozenn GALLAIS, post-doctoral fellow 4 years & 10 months

172

Team 12 "Epithelia dynamics and mechanics - polarity, division and fate acquisition" Leader: Roland LE BORGNE

173

2.1. Team presentation

Asymmetric cell division (ACD) is a conserved mechanism by which cell fate diversity is generated during development and throughout adult life. How a cell can produce two daughter cells with different identities and how defects in this asymmetry contribute to diseases are the fundamental questions we study using the Drosophila adult sensory organ (SO) lineage as a model system. SO Precursors (SOP) are epithelial cells present in a single layer neuroepithelium on the dorsal thorax of Drosophila. SOP undergoes a series of four ACD in which a mother cell gives rise to two daughter cells. At each division, the acquisition of cell identity is controlled by the differential activation of Notch. Notch is activated by Delta (Dl) present on the surface of adjacent cells. Our work contributed to show that sorting of N and Dl along the apico-basal axis at cytokinesis is important for proper N activation. We also identified novel regulators of N signaling including membrane traffic regulators and regulators of epithelial cell cytokinesis. This led us to propose a model according which at cytokinesis Notch is activated at the level of the novel E-Cad-based adhesive contact that assembles between SOP daughter cells. We are currently developping cutting edge genetic, light and electron microscopy together with biophysical approaches to investigate the interplay between the mechanics of epithelial cell cytokinesis and the spatio-temporal control of Notch activation in flies, and ultimately in organoid model system. On a long term, we aim to understand how deregulation of these mechanisms could contribute to tumorigenesis.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

LE BORGNE Roland DR1 CNRS GICQUEL Isabelle IE1 UR1 LE BRAS Stéphanie MCU UR1 KOLOTUEVA Irina IR2 CNRS (50%) PINOT Mathieu CR2 CNRS (from 10/2015)

2.2.2.2. Temporary staff

PINOT Mathieu Post-doc (until 09/2015) BELLEC Karen PhD student (2014-2017) UHART Perrine IE DANIEL Emeline PhD student (2014-2017)

2.2.3. Achievements

174

2.2.3.1. Scientific achievements

1) Spatio-temporal regulation of Notch activation by membrane trafficking

Using a compartmentalized antibody uptake assay, we proposed that Neuralized-dependent transcytosis regulates the signaling activity of Delta by relocalizing Delta from a membrane domain where it cannot interact with Notch to another membrane domain where it can bind and activate Notch (Benhra et al, MBoC, 2010). To elucidate the intracellular routes followed by Notch and ligands during binary cell fate acquisition, we next performed a RNAi screen and identified 20 new regulators including the AP-1 adaptor complex and the septins (Le Bras et al, J. C. Sci. 2012).

Using a combination of in vitro, ex vivo and in vivo approaches, we also observed that Delta can be sorted into exosome-like vesicles and could regulate long distance Notch signaling in vitro. Whether ELV have a functional significance in vivo remains unsolved (Monier et al, unpublished observations). Similar observations were made for the morphogen Wingless in collaboration with the team of J.-P. VINCENT, NIMR, London (Beckett et al, Traffic 2013).

2) Role of AP-1 in Notch signaling and cell-cell adhesion

We identified AP-1 complex as a negative regulator of Notch signaling. It does so by regulating the endocytic recycling of Notch and its cofactor Sanpodo towards newly formed E-Cadherin adhesive contacts at the SOP daughter cell interface (Benhra et al, Curr. Biol., 2011). We further show that AP-1 acts through Numb and demonstrate that Numb inhibits Sanpodo recycling rather than endocytosis as previously thought (Cotton et al, Curr. Biol. 2013). This activity is tightly controlled by cell cycle. Together with the group of C. PRIGENT (IGDR, Rennes), we found that the mitotic kinase Aurora A phosphorylates Numb and controls it ability to interact with the AP-1 complex (Cotton et al, in revision). On a side project, in collaboration with J. BRILL (U. Toronto), we found that another evolutionarily conserved function of AP-1 is to control secretory granule biogenesis (Burgess et al, MBoC, 2011). In addition, we found that AP-1 regulates E-Cad trafficking in epithelial cells and in female germline cysts where sustained E-Cad dependent adhesion organizes a microvilli meshwork and ensures proper attachment of ring canals (a remnant from incomplete cytokinesis) to plasma membrane (Loyer et al, accepted for publication at PNAS).

3) Epithelial cell cytokinesis and cell mechanics

How adhesive contacts with neighbors affect epithelial cell cytokinesis was unknown. We reported that, septins (identified in the screen (Le Bras et al, J. C. Sci. 2012)) are specifically required for planar (but not orthogonal) cytokinesis. During planar cell division, cytokinetic furrowing initiates basally, resulting in a contractile ring displaced toward the adherens junction (AJ). The formation of new AJ between daughter cells requires the disengagement of E-Cadherin complexes between mitotic and neighboring cells at the cleavage furrow, followed by the assembly of E-Cadherin complexes on the daughter-daughter interface. The strength of adhesion with neighbors directly impacts both the kinetics of AJ disengagement and the length of the new AJ. Loss of septins causes a reduction in the contractility of the actomyosin ring and prevents local disengagement of AJ in the cleavage furrow. By modulating the strength of tension induced by neighbors, we uncovered a mechanical function for septins to overcome the extrinsic tension induced by neighboring interphasic cells (Founounou et al, Dev. Cell 2013; Le Bras and Le Borgne, J. C. Sci. 2014). This pioneer work opened new avenue of research on epithelial cell mechanics and Notch signaling (see project).

4) Biophysical approaches

Upon arrival in Rennes, I teamed up with physicists (Z. GUEROUI) and chemists (V. MARCHI-ARTZNER) in order to develop novel methodological approaches for spatio- temporal control of signaling activities (Dif et al, JACS 2009). Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signaling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. As a proof of concept, we have shown that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signaling pathway that regulates the cell cytoskeleton (Hoffmann et al, Nat. Nanotech. 2013). This pioneer quest is at the basis of the novel methodological approaches including optogenetics we are currently developing.

175

2.2.3.2. Scientific dissemination and influence

N/A

2.2.3.3. Interaction with the economic, social and cultural environment

Maturation Programme Rennes Métropole/SATT Ouest ‘inDroso functional genomics’ based on the development of genome editing methodology using CRISPR/Cas9 methodology (2015-2016).

2.3. Projects, scientific strategies & perspectives (5 years)

Our project is built on our past achievements. Our goal is to decipher the spatio-temporal control of Notch activation following asymmetric cell division of the sensory organ precursor (SOP) in Drosophila melanogaster. To this aim we develop a set of novel methodologies and approaches to study the interplay between epithelial cell division and the establishment of the Notch signaling platform during cytokinesis. In 2013, a turn to biophysical approaches was made through collaboration with the team of Dr.TOYAMA, Institute of Mechanobiology (Singapore) and the hiring of a biophysicist in 2014 (M. PINOT). This research proposal is implementing biophysical approaches to study the role of cellular mechanics in epithelial cell cytokinesis and mechanotransduction in Notch signaling following asymmetric cell division.

1- Methodological developments

Genome editing (I. GICQUEL + P. UHART): Over the past two years, we have adapted genome-editing tools based on CRISPR/Cas9 technology. On a routine manner, we are generating knockout, knock-In, introducing point mutations, and gene tagging at the locus. Part of our academic knowledge has been transferred to a Business Unit named inDroso that proposes these services to academic laboratories. We will continue to develop vectors and improve the methodology to develop novel probes suitable for our quantitative and non-invasive time lapse microscopy (linked to task 3).

Optogenetics (I. GICQUEL): One of the major challenges in the postgenomic era is to decipher the spatiotemporal dynamics of signaling pathways. A limitation when analysing the consequence of the loss-of-function of a gene product is the temporal delay. Typically, in every model system, RNAi and/or mutant phenotypes are analyzed 2 to 7 days after induction of gene silencing or mutation. That implies that there is a progressive protein loss over several days while the kinetics of activity of the gene product involved in membrane traffic or signalling is ranging from seconds to minutes time scales, not days Therefore, it is always difficult to decipher whether the observed phenotypes are a direct or an indirect consequence of gene product inactivation (i.e. be able to identify the real target at the cause of the phenotype). Alternatively, cells can adapt from the progressive loss of protein and develop new strategies to compensate for the lack of the protein of interest. We develop optogenetic tools to conduct spatio-temporally controlled gene product inactivation. This approach is based on the ability of Arabidposis thaliana crytpochrome 2 to dimerize with CIBN upon light activation at seconds time scale and in a reversible manner. The idea is to perform knockside away experiments of membrane traffic regulators (AP-1, cell fate determinants Numb and Neur, and novel regulator identified in our screen (Le Bras et al, 2012)). A second development consists in inducing the controlled membrane localization of enzymes such as lipid kinases or phosphatases (linked to task 4-2).

Electron microscopy (I. KOLOTUEVA): The major goal of Transmission Electron Microscopy (TEM) characterization is to obtain the resolution required for understanding of the cellular processes on ultrastructural level. Automated serial reconstruction of a given tissue using Serial Block Face Scanning EM microscopy enables to analyze relatively large volumes of tissue with the necessary resolution. We are using this method to characterize different steps of cell division as well as to attempt to recognize the SOP cells in the notum wild type and mutant animals. We are equally interested in the Correlative light to electron microscopy (CLEM) analysis applied to mosaic studies in Drosophila. CLEM enables precise localization of the same region of interest (ROI), cell or organelle while changing the observation modality. Array tomography was introduced several years ago as a tool for simultaneous localization of multiple fluorescently-labeled antibodies in synaptic neurons. This technique is currently developed and shall permit the localization of fluorescence in TEM- like treated samples. This will help to correlate the fluorescent signal in SOP and mosaic samples and the ultrastructure of these particular cells (linked to tasks 2-5).

176

2- Cytokinesis and abscission of epidermal cells: E. DANIEL, I. KOLOTUEVA

As a follow up of our study on septins in epithelial cell cytokinesis (Founounou et al, 2013), we are investigating how the septate junctions (the functional ortholog of tight junctions in vertebrates) are remodeled to preserve the paracellular barrier function (i.e. how tissue integrity and chemical barrier function of epithelia is preserved throughout cytokinesis), as well as the late steps of cytokinesis, the abscission. We perform non-invasive, quantitative confocal live imaging of various polarity markers, components of adhesion complexes and cytokinesis machinery tagged with fluorescent reporters and expressed at physiological levels (micrometer resolution). In parallel, large data set of EM images are collected to reconstitute the 3D volume of dividing cells at the nanometer resolution. This first step will enable us to describe the cytokinesis to abscission process of epithelial cells. Our preliminary results indicate that the midbody that is forming apically (Founounou et al, 2013, Hertzerg et al, 2013, Guillot and Lecuit, 2013) mature into an intercellular bridge as it migrates basally within the septate junctions and progresses towards abscission. The next step consists in investigating the role of evolutionarily conserved ESCRT machinery (snf7), and that of components of septate junction durin g epithelial cell abscission. This task will identify the key regulators of cytokinesis of epithelial cells that divide symmetrically. It is a prerequisite for a comparative analysis with the cytokinesis of SOP dividing asymmetrically and exhibiting binary cell fate decision during cytokinesis.

3- Regulation of SOP cytokinesis and abscission-link to Notch activation: K. BELLEC, E. DANIEL, I. KOLOTUEVA, S. LE BRAS, P. UHART

Work from several groups (H. BELLEN, M. GONZALEZ-GAITAN, J. KNOBLICH, F. SCHWEISGUTH) including ours has pinpoint that Notch signaling takes place during cytokinesis. Several non-mutually exclusive models involves distinct modalities for Notch activation: activation from asymmetrically inherited Sara endosomes (M. GONZALEZ-GAITAN, J. KNOBLICH), activation at the basal interface separating SOP daughter cells (F. SCHWEISGUTH) and/or activation at the apical interface (H. BELLEN and R. LE BORGNE).

In order to directly address when and where the productive ligand/receptor activation takes place during SOP cytokinesis, we will analyzed the epithelial cell polarity, investigate the role of membrane traffic regulators, and that of mitotic kinases.

3-1 Dynamics of cell polarity markers throughout SOP division, define the active pool of Notch Live imaging and EM approaches described in task 2 will be applied to SOP mitosis. This task will enable to define epithelial cell polarity transmission, polarity of the novel SOP daughter cell interface, kinetics of formation and composition of the novel adhesive interface that we hypothesize serves as a launching platform for Notch activation. Development of IEM will help further characterizing. We will genome edit (task 1) the Notch and Delta loci using classical fluorescent protein (FP), photoactivable FP and bifunctional FP probes. These reagents should help defining when and where the ligand and receptor interact at the exit of SOP mitosis and define which pool of plasma membrane Notch is in fine translocated within the nucleus and acts as a transcriptional co-activator.

3-2 membrane traffic regulators Our genetic screen identified various candidates of which the invalidation by dsRNA leaded to steady- state Notch and/or Delta subcellular localization defects correlated with adult loss or gain of Notch signaling (Le Bras et al, 2012). Among them, we will first concentrate on CG7787, a putative RabGEF. Time-lapse imaging, genome editing to generate mutants and gene tagging at the locus, will be employed to study the functions of CG7787 in Notch/Delta trafficking and signaling activity.

3-3 AurA, asymmetric cell divisions and control of Notch activation (collaboration with C. PRIGENT) The mitotic kinase AurA behaves as an oncogene (symmetric cell division) or as a tumor suppressor (asymmetric cell division) depending of the context. AurA regulates asymmetric cell division in part by controlling cell polarity via phosphorylation of the Par Complex. We found that AurA also regulate Notch signaling by phosphorylating Numb in mitosis, thereby regulating the endocytic trafficking of Notch/Sanpodo. By adopting a KO/KI approach on AurA and Numb, we plan to determine the respective roles of AurA in symmetric versus asymmetric cell division, and separate the activity of AurA on Par complex versus Numb (phosphomimetic or phospho-dead versions).

4- Role of mechanical forces in epithelial cell cytokinesis and fate acquisition: M. PINOT, international collaboration with Y. TOYAMA (Singapore)

Notch signaling and mechanical forces are intrinsically linked: (1) signaling takes place in 177

epithelial cells that are subjected to tensile forces transmitted by the adherens junctions, (2) tensiles forces are applied to novel membrane interface at cytokinesis ((our data), (3) forces are associated to membrane deformability during ligand endocytosis, (4) pulling forces are exerted on Notch and induce conformational change needed for receptor activation (Weinmaster’s lab), (5) and acto-myosin cytoskeleton dynamics that are source of tensile forces are different in SOP versus epidermal cells (our data). By multi-disciplinary approaches coupling fly genetics, cell biology and biophysical methodologies consisting of nano-laser ablation techniques, micro-rheology using optical tweezers and the use of FRET- based tension sensors, we propose to study the link between mechanical forces and Notch signaling by: (a) Characterizing the mechanical properties of the interfaces resulting from the asymmetric division and exhibiting Notch signaling; (b) analyzing the mechanical distinction with interfaces resulting from symmetrical division exhibiting no signaling; (c) studying the role of regulators of Notch signaling, of E- cadherin trafficking and of acto-myosin network remodeling in the control of mechanical properties of novel adhesive interfaces; (d) analyzing the mechanical characterization of the influence of a new interface on tissue reorganization. These approaches will be first performed on Drosophila epithelia, then applied to 3D epithelial cell cultures in which Notch signaling regulates cell fates (spheroids/organoids). In parallel, using genome editing tools and optogenetic approach (task 1), we wish to modulate phosphoinositide dynamics throughout cytokinesis. Indeed PIPs are essential for cell polarity establishment and maintenance, for completion of cytokinesis, and RNAi screen identified kinases and phosphatases involved in PIPs metabolism as potential regulators of Notch signaling.

Perspectives

These four tasks are aimed at deciphering the interplay between cell polarity, membrane trafficking, cell cycle and cellular mechanics in Notch-dependent fate acquisition. Based on the importance of Notch signaling in human pathology, including tumorigenesis, on a medium term, we aim to develop a genetically tractable mammalian cell system setup such as stem-cell based organoid to further investigate this interplay in a pathological context.

2.4. Collaboration

2.4.1. Within the IGDR

Dr. C. PRIGENT, Cell Cycle team (role of Aurora in symmetric versus asymmetric cell divisions: isolated cells, epidermal cells, sensory organ precursors, larval neuroblasts dividing in a stem cell mode- L. VAUFFREY, PhD student) - Dr. M. TRAMIER, R&D Quantitative Fluorescence Microscopy team (use of FAST- FLIM for the E-Cadherin-based FRET tension-sensor [linked to task 4, M. PINOT], investigating the role of AurA on mitochondria physiology in Drosophila- G. BERTOLIN, Post-doc in M. TRAMIER’s lab).

2.4.2. Other collaborations

International Dr. V. AULD (Department of Zoology/Cell Biology, Life Sciences Center, University of British Columbia, Vancouver, Canada): role of septate junctions in epithelial cell cytokinesis [impact on task 2] - Dr. U. TEPASS, University of Toronto, Canada: Role of AP-1 in basolateral membrane protein targeting [impact on task 3] - Dr. Y. TOYAMA, Institute For Mechanobiology, NUS, Singapore: Tensile forces and cell fate acquisition (Merlion Project, 2014-2016) [impact on task 4].

2.5. SWOT Analysis STRENGTHS Team internationally recognized as judged by the level of publications, invitations to meeting, selection of abstracts in key meetings/Conferences Team members are expert in their respective areas, highly motivated and focused The move to biophysical approaches and investigating the link between the mechanical properties of novel membrane interface and Notch signaling is timely made (hiring of M. PINOT, CR2 CNRS) Network of collaborators

178

WEAKNESSES Fundings need to be secured, still a long way to go before reaching the level of ERC funding Low attractiveness for high profile post-doctoral fellows Stabilize the task force (linked to funding) lack of mathematical modeling OPPORTUNITIES The turn to biophysical approach is timely made EM approaches Development of genome editing and optogenetic THREATS Field is competitive, no time for dispersion; the project must remain highly focused EM development (tedious and time consuming) relies on one person.There is a need to train/hire predocs and post-docs on this key superesolution microscopy approach.

179

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Benhra, N., Vignaux, F., Dussert, A., Schweisguth, F., and Le Borgne, R. (2010). Neuralized Promotes Basal to Apical Transcytosis of Delta in Epithelial Cells. Molecular Biology of the Cell 21 (12), 2078-2086. Cited in Faculty of 1000 Biology (IF: 5)

2011 Benhra, N., Lallet, L., Cotton, M., Le Bras, S., Dussert, A., and Le Borgne, R. (2011). AP-1 controls the trafficking of Notch and Sanpodo toward E-Cadherin junctions in sensory organ precursors. Current Biology 21 (1), 87-95. Cited in Faculty of 1000 Biology (IF: 10) Burgess J, Jauregui M, Tan J, Rollins J, Lallet S, Leventis PA, Boulianne GL, Chang HC, Le Borgne R, Krämer H, Brill JA. (2011). AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila. Molecular Biology of the Cell 22 (12), 2094-105. (IF: 5) Le Bras, S., Loyer, N. and Le Borgne, R. (2011) The multiple facets of ubiquitination in the regulation of Notch signaling pathway. Traffic 12(2), pp 149-61 (Review) (IF: 5) Founounou, N. and Le Borgne, R. (2011) Tissue polarity: PCP inheritance ensured by selective mitotic endocytosis. Current Biology 21 (18), R690-2 (Dispatch) (IF: 10)

2012 Le Bras, S., Rondanino, C., Kriegel-Taki, G., Dussert, A. and Le Borgne, R. (2012). Genetic identification of intracellular trafficking regulators involved in Notch-dependent binary cell fate acquisition following asymmetric cell division. Journal of Cell Science 125 (Pt 20), pp 4886-901 (IF: 6)

2013 Cotton, M., Benhra, N., and Le Borgne, R. (2013) Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor. Current Biology 23 (7), pp 581-7.Comment in Current Biology 23 (7) R270-2 (IF: 10) Founounou, N., Loyer, N. and Le Borgne, R. (2013) Septins regulate the contractility of the actomyosin ring to enable adherens junction remodeling during cytokinesis of epithelial cells. Developmental cell 24 (3), pp242-255. Cited in Faculty of 1000 Biology & comment in Developmental Cell 24(4) 336-8 (IF: 13) Hoffmann,C., Mazari, E., Lallet, S., Le Borgne, R., Marchi-Artzner, V., Gosse, C. and Gueroui, Z. (2013) Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles. Nature Nanotechnology 8 (3), pp 199-205 (IF: 33) Beckett K, Monier S, Palmer L, Alexandre C, Green H, Bonneil E, Raposo G, Thibault P, Le Borgne R., Vincent JP. (2013) Drosophila S2 Cells Secrete Wingless on Exosome-Like Vesicles but the Wingless Gradient Forms Independently of Exosomes. Traffic 14 (1), pp 82-96 (IF: 5)

2014 Le Bras S. and Le Borgne, R. (2014) Epithelial cell division – multiplying without losing touch. J. Cell Science 127 (24), 5127-37. Commentary (IF: 6)

2015 Loyer, N., Kolotuev, I., Pinot, M. and Le Borgne, R. (2015) Drosophila E-Cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth, accepted for publication at PNAS (IF: 9.7)

2. Patents (with licence)

N/A

3. Conferences (actual team members)

- International

2010 Roland LE BORGNE: The molecular and developmental biology of Drosophila-2010 Crete Drosophila meeting (Chiana, Crete, 20-26th June), Selected Speaker 180

Roland LE BORGNE: PepCon Conference, (Beijing, China, 20-23 March) Invited speaker and Chairman

2011 Najate BENHRA: Annual Drosophila Research Conference, San Diego, USA, March 30-April 3, Poster Roland LE BORGNE, Nabila FOUNOUNOU: EMBO Worshop on Septins, St Goar (Germany, March 6-9), 1 Talk (selection on abstract), 1 Poster Stéphanie LE BRAS, Nabila FOUNOUNOU, Mathieu COTTON: CCCD (cell cycle cancer and development) SBCF meeting Saint Malo, 3 Posters

2012 Roland LE BORGNE: The Notch meeting (Athens), Invited speaker Nabila FOUNOUNOU: Annual Drosophila Research Conference (Chicago), Talk (selection on abstract) Roland LE BORGNE: ESF- EMBO Conference on Cell Polarity and Traffic Membrane, Pulstuk, Poland, Talk (selection on abstract)

2013 Roland LE BORGNE: The Notch meeting (Athens), Invited speaker Roland LE BORGNE: Gordon Research Conference, Cell Contact and Adhesion (Lucca, Italy), Talk (selection on abstract) Roland LE BORGNE, Nabila FOUNOUNOU: Annual Drosophila Research Conference (Washington), 2 Posters Nicolas LOYER: Santa Barbara Advanced School of Quantitative Biology ‘New Approaches to Morphogenesis-Live Imaging and Quantitative Modeling’- Selected on application

2014 Stéphanie LE BRAS: The Notch Meeting Athens (Greece), Selected speaker Roland LE BORGNE: 19th The molecular and developmental biology of Drosophila-2014 Crete Drosophila meeting (Chiana, Crete, 22-28th June), Selected Speaker Roland LE BORGNE: IMP-Institute of Molecular Biotechnology, Vienna (Autria), Invited by J. KNOBLICH Stéphanie LE BRAS: ESF- EMBO Conference on Cell Polarity and Traffic Membrane, Pulstuk, Poster Nicolas LOYER, Irina KOLOTUEVA: 55th American Drosophla Research Conference, San Diego, 1 Talk selected from abstract, 1 Poster

2015 Roland LE BORGNE; Mathieu PINOT: 14th EMBO Endocytosis Conference- The multidisciplinary era of endocytic mechanics and functions, Mandelieu la Napoule, France, Invited Speaker, 1 Poster Roland LE BORGNE: University of Dundee, College of Life Sciences (Scotland), Invited by Dr. J. JANUSCHKE Roland LE BORGNE, Karen BELLEC: The Notch meeting, Athens, Organizing committee and Invited Speaker, 1 Poster Emeline DANIEL, Roland LE BORGNE: 56th American Drosophla Research Conference, Chicago, 1 Talk selected from abstract, 1 Poster

- National

2010 Roland LE BORGNE: Curie Development (Paris), Invited by Y. BELLAÏCHE

2012 Roland LE BORGNE, Nicolas LOYER: 1st joint meeting SBCF-SFBD ‘When Development meets Cell Biology’, (Montpellier), Invited speaker, 1 Poster Roland LE BORGNE: Institut Jacques Monod (Paris), Invited by A. GUICHET Roland LE BORGNE: IBDML (Marseille), Invited by L. KODJABACHIAN

2013 Roland LE BORGNE: Institut de Recherche Servier, ‘apport de la Drosophile dans la recherche sur le cancer’, Invited by B. LOCKART Roland LE BORGNE: Institut Pasteur (Paris), Invited by A. ECHARD

181

Roland LE BORGNE: Centre de Biologie du Développement (Toulouse), Invited by S. PLAZA and F. PAYRE Roland LE BORGNE: GRED (Clermont Ferrand), Invited by V. MIROUSE Roland LE BORGNE: Laboratoire de Biologie du Développement (Paris), Invited by M. GHO

2014 Roland LE BORGNE: Institut Albert Bonniot, Grenoble (France), Invited by C. ALBIGES-RIZO

2015 Roland LE BORGNE: Drosoph’Ile de France Day, Paris, Invited Speaker

4. Funding

2008-2010 Association pour la Recherche sur le Cancer (ARC-subvention fixe) 50 k€ 2008-2011 Région Bretagne – ARED 90 k€ 2009-2011 INCA (Partners: M. LABOUESSE, G. RAPOSO) 110 k€ 2009-2010 Université de Rennes 1 40,8 k€ 2009-2012 ANR PNANO (Partners: Z.GUEROUI, V. MARCHI-ARTZNER, C. GOSSE) 100 k€ 2010-2011 CNRS – SDV 60 k€ 2010-2011 Université de Rennes 1 40,8 k€ 2011-2012 La Ligue contre le cancer comité 35 25 k€ 2012-2015 ANR blanc SVSE2-ApiNotch (Porteur) 430 k€ 2012-2015 ANR blanc SVSE5- KinBioFRET (Partners: M. TRAMIER & C. PRIGENT, IGDR) 20 k€ 2014-2016 Merlion Project- collaborative project with Dr Y. TOYAMA, NUS-MBI, Singapore 30 k€ 2014-2016 FRM- Projet Innovant (genome editing and optognetics) 90 k€ 2014-2017 Région Bretagne 45 k€ 2014-2019 Equipe Labéllisée Ligue Nationale contre le Cancer 50 k€/ year 2015- SATT maturation project inDroso 64.5 k€ 2015 BIOSIT 8 k€ 2015 Université de Rennes 1 10 k€

5. Training Teaching Stéphanie LE BRAS: As an associate professor, teaches 192h/year included 25% in the master SCMV of Rennes 1 University. Creation and supervision of two Master 1 teaching modules: Eukaryotic genetic models (15-35 students) and Integrative Cellular Biology (30-40 students), management of internship offers in public and private academic research in France and abroad (https://etudes.univ-rennes1.fr/master-scmv/themes/Stages/Stages_proposes) and development of a professional network alumni through LinkedIn. Invited teacher (4 hr/r year) in Master 2 teaching module (Stem Cells and Therapy), European Genetics Master, Paris VI. Licence 2011 - Margot MORIN, L2 (1 month) 2013 - Jules LAVALOU, L3 (6 weeks) 2014 - Anne-Sophie LANCELOT, L1 (1 week) - Bénédicte LEFEVRE, L2 (1 month) 2015 - Anne-Sophie LANCELOT, L2 (3 weeks) - Thomas ESMANGART de BOURNONVILLE, L3 (6 weeks) Master 2011 - Sylvain BERTHO, M2 (6 months) 2013 - Karen BELLEC, M1 (2 months) - Yacine KHELIF, M1 (2 months) - Emeline DANIEL, M2 (6 months) 2014 - Guillaume JACQUEMIN, M1 (2 months) 2015 - Marion DAUDE, M1 (2 months) PhD 23/09/2010: Najate BENHRA – Dir. Thèse Roland LE BORGNE – currently post-doc in Marco MILAN’s lab (Barcelona) 28/11/2012: Nabila FOUNOUNOU - Dir. Thèse Roland LE BORGNE – currently post-doc Marek MLODZIK’s lab (New York) 16/10/2014: Nicolas LOYER - Dir. Thèse Roland LE BORGNE – currently post-doc in Jens JANUSCHKE’s lab (Dundee) Since 10/2014: - Emeline DANIEL - Dir. Thèse Roland LE BORGNE - Karen BELLEC - Dir. Thèse Stéphanie LE BRAS & Roland LE BORGNE Post-doctorants 2009-2013: Mathieu COTTON, post-doctoral fellow 4 years & 4,5 months - currently looking for a position 2009-2011: Christine RONDANINO, post-doctoral fellow 1 year & 10,5 months - GreD, Clermont Ferrand 2014-2015: Mathieu PINOT, post-doctoral fellow 1 year & 2,5 months - hired CR2 CNRS in 10/2015

182

CDD 2009-2010 & 2013-2014: Aurore DUSSERT, Engineer CNRS, contract 1 year & 6 months & contract 1 year. 2014-2016: Perrine UHART, Engineer CNRS, contract 2 years.

183

Team 13 "Membrane traffic and polarity in C. elegans" Leader: Grégoire MICHAUX

185

2.1. Team presentation

Without food absorption animal organisms cannot survive. Our goal for the coming years will be to identify the mechanisms required for the maintenance of the absorption function at the apical membrane in intestinal epithelial cells. We will achieve this goal by focussing on the poorly characterised interactions between membrane traffic factors, the apical PAR/Cdc42 module and the brush border structural components. We will use C. elegans as a model organism and exploit our results to better understand rare congenital absorption disorders. This project is a development of our previous focus on the control of apical trafficking in intestinal epithelial cells.

To this aim we will exploit recently developed tools such as knock-in insertions of fluorescent reporters using the CRISPR-Cas9 technology to observe endogenous expression levels in vivo, a degron system allowing efficient and rapid in vivo depletion of specific factors (Armenti et al, Development, 141- 4640), and our newly developed protocol for C. elegans immuno electron microscopy (IEM) (Nicolle et al, Traffic, in press). We will also develop tools to quantitatively measure food absorption in vivo.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

MICHAUX Grégoire CR1 CNRS NICOLLE Ophélie IE2 UR1 PACQUELET Anne CR2 INSERM

2.2.2.2. Temporary staff

GILLARD Ghislain PhD student (2012-2016)

2.2.3. Achievements

2.2.3.1. Scientific achievements 186

A) Control of apical trafficking in epithelial cells

- Membrane traffic and polarity maintenance in the intestine; Shafaq-Zadah et al, Development, 2012; Michaux et al, in revision. Understanding how the polarity of epithelial tubes is maintained is essential. We found that the clathrin adaptor AP-1 is required for epithelial polarity maintenance by controlling the polarised distribution of CDC-42 and the apical PAR module. Upon AP-1 depletion, lateral mislocalisation of CDC-42 and the PAR module induces the conversion of the lateral membrane into an apical membrane with the formation of microvilli-containing ectopic lumens. Our results demonstrates that AP-1 controls an apical trafficking pathway required for the maintenance of epithelial polarity in vivo in a tubular epithelium. => Contrary to the commonly accepted model, we showed that AP-1 does not control only basolateral trafficking but also apical sorting, and that this function is dominant in the intestine. Similar results obtained in the mouse intestine (Hase et al, Gastroenterology, 2013) confirmed this new AP-1 function. => Our work also led to translational research in collaboration with Dr Frank Ruemmele (Hôpital Necker-Enfants Malades/Institut Imagine) and André Le Bivic (IBDM, Marseille) to better characterise defects associated with Microvillus Inclusion Disease, a rare congenital absorption disorder leading to the formation of intracellular ectopic lumens (Michaux et al, in revision).

- Apical sorting of E-cadherin in the epidermis; Gillard et al, Development, 2015. E-cadherin is the main component of adherens junctions in multicellular organisms. Its localisation is often strongly polarised along the apico-basal axis. However the mechanisms required for its polarised distribution are largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epidermal cells. We found that clathrin, its adaptor AP-1, the AP-1 interactor SOAP-1/p200 and the small GTPase Rab11 are all required for E- cadherin apical sorting. We also showed that this function of AP-1 in E-cad apical delivery is essential during elongation, the final morphogenetic step of embryogenesis. We propose that a molecular pathway including SOAP-1, AP-1, clathrin and RAB-11 controls the apical delivery of E-cadherin and morphogenesis. The only other candidate identified in the screen is a small GTPase of the Rab family which is currently being characterised in C. elegans. We will also investigate its function in mammalian cells in collaboration with the group of Franck PEREZ (Institut Curie, Paris) using its RUSH protocol to characterise membrane trafficking. => This work led to the identification of the first factors controlling E-cad apico-basal distribution in vivo with a systematic physiological impact on morphogenesis.

B) Coupling the positions of the mitotic splindle and the cytokinetic furrow

- PAR-4/LKB1 and anillin function in coupling mitotic spindle and cytokinetic furrow positions; Pacquelet et al, accepted. Mitotic spindle and polarized myosin can both determine the position of cytokinetic furrowing. However, how cells coordinate both activities to ensure that cleavage occurs through the central spindle is unknown. In C. elegans, myosin localizes to the anterior while the mitotic spindle localizes to the posterior of the one-cell embryo. We found that in the absence of both PAR-4/LKB1 and anillin ANI-1, myosin accumulation at the anterior cortex increases and induces a strong shift of the cytokinetic furrow towards the anterior while the spindle remains at its posterior position. This uncoupling between spindle and furrow positions demonstrates that PAR-4/LKB1 and anillin play an essential role in preventing asymmetrically localized myosin from shifting the cytokinetic furrow away from the central spindle. Our results indicate that the ability of cells to use both spindle and myosin to induce cytokinesis furrowing must be accompanied by a tight regulation of myosin to prevent the uncoupling of spindle and furrow positions. => We uncovered a new mechanism controlling myosin activity during the positioning of the cytokinetic furrow.

2.2.3.2. Scientific dissemination and influence

Grégoire MICHAUX: Board member of the IGDR PhD program - Coordinator of the IGDR Cell and Developmental Biology department.

187

2.2.3.3. Interaction with the economic, social and cultural environment

Grégoire MICHAUX: Board member of the French Society for Cell Biology (SBCF) since 2008 - SBCF newsletter and website editor.

2.3. Projects, scientific strategies & perspectives (5 years)

The functional and higly specialised apical membrane of intestinal cells is necessary for food absorption. Our goal is to identify the mechanisms required for the maintenance of the absorption function at the apical membrane. We will achieve this goal by focussing on the poorly characterised interactions between membrane traffic factors, the apical PAR/Cdc42 module and the brush border structural components, using C. elegans as a model organism.

A) Genetic control of intestinal polarity and food absorption

Our first goal will be to characterise the role of membrane traffic in the maintenance of epithelial polarity and more specifically of the apical pole of intestinal cells. To that aim we have performed several in vivo RNAi screens in the intestine of C. elegans to identify new membrane traffic factors required for the localisation of the apical PAR module and Cdc42. Other genes have been identified by other groups or are mutated in rare human diseases affecting intestinal absorption. Altogether these genes define a network of factors which have a strong role in the maintenance of apical polarity and/or food absorption. These factors are implicated in many trafficking steps including cargo sorting (AP-1 and glycosphingolipids/GSL), vesicle transport (Rab8, Rab11, Myo5B) or acidification (V-ATPase) and membrane fusion (Munc18-2, Nsf, Syntaxin-3) and are all required to control the localisation of apical polarity determinants and microvilli proteins. However they do not all have the same function. For instance AP-1 do not affect the apical localisation of polarity determinants but it prevents their lateral accumulation; in contrast Rab11 is strictly required for the apical localisation of these polarity determinants (Shafaq-Zadah et al, Development, 139-2061). To better analyse the function of these genes we will perform a series of functional studies including localisation of polarised and intracellular markers, genetic interactions and phenotype analysis by EM. In parallel we will exploit new tools to quantitatively investigate food absorption in vivo as a functional test for the integrity of the absorption function of the intestine (Gomez-Amaro et al, Genetics, in press). This will lead to the characterisation of the networks formed by these factors and help us to understand how they participate to the robustness of epithelial polarity maintenance essential for intestinal absorption.

B) Maintenance of intestinal absorption: the role of a Sec1/Munc18 protein

One of the genes we identified as required for PAR-6 and CDC-42 apical localisation in the C. elegans intestine could be the functional homolog of Munc18-2, a member of the Sec1/Munc18 family which is mutated in type 5 familial hemophagocytic lymphohistiocytosis (FHL5; Côte et al, J Clin Invest, 119-3765; zur Stadt et al, Am J Hum Genet, 85-482). This genetic disorder is characterised by immune and absorption defects. The latter phenotype was recently associated with disorganisation of microvilli and abnormal staining of non-essential apical markers (Stepensky et al, Pediatr Blood Cancer, 60-1215). We will characterise the function of Munc18-2 both in C. elegans and in human tissues. First we will characterise Munc18-2 function in C. elegans by genetic and biochemical approaches. We will analyse its loss of function phenotype by fluorescence and electron microscopy, identify the cargos dependent on Munc18-2 and the membrane traffic factors which could interact with it, such as the functional homolog of syntaxin-3 which is mutated in MVID (Wiegerinck et al, Gastroenterology, 147-65) and is known to interact with Munc18-2 (Riento et al, J Cell Sci, 111-2681). We will then exploit the results obtained in C. elegans to help characterise absorption defects in patients carrying mutations in Munc18-2 as we have done for other absorption disorders (Michaux et al, in revision). In that perspective we have established a collaboration with the group of Geneviève de SAINT BASILE (Institut Imagine/Hôpital Necker-Enfants Malades) who is working on FHL5 and Munc18-2 in a mouse model and in human tissues.

C) Interactions between apical polarity determinants and brush border components

The formation and maintenance of the absorptive apical membrane of the intestine has been mostly studied by focussing on the brush border (i.e. microvilli) and the role of actin and its interactors (reviewed in Crawley et al, J Cell Biol, 207-441). Meanwhile recent work has revealed an essential but poorly characterised function for membrane traffic in the localisation of the apical PAR/Cdc42 module (reviewed in Apodaca, Nat Cell Biol, 14-1235) and of the brush border (Crawley et al, J Cell Biol, 207- 441). However their relationships are not well understood and we aim to better characterise the 188

interactions between the apical PAR/Cdc42 module and brush border components. Because the precise localisation of the apical PAR/Cdc42 module with respect to the brush border is not known we will perform colocalisation studies of these factors. At the light microscopy level we will use CRISPR/Cas9- mediated knock-in insertion of GFP or mCherry; at the ultrastructural level we will perform IEM, the ultimate super-resolution technique (Nicolle et al, Traffic, in press); correlative light electron microscopy (CLEM) will also be exploited with the method of correlative array tomography developped with the help of Irina KOLOTUEVA. Physical interactions between polarity proteins and brush border components will be tested by biochemistry. To examine the role of the PAR/Cdc42 module in the maintenance of the brush border we will exploit a degron system allowing a fast and very efficient targeting of endogenous genes (Armenti et al, Development, 141, 4640) at any time during development and in adults. The role of other polarity proteins such as PAR-4/LKB1 and PAR-5 (Winter et al, Nat Cell Biol, 14-666) which could have a role in polarity maintenance will also be investigated. This work, which will elucidate the interactions between polarity determinants and brush border components, could then be transferred to mammalian models and help to better understand genetically-based human absorption disorders.

Conclusion

Together with our previous work and the screens we have already performed, this project will lead to an in depth understanding of the genetic and molecular networks required for the maintenance of an absorptive membrane in enterocytes. It will also contribute to characterise absorption disorders in patients in close collaboration with groups (Geneviève de SAINT BASILE) and physicians (Frank RUEMMELE) of Institut Imagine/Hôpital Necker-Enfants Malades.

2.4. Collaboration

2.4.1. Within the IGDR

Jacques PECREAUX (Signal analysis) - Roland LE BORGNE (AP-1 and E-cadherin trafficking).

2.4.2. Other collaborations

National Frank RUEMMELE, Institut Imagine, Hôpital Necker-Enfants Malades, Paris & André LE BIVIC, IBDM, Marseille (Polarity and membrane traffic in Microvillus Inclusion Disease. Michaux*... Le Bivic... Ruemmele, in revision. *corresponding author) - Geneviève de SAINT BASILE, Institut Imagine, Paris. (Polarity and membrane traffic in Familial Hemophegocytic Lymphohistiocytosis) - Franck PEREZ, Institut Curie, Paris (Membrane traffic in mammalian models / RUSH protocol).

International Dan CUTLER, LMCB, London ( Whole genome RNAi screening to identify new AP-1 interactors. Michaux* ... Cutler*, J Thromb Haemost. 9, 392-401. *Corresponding authors).

2.5. SWOT Analysis STRENGTHS Fundamental to translational Expertise in polarity and membrane traffic Integration in IGDR WEAKNESSES Size of the team International recognition OPPORTUNITIES Transfer to the clinic Potential application (food absorption)

189

THREATS Level of funding Competition

190

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Kumar S, Aninat C, Michaux G, Morel F (2010). Anticancer drug 5-fluorouracil induces reproductive and developmental defects in Caenorhabditis elegans. Reprod Toxicol. 29, 415-20. (IF 2.8)

2011 Michaux G*, Dyer CEF, Nightingale TD, Gallaud E, Nurrish S, Cutler DF* (2011). A role for Rab10 in Von Willebrand Factor release discovered by an AP-1 interactor screen in C. elegans. J Thromb Haemost. 9, 392-401. *Corresponding authors. (IF 5.6)

2012 Britto R, Sallou O, Collin O, Michaux G, Primig M, Chalmel F (2012). GPSy: a cross-species gene prioritization system for conserved biological processes-application in male gamete development. Nucleic Acids Res, 40, W458-65. (IF 8.8) Shafaq-Zadah M, Brocard L, Solari F, Michaux G (2012). AP-1 is required for the maintenance of apico-basal polarity in the C. elegans intestine. Highlighted in "In this issue" section. Development, 139, 2061-2070. (IF 6.3)

2015 Gillard G, Shafaq-Zadah M, Nicolle O, Damaj, R, Pécréaux J, Michaux G (2015). Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells. Development, 142:1684-1694. Journal cover. (IF 6.3) Nicolle O, Burel A, Griffiths G, Michaux G*, Kolotuev I* (2015). Adaptation of cryo-sectioning for immuno-EM-labeling of asymmetric samples: C. elegans case study. Traffic, in press. *Corresponding authors. (IF 4.6) Pacquelet A, Uhart P, Tassan JP, Michaux G (2015). PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division. J Cell Biol, accepted. (IF 9.8).

2. Patents (with licence)

N/A

3. Conferences (actual team members)

- International

2011 Massiullah SHAFAQ-ZADAH: BSCB-BSDB "Joint spring meeting", Kent university, UK. Selected speaker. Grégoire MICHAUX: "C. elegans international meeting", Los Angeles, USA. Selected speaker.

2012 Grégoire MICHAUX: ESF/EMBO "Cell polarity and membrane traffic", Pultusk, Poland. Selected speaker. Grégoire MICHAUX: Seminar at LMCB, London, UK. Invited by Dan CUTLER.

2013 Anne PACQUELET: "EMBO meeting", Amsterdam, Netherlands. Selected speaker.

2014 Grégoire MICHAUX: ESF/EMBO "Cell polarity and membrane traffic", Pultusk, Poland. Selected speaker. Grégoire MICHAUX: Seminar at the Imagine Institute - Invited by Matias SIMONS. Ghislain GILLARD: ASCB meeting, Philadelphia, USA. SBCF-ASCB prize. Ghislain GILLARD: Seminar at the Duke University Cell Biology Department. Invited by Michel BAGNAT.

- National

191

2011 Grégoire MICHAUX: SFBD-BSDB joint Meeting, Nice, France. Selected speaker.

2012 Grégoire MICHAUX: Seminar at IBDM, Marseille. Invited by André LE BIVIC. Anne PACQUELET: Seminar at the CGΦMC. Invited by Frédéric MOREL.

2013 Anne PACQUELET: Ver Midi meeting, Lyon. Selected speaker.

2014 Grégoire MICHAUX: SBCF "Building the cell" meeting, Paris, France. Selected speaker. Ghislain GILLARD: Ver Midi meeting, Paris. Selected speaker. Grégoire MICHAUX: Seminar at the CGΦMC. Invited by Kathrin GIESELER.

2015 Anne PACQUELET: Ver Midi meeting, Paris. Selected speaker. Ophélie NICOLLE: RCCM meeting, Université de Corse. Invited speaker.

4. Funding

2009-2011 INSERM 50 k€ 2010 Avenir + 30 k€ 2011 Avenir + 30 k€ 2012 Ligue contre le cancer 22 k€ BIOSIT microscopy 10 k€ 2013 Ligue contre le cancer 25 k€ AVIESAN Rare diseases 15 k€ 2014 Ligue contre le cancer 24 k€ 2015 Rare Diseases Foundation 20 k€ Ligue contre le cancer 23 k€ Ligue contre le Cancer - 4th year PhD (Ghislain GILLARD) 30 k€

5. Training Licence 2011 - Guillaume JACQUEMIN, L1 University Rennes 1 (2 months) 2012 - Marine LEHUE, L3 University Rennes 1 (6 weeks) 2013 - Nadège MOREAU, L3 University Rennes 1 (6 weeks) 2014 - Markus HECK, Erasmus internship (5 months) BTS 2010 - Virginie VION, BTS Angers Master 2010 - Emmanuel GALLAUD, M2 University Rennes 1 (5 months) 2011 - Ghislain GILLARD, M1 University Rennes 1 (2 months) - Clémence KERGOULAY, M1 University Rennes 1 (2 months) - Aurélien PERRIN, M2 University Rennes 1 (5 months) 2012 - Emeline DANIEL, M1 University Rennes 1 (2 months) 2013 - Sophie ROSE, M1 University Rennes 1 (2 months) - Romain JUGELE, M1 University Rennes 1 (2 months) 2014 - Perrette QUERIC, M1 internship (4 months) - Perrine UHART, M2 University Rennes 1 (5 months) 2015 - Marjorie PONNAVOY, M2 University Rennes 1 (5 months) - Thomas LE GAL, M2 University Rennes 1 (5 months) PhD 12/01/2012: Massiullah SHAFAQ-ZADAH – Dir. Thèse Grégoire MICHAUX- Currently postdoc at Institut Curie, France. Since 10/2012: Ghislain GILLARD – Dir. Thèse Grégoire MICHAUX Post-doctorants 2008-2010: Raghida DAMAJ, post-doctoral fellow 1 year & 10,5 months 2008-2010: Alexander HOLMES, post-doctoral fellow 2 years & 8,5 months

192

Team 14 "Integrated functional genomics and biomarkers" Leader: Jean MOSSER

193

2.1. Team presentation

The research interests of the team has revolved around two themes focusing on the understanding of the genetic and epigenetic mechanisms linked to the development of somatic or constitutional genetic diseases. In sporadic cancers (glioblastoma), we look for the molecular elements associated with tumor heterogeneity in order to improve the individualization of patient care. In the case of cancer arising as a complication of a genetic disease (hemochromatosis), we look for genetic factors modifying the HFE- hemochromatosis penetrance. This translational project will contribute to the tumor molecular stratification and to the individualization of patient care. For our projects, we benefit from regional, national and European cohorts. We also rely on the Human Health Genomic Platform (BIOSIT) that we have in charge at the scientific level. Therefore we use and develop tailored "omic" approaches required for our research. For instance, technological innovations such as Next Generation Sequencing and genotyping of circulating DNA are developed to allow patient monitoring on liquid biopsies, to assess response to therapy and to anticipate relapses or treatment failures.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

LIEVRE Astrid PU-PH2 UR1-CHU AVRIL Tony IR CRLCC (50%) MONNIER Annabelle MCU UR1 BELLIER Rachel TCN UR1 MOSSER Jean PU-PH1 UR1-CHU DENOUAL Florent IR CHU (50%) QUILLIEN Véronique PH CRLCC (30%) ETCHEVERRY Amandine IR CHU (50%) VAULÉON Elodie PH CRLCC (15%) LESPAGNOL Alexandra IR CHU (50%) AUBRY Marc IR2 UR1 (20% UMS BIOSIT) SURBLED Cyrille ADTRF UR1

2.2.2.2. Temporary staff

N/A

2.2.2.3. Permanent staff who left the team during the contract de TAYRAC Marie MCU-PH2 UR1-CHU (until 05/2015) 194

2.2.3. Achievements

2.2.3.1. Scientific achievements

During this former contracting period, our team was involved in understanding the genetics of HFE-hemochromatosis, an issue that has considerably evolved over the last decade. HFE- hemochromatosis is seen as a rare disease with variable penetrance, constituting a complex multifactor genetic trait. In this context, our plan was to identify HFE-hemochromatosis modifier genes by a GWAS approach. At the end of this work, this thematic has been stopped in order to exclusively focus on the molecular genetics of solid tumors. For this purpose, we have initiated regional and national collaborative studies on the genetics and epigenetics of glioblastoma, a severe primary brain tumor, which has become our major team project. The reason for that change is the necessity to be in phase with our new clinical activity: the somatic molecular genetic characterization of solid tumors in a context of routine diagnostic but also of diagnostic innovation linked to clinical research.

Our main achievements during the past 5 years concern this two thematics: genetic of HFE- hemochromatosis and molecular characterization of solid tumors with a focus on GBM.

Such genetic studies needs the availability of large cohorts of patients. We have centralized the constitution of 3 large cohorts:

- an international cohort of more than 1000 unrelated patients with C282Y HFE-hemochromatosis useful to identify modifier genes of iron overload

- a national cohort of more than 400 fully annotated GBM patients required to characterize the GBM molecular heterogeneity in terms of prognosis and response to therapy

- and a large regional cohort of more than 3000 solid tumor FFPE samples in order to improve the theranostic molecular stratification of solid tumors

These cohorts have been used to perform genetic and omic studies :

- A GWAS, the largest performed so far in unselected HFE-HH patients, identified the rs3811647 polymorphism in the TF gene (transferrin) as the only SNP significantly associated with iron metabolism through serum transferrin and iron levels. (de Tayrac M et al., Journal of hepatology [IF 10.4]).

- In order to improve the molecular stratification of High Grade Glioma or GBM in relation with tumor aggressiveness (prognosis) and response to the standard therapy, we use "omic" approaches to construct prognostic or predictive model potentially useful for clinical practice. We reported a robust risk estimation model based on the weighted expression level of 4 genes (CHAF1B, PDLIM4, EDNRB, and HJURP) in high-grade glioma. This 4-gene signature provides an independent risk score strongly associated with the outcome of patients with high-grade gliomas. (de Tayrac, Aubry et al. Clin Cancer Res 2011;17(2); 317–27 [IF 8.2]; de Tayrac et al. PLoS One 2013;8(9):e73332 [IF 3.5]). We further focused our attention on GBM only and performed a methylome analysis of about 400 homogeneously treated GBM, which led to the characterisation of a new marker: the methylation status of the DGKI gene promoter which identifies among potentially responder GBMs (MGMT promoter methylated GBM) those who will not respond to the standard treatment based on an alkylating drug, the temozolomide. The assessment of DGKI methylation status therefore improves the conventional MGMT stratification of GBM patients receiving standard treatment and can be of help in the interpretation of published or ongoing clinical trial outcomes. It can also be used to refine patient recruitment in the future. Eventually, this marker may constitute a potential target for epigenetic drugs (Etcheverry A et al. BMC Genomics, 2010, 11(1):701. [IF 4.1]; PloS one 2014,9,9,e104455, [IF 3.5]).

By cellular - functional analyses, we have characterized GBM stem-like cells and identified putative targets for immunotherapies. We have derived primary glioblastoma cell lines derived either as adherent cells in the presence of serum (n=11) or as neurospheres enriched in cancer stem cells (CSC; n=12). We have shown that CSCs, which are considered to play an important role in disease recurrence, are suitable target cells for immunotherapy. We have conducted a proteomic analysis of these cell lines and have shown that each type of cell line display different GBM-specific features, highlighting that these two types of culture are complementary tools for drug screening. (Avril et al, Brain pathol, 2012; 22(2);159-74 [IF 4.3]; Collet et al. Journal of proteomics,110,7-19,2014 [IF 3.9]) 195

Finally, Our research expertise has implications in the improvement the molecular diagnosis (stratification) of solid tumors:

- We demonstrated that pyrosequencing is the best test to assess the MGMT promoter methylation in GBM. Now this technics is considered as the reference method for routine MGMT methylation assessment in France.

- And we have transferred Next Generation Sequencing (NGS) as a routine technology to test theranostic somatic biomarkers for the molecular diagnosis of solid tumors. In 2014, we were the first lab in France (belonging to INCa – French National Cancer Institute – molecular genetic platform network) to implement NGS for routine diagnosis of solid tumor.

2.2.3.2. Scientific dissemination and influence

Conference invitations: - 9th Meeting of the European Association of Neurooncology, Maastricht, The Netherlands September 16-19, 2010. - Journée inter-cancéropôles : Les glioblastomes, Marseille, 22/02/2012 - 1ere journée éducationnelle de l’ANOCEF, Paris 14/11/2013 - Ateliers “characterisation moléculaire tumorale à haut debit”, GFCO, Paris, 27-28/03/2015 - Congres annuel de l’ANOCEF, Amiens, 19-20/06/2015

Member of scientific societies: - ANOCEF - Association des Neuro-Oncologue d'Expression Française - ANPMCB – CNBBMM - Le Collège National de Biochimie et de Biologie Moléculaire Médicale

Member of National working group: - INCa NGS Working group - INCa bioinformatics network in NGS transfer on cancer molecular diagnostic

Regional networks: - Canceropôle Grand Ouest Réseau Epigénétique - REPICGO, - Canceropôle Grand Ouest Réseau Gliome Grand Ouest – REGGO

Expertise – evaluation: - Member of the national evaluation comity of the training program for Translational Cancer Research (AVIESAN – INCa), 2015 - Member of the scientific evaluation comity of the "Groupement Interrégional de Recherche Clinique et d’Innovation Grand Ouest" - Reviewer for Plos one

2.2.3.3. Interaction with the economic, social and cultural environment

Our expertise in genetics and genomics, acquired since several years, has lead to the improvement of cancer affected patients care. Indeed, we now provide a NGS based genotyping of one hundred genetic targets for the weekly molecular stratification of patients affected by non-small lung cancer, metastatic colorectal cancer, melanoma and glioma. Therefore, about 2600 patients a year benefit from this testing which permit the choice of the targeted therapy strategy or, if any, patient enrolment in adequate clinical trials such as the AcSe crizo and vemurafenib national protocols. Thus, our diagnostic innovation in cancer molecular diagnostic has direct social (patient care improvement) and economical (therapy strategy definition) impacts.

We have also develop partnerships with private companies: - Industrial Contract with the Roche laboratory: ANTiCIPe project "Advancing monitoring of NSCLC Treated with EGFR TKI: Molecular diagnosis is circulating tumor DNA" - Partnership with Biotrial company (CRO) for the development of xenograft models with primary glioblastoma cell lines. - Partnership with the AstraZeneca laboratory for the formation of members of other INCa platform to the free circulating tumor DNA testing.

196

2.3. Projects, scientific strategies & perspectives (5 years)

We propose a new name for the team: Translational Oncogenomics, a name that corresponds better with our team activities. • The members belonging to the Rennes anticancer Centre Eugène Marquis (T. AVRIL, V. QUILLIEN, E. VAULEON) will leave the team in 2017, due to a scientific policy reorientation of the Centre. • The team will integrate (1) F. DENOUAL and A. LESPAGNOL, members from the Medical Genomic and Somatic Cancer Genetic Labs (University Hospital Centre [CHU] from Rennes) and (2) Astrid LIEVRE nominated as PU-PH2 in gastroenterology in 2015 in Rennes. She is expert in digestive oncology and will initiate a similar scientific genomic project applied to digestive cancers. She will also be implicated in the coordination of an in site clinic-biological oncodigestive care framework in Rennes (FHU CAMIn: Hospital-University Federation "CAncer, Microenvironnement et Innovation"). • We will continue our working in tight collaboration with Marc AUBRY (Research Engineer in Bioinformatics, Genomic Health Platform from Rennes, BIOSIT).

Our project is within the scope of the improvement of solid tumors personalized medicine by using and identifying new relevant genetic biomarkers. By improving the tumor molecular stratification and by contributing to the individualization of patient care, this translational project will lead to an impact on the solid tumor diagnosis and prognostication. Technological innovations such as Next Generation Sequencing and genotyping circulating DNA will allow patient monitoring on liquid biopsies to assess response to therapy and to anticipate relapses or treatment failures.

Functional Genomic expertise acquired by the team and relying on the Genomic health platform has been applied to the study of glioblastoma (GBM). GBM is the most frequent and the most aggressive primary brain tumor in adult. The standard therapy associates brain surgery, radiotherapy and alkylating chemotherapy. But the prognosis remains dismal (survival median = 15 months). There is no efficient therapy yet. New strategy has to be proposed, which may rely on knowledge on GBM predispositions, on epigenetic mechanisms, on intra tumor heterogeneity characteristics, or on the microenvironment analysis (tumor progenitor cell renewal or tumor immune infiltration characteristics).

1- The development or the use of omic and cellular approaches will characterize new key somatic markers and putative therapeutic targets.

Starting from the promising data of DGKI promoter methylation status (which is an unfavorable epigenetic signature), we want to further explore the GBM methylome, genome and transcriptome with two objectives:

First, we want to precise the role and the prognostic vale of DGKI in an omic context. And we plan to: - Complete the molecular annotation of 120 GBMs (at the transcriptome, genome, methylome levels) - Compare the prognostic value of DGKI with others known molecular biomarkers - Identify other potentially co-methylated promoters that belong to the DGKI methylation class - Functionally annotate the expression signature that is significantly associated with this DGKI-class

Then, we plan to go further in tight collaboration with the group of Pierre François CARTRON (INSERM U892, Nantes), which has a recognized expertise in the field of molecular epigenetics. The second objective is to develop a potential DGKI-selective epigenetic drug. For that, our research will: - in silico identify and in vitro validate [Dnmt/TF] complexes responsible for the methylation of DGKI - identify all the targets of the validated [Dnmt/TF] complexes (by ChIP-seq) - and identify small molecules able to disrupt the [Dnmt/TF] complexes and reverse the unfavorable epigenetic mark.

This project will (1) validate the predictive value of the DGKI methylation status or the DGKI related methylome class, (2) propose an optimal biomarker set for prognostication and therapy response prediction in GBM, and (3) identify a epidrug able to reverse the unfavorable DGKI methylation mark.

197

Besides, the GBM intra tumor heterogeneity is poorly described. However its molecular characterization is challenging given the systematic tumor recurrence starting from disseminated cells at the site of neurosurgery. Therapy Improvement needs biomarkers of tumor infiltration. Therefore we are conducting integrated "omic" analyses of 4 distinct tumor zones (necrosis, tumor, interface and normal peripheral brain) from 10 unrelated GBM patients. Our preliminary results described an expression signature underlying the intratumor gradient from the core to beyond the margin of the tumors. Now we plan (1) to test an epigenetic hypothesis that may control this expression signature and (2) to functionally study new genes of interest within this expression signature. These genes will be inactivated or overexpressed within GBM cell lines in order to test for induced cell phenotypes. For genes coding transcription factor we will subsequently perform ChIP-Seq to decipher the regulation network that is potentially associated with cell behaviour modifications (growth, proliferation, apoptosis or migration) induced by transcription factors belonging to this expression gene signature.

2-Technological innovations such as Next Generation Sequencing (NGS) and genotyping cell-free circulating tumor DNA will improve patients care and allow patient monitoring on liquid biopsies to asses the response to therapy and to anticipate relapses or treatment failures. The Rennes hospital somatic genetic laboratory is involved in the weekly molecular theranostic diagnosis of solid tumors with now 2800 samples per year. This lab is the first in France to use targeted-NGS as a routine method for the genotyping of solid tumors on a dedicated gene panel. This custom panel includes 100 putative predictive targets distributed within 20 key cancer genes. It is not only useful for the routine diagnosis but also for clinical research (clinical trail enrolment based on molecular stratification). For glioma prognostic assessment and to ensure optimal patient enrolment in clinical trials we plan to adapt the tumor solid gene panel composition by including new useful markers (TERT, H3.3A…). We will also include in the gene panel biomarkers of resistance to targeted therapy (successive generation of EGFR-TKI, ALK-TKI, BRAF- Inhibitors for instance). We want then to develop and to validate a NGS based screening protocol on plasma free DNA and eventually propose this procedure for the longitudinal monitoring of patients affected by GBM, lung cancer, colon cancer and melanoma. This approach may be transferred to patient experiencing a therapeutic escape for all types of solid tumors.

For all these on-going research projects linked to diagnostic innovation we have several collaborators and partners:

2.4. Collaboration

2.4.1. Within the IGDR

Catherine ANDRE team - Véronique DAVID team - Marie-Dominique GALIBERT team.

2.4.2. Other collaborations

Rennes Caroline ABADIE, Oncogénétique, Centre Eugène Marquis, Rennes - Hervé LÉNA, Pneumologie, CHU de Rennes - Dominique LAVENIER, IRISA, INRIA, Université de Rennes 1.

National Philippe MENEI, Inserm U646, Chu d’Angers, Canceropole Grand-Ouest - Pierre-François CARTRON, Inserm U892, Nantes, Canceropole Grand-Ouest - Jean-Yves DELATTRE, CRICM UMR-S975, GH Pitié -Salpetriere, Paris - Dominique FIGARELLA, CRO2, UMR 911 Université d’Aix-Marseille, AP-HM Timone Marseille.

International Xiang ZHANG, Department of Neurosurgery, Xijing HospitalFourth Military Medical University, China - Stephan SAIKALI, Hôpital de l’enfant Jesus, CHU de Quebec, Canada

2.5. SWOT Analysis STRENGTHS Strong expertise in Genomics Equipment & IT solution National Networks 198

Translational approach WEAKNESSES Expertise in functional analysis to be improved Weak attractiveness for Post-doc / PhD students Lack of international collaborations Communication strategy - AERES B OPPORTUNITIES Collaboration for functional studies Leading physician in the team THREATS Funding High competition: TCGA consortium

199

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Avril T, Saikali S, Vauléon E, Jary A, Hamlat A, De Tayrac M, Mosser J, Quillien V. Distinct effects of human glioblastoma immunoregulatory molecules programmed cell death ligand-1 (PDL-1) and indoleamine 2,3-dioxygenase (IDO) on tumour-specific T cell functions. J. Neuroimmunol., 2010, 225(1-2):22-33. [IF 2.8] De Tayrac M, Mosser J. Catégorisation des glioblastomes : typologie et profilage du génome. [Classification of glioblastoma and genome profiling]. Neurochirurgie, 2010, 56(6):464-466. [IF 0.4] Etcheverry A, Aubry M, De Tayrac M, Vauléon E, Boniface R, Guénot F, Saikali S, Hamlat A, Menei P, Quillien V, Mosser J. DNA methylation in glioblastoma: impact on gene expression and clinical outcome. BMC Genomics, 2010, 11(1):701. [IF 4.1] Gandemer V, Aubry M, Roussel M, Rio AG, De Tayrac M, Vallée A, Mosser J, Ly-Sunnaram B, Galibert MD. CD9 expression can be used to predict childhood TEL/AML1-positive acute lymphoblastic leu kemia: Proposal for an accelerated diagnostic flowchart. Leuk. Res., 2010, 34(4):430-437. [IF 2.7] Jaillard S, Drunat S, Bendavid C, Aboura A, Etcheverry A, Journel H, Delahaye A, Pasquier L, Bonneau D, Toutain A, Burglen L, Guichet A, Pipiras E, Gilbert-Dussardier B, Benzacken B, Martin-Coignard D, Henry C, David A, Lucas J, Mosser J, David V, Odent S, Verloes A, Dubourg C. Identification of gene copy number variations in patients with mental retardation using array-CGH: Novel syndromes in a large French series. Eur. J. Med. Genet., 2010, 53(2):66-75. [IF 1.5] Milet J, Le Gac G, Scotet V, Gourlaouen I, Thèze C, Mosser J, Bourgain C, Deugnier Y, Ferec C. A common SNP near BMP2 is associated with severity of the iron burden in HFE p.C282Y homozygous patients: A follow-up. Blood Cells Mol. Dis., 2010, 44(1):34-37. [IF 2.3] Monnier A, Liverani S, Bouvet R, Jesson B, Smith JQ, Mosser J, Corellou F, Bouget FY. Orchestrated transcription of biological processes in the marine picoeukaryote Ostreococcus exposed to light/dark cycles. BMC Genomics, 2010, 11(1):192. [IF 4.1] Mouchet N, Adamski H, Bouvet R, Corre S, Courbebaisse Y, Watier E, Mosser J, Chesné C, Galibert MD. In vivo identification of solar radiation-responsive gene network: role of the p38 stress-dependent kinase. PLoS ONE, 2010, 5(5):e10776. [IF 3.5] Vauléon E, Avril T, Collet B, Mosser J, Quillien V. Overview of cellular immunotherapy for patients with glioblastoma. Clin. Dev. Immunol., 2010, 2010:689171. [IF 2.9]

2011 De Tayrac, Marie*; Aubry, Marc*; Saïkali, Stephan; Etcheverry, Amandine; Surbled, Cyrille; Guénot, Frédérique; Galibert, Marie-Dominique; Hamlat, Abderrahmane; Lesimple, Thierry; Quillien, Véronique; Menei, Philippe; Mosser, Jean. A 4-gene signature associated with clinical outcome in high-grade gliomas, Clinical Cancer Research,17,2,317- 327,2011 [IF 8.2] Collet, Brigitte; Guitton, Nathalie; Saïkali, Stephan; Avril, Tony; Pineau, Charles; Hamlat, Abderrahmane; Mosser, Jean; Quillien, Véronique. Differential analysis of glioblastoma multiforme proteome by a 2D-DIGE approach, Proteome Sci,9,1,16,2011 [IF 1.9] Dubourg C, Sanlaville D, Doco-Fenzy M, Le Caignec C, Missirian C, Jaillard S, Schluth-Bolard C, Landais E, Boute O, Philip N, Toutain A, David A, Edery P, Moncla A, Martin-Coignard D, Vincent-Delorme C, Mortemousque I, Duban-Bedu B, Drunat S, Beri M, Mosser J, Odent S, David V, Andrieux J. Clinical and molecular characterization of 17q21. 31 microdeletion syndrome in 14 French patients with mental retardation, European journal of medical genetics,54,2,144-151,2011 [IF 1.5] Avril, Tony; Vauleon, Elodie; Tanguy-Royer, Séverine; Mosser, Jean; Quillien, Véronique. Mechanisms of immunomodulation in human glioblastoma, Immunotherapy,3,4s,42-44,2011 [IF 3.4] Jaillard S, Andrieux J, Plessis G, Krepischi AC, Lucas J, David V, Le Brun M, Bertola DR, David A, Belaud-Rotureau MA, Mosser J, Lazaro L, Treguier C, Rosenberg C, Odent S, Dubourg C. 5q12. 1 deletion: delineation of a phenotype including mental retardation and ocular defects, American Journal of Medical Genetics Part A,155,4,725-731,2011 [IF 3] Quillien, Véronique; Vauléon, Elodie; Saikali, Stephan; Lesimple, Thierry; Hamlat, Abderrahmane; Etcheverry, Amandine; Mosser, Jean. [MGMT analysis in gliomas], Bulletin du cancer,98,3,291-303,2011 [IF 0.6]

2012 Avril, Tony; Vauleon, Elodie; Hamlat, Abderrahmane; Saikali, Stéphan; Etcheverry, Amandine; Delmas, Caroline; Diabira, Sylma; Mosser, Jean; Quillien, Véronique. Human Glioblastoma Stem-Like Cells are More Sensitive to

200

Allogeneic NK and T Cell-Mediated Killing Compared with Serum-Cultured Glioblastoma Cells, Brain Pathology,22,2,159-174,2012 [IF 4.4] Clavreul, Anne; Etcheverry, Amandine; Chassevent, Agnès; Quillien, Véronique; Avril, Tony; Jourdan, Marie-Lise; Michalak, Sophie; François, Patrick; Carré, Jean-Luc; Mosser, Jean et al. Isolation of a new cell population in the glioblastoma microenvironment, Journal of neuro-oncology,106,3,493-504,2012 [IF 2.8] Doyard, Mathilde; Fatih, Nadia; Monnier, Annabelle; Island, Marie-Laure; Aubry, Marc; Leroyer, Patricia; Bouvet, Regis; Chalès, Gérard; Mosser, Jean; Loréal, Olivier. Iron excess limits HHIPL-2 gene expression and decreases osteoblastic activity in human MG-63 cells, Osteoporosis International,23,10,2435-2445,2012 [IF 4.2] Eimer S, Dugay F, Airiau K, Avril T, Quillien V, Belaud-Rotureau MA, Belloc F. Cyclopamine cooperates with EGFR inhibition to deplete stem-like cancer cells in glioblastoma-derived spheroid cultures. Neuro Oncol. 2012 Dec;14(12):1441-51. [IF 5.3] F. Godey, J. Leveque, P. Tas, G. Gandon, P. Poree, H. Mesbah, V. Lavoue, V. Quillien and C. B. Athias Sentinel lymph node analysis in breast cancer: contribution of one-step nucleic acid amplification (OSNA). Breast Cancer Res Treat.2012, 131: 509-16. [IF 4.2] H. Pere, C. Tanchot, J. Bayry, M. Terme, J. Taieb, C. Badoual, O. Adotevi, N. Merillon, E. Marcheteau, V. R. Quillien, C. Banissi, A. Carpentier, F. Sandoval, M. Nizard, F. Quintin-Colonna, G. Kroemer, W. H. Fridman, L. Zitvogel, S. P. Oudard and E. Tartour Comprehensive analysis of current approaches to inhibit regulatory T cells in cancer (2012). Oncoimmunology. 2012, 1: 326-333. [IF 6.2] V.Quillien, A. Lavenu, L. Karayan-Tapon, C. Carpentier, M. Labussiere, T. Lesimple, O. Chinot, M. Wager, J. Honnorat, S. Saikali, F. Fina, M. Sanson and D. Figarella-Branger Comparative assessment of 5 methods (methylation-specific polymerase chain reaction, MethyLight, pyrosequencing, methylation-sensitive high-resolution melting, and immunohistochemistry) to analyze O6-methylguanine-DNA-methyltranferase in a series of 100 glioblastoma patients. Cancer. 2012,118: 4201-11. [IF 4.9] Vauléon, Elodie; Tony, Avril; Hamlat, Abderrahmane; Etcheverry, Amandine; Chiforeanu, Dan C; Menei, Philippe; Mosser, Jean; Quillien, Véronique; Aubry, Marc. Immune genes are associated with human glioblastoma pathology and patient survival, BMC medical genomics,5,1,41,2012 [IF 3.9] E. Vauleon, H. Mesbah, D. Gedouin, I. Lecouillard, G. Louvel, A. Hamlat, L. Riffaud, B. Carsin, V. Quillien, O. Audrain and T. Lesimple Retrospective analysis of 24 recurrent glioblastoma after chemoradiation and treated with nitrosoureas or irinotecan and bevacizumab. Cancer. 2012, 99: 121-126. [IF 4.9]

2013 Avril, Tony; Hamlat, Abderrahmane; Reste, Le; Mosser, Jean; Quillien, Veronique. Tumor migration of human glioblastoma is modulated by the molecule CD90 (thy-1), neuro-oncology,15,12-12,2013 [IF 5.3] Avril, Tony; Vauleon, Elodie; Hamlat, Abderhamman; Mosser, Jean; Quillien, Veronique. Flow cytometry analysis of tumor-infiltrating cells in a large series of glioblastoma patients: impact of lymphocyte infiltration on survival, Neuro- Oncology,15,3,65-66,2013 [IF 5.3] De Tayrac, Marie; Saikali, Stephan; Aubry, Marc; Bellaud, Pascale; Boniface, Rachel; Quillien, Véronique; Mosser, Jean. Prognostic Significance of EDN/RB, HJURP, p60/CAF-1 and PDLI4, Four New Markers in High-Grade Gliomas, PloS one,8,9,e73332,2013 [IF 3.5] Lemée JM, Com E, Clavreul A, Avril T, Quillien V, de Tayrac M, Pineau C, Menei P. Proteomic analysis of glioblastomas: what is the best brain control sample? J Proteomics. 2013 Jun 24;85:165-73 [IF 3.9]

2014 Beau-Faller M, Blons H, Domerg C, Gajda D, Richard N, Escande F, Solassol J, Denis MG, Cayre A, Nanni-Metellus I, Olschwang S, Lizard S, Piard F, Pretet JL, de Fraipont F, Bièche I, de Cremoux P, Rouquette I, Bringuier PP, Mosser J, Legrain M, Voegeli AC, Saulnier P, Morin F, Pignon JP, Zalcman G, Cadranel J. A Multicenter Blinded Study Evaluating EGFR and KRAS Mutation Testing Methods in the Clinical Non–Small Cell Lung Cancer Setting—IFCT/ERMETIC2 Project Part 1: Comparison of Testing Methods in 20 French Molecular Genetic National Cancer Institute Platforms, The Journal of Molecular Diagnostics,16,1,45-55,2014 [IF 4] Clavreul A, Etcheverry A, Tétaud C, Rousseau A, Avril T, Henry C, Mosser J, Menei P. Identification of two glioblastoma-associated stromal cell subtypes with different carcinogenic properties in histologically normal surgical margins. J Neurooncol. 2014 Dec 13 [IF 2.8] Clavreul A, Guette C, Faguer R, Tétaud C, Boissard A, Lemaire L, Rousseau A, Avril T, Henry C, Coqueret O, Menei P. Glioblastoma-associated stromal cells (GASCs) from histologically normal surgical margins have a myofibroblast phenotype and angiogenic properties. J Pathol. 2014 May;233(1):74-88 [IF 7.3] Collet, Brigitte; Avril, Tony; Aubry, Marc; Hamlat, Abderrahmane; Le Reste, Pierre-Jean; Chiforeanu, Dan; Vauleon, Elodie; Mosser, Jean; Quillien, Véronique. Proteomic analysis underlines the usefulness of both primary adherent and stem-like cell lines for studying proteins involved in human glioblastoma, Journal of proteomics,110,7-19,2014 [IF 3.9] Marie de Tayrac, Marie-Paule Roth, Anne-Marie Jouanolle, Hélène Coppin, Gérald le Gac, Alberto Piperno, Claude 201

Férec, Sara Pelucchi, Virginie Scotet, Edouard Bardou-Jacquet, Martine Ropert, Régis Bouvet, Emmanuelle Génin, Jean Mosser*, Yves Deugnier*. Genome-wide association study identifies TF as a significant modifier gene of iron metabolism in HFE hemochromatosis, Journal of hepatology, 2014 Oct 18. pii: S0168-8278(14)00778-8. [IF 10.4] Duval C, De Tayrac M, Sanschagrin F, Michaud K, Gould Pv, Saikali S. ImmunoFISH is a reliable technique for the assessment of 1p and 19q status in oligodendrogliomas. PLoS ONE, 2014, 9(6):e100342. doi: 10.1371/journal.pone.0100342 [IF 3.5] Etcheverry A*, Aubry M*, Idbaih A, Vauleon E, Marie Y, Menei P, Boniface R, Figarella-Branger D, Karayan-Tapon L, Quillien V, Sanson M, de Tayrac M, Delattre JY, Mosser J. DGKI Methylation Status Modulates the Prognostic Value of MGMT in Glioblastoma Patients Treated with Combined Radio-Chemotherapy with Temozolomide, PloS one,9,9,e104455,2014 [IF 3.5] Kammerer-Jacquet, SF; Belaud-Rotureau, MA; Oger, E; Verhoest, G; Lespagnol, A; Edeline, J; Jaillard, S; Laguerre, B; Vauleon, E; Mosser, J; Nomogram for Predicting Resistance to Sunitinib in Patients with Metastatic Clear Cell Renal Carcinoma, laboratory investigation,94,238A-238A,2014 [IF 3.8] Norgren N, Olsson M, Nyström H, Ericzon BG, De Tayrac M, Genin E, Planté-Bordeneuve V, Suhr OB. Gene expression profile in hereditary transthyretin amyloidosis: differences in targeted and source organs. Amyloid, 2014, 21(2):113- 119. doi: 10.3109/13506129.2014.894908 [IF 2.5] Quillien, Véronique; Lavenu, Audrey; Sanson, Marc; Legrain, Michèle; Dubus, Pierre; Karayan-Tapon, Lucie; Mosser, Jean; Ichimura, Koichi; Figarella-Branger, Dominique. Outcome-based determination of optimal pyrosequencing assay for MGMT methylation detection in glioblastoma patients, Journal of neuro-oncology,116,3,487-496,2014 [IF 2.8] Ricordel, C; Labalette-Tiercin, M; Lespagnol, A; Kerjouan, M; Dugast, C; Mosser, J; Desrues, B; Léna, H. EFGR-mutant lung adenocarcinoma and Li-Fraumeni syndrome: Report of two cases and review of the literature, Lung Cancer,2014 [IF 3.7]

2015 Aubry M*, de Tayrac M*, Etcheverry A, Clavreul A, Menei P, Mosser J. From the Core to beyond the Margin: a genomic picture of glioblastoma intratumor heterogeneity. OncoTarget, 2015, sous-presse [IF 6.6] Bardou-Jacquet E, de Tayrac M, Mosser J, Deugnier Y. GNPAT variant associated with severe iron overload in HFE hemochromatosis. Hepatology. 2015 Apr 18. doi: 10.1002/hep.27854. [IF 11.19] Barlesi F, Mazieres J, Merlio JP, Debieuvre D, Mosser J, Lena H, Ouafik L, Besse B, Rouquette I, Westeel V, Escande F, Monnet I, Lemoine A, Veillon R, Blons H, Audigier-Valette C, Bringuier PP, Lamy R, Beau-Faller M, Pujol JL, Sabourin JC, Penault-Llorca F, Denis MG, Lantuejoul S, Morin F, Tran Q, Missy P, Langlais A, Milleron B, Cadranel J, Soria JC, Zalcman G. Routine molecular profiling of cancer: results of a one-year nationwide program of the French Cooperative Thoracic Intergroup (IFCT) for advanced non-small cell lung cancer (NSCLC) patients. Lancet. 2015, accepted for publication [IF 45,22] Lemée JM, Clavreul A, Aubry M, Com E, de Tayrac M, Eliat PA, Henry C, Rousseau A, Mosser J, Menei P. Characterizing the peritumoral brain zone in glioblastoma: a multidisciplinary analysis. J Neurooncol. 2015 Mar;122(1):53-61 [IF 2.8]. Mouden C, De Tayrac M, Dubourg C, Rose S, Carre W, Hamdi-Rozé H, Babron Mc, Akloul L, Héron-Longe B, Odent S, Dupé V, Giet R, David V. Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings. PLoS ONE, 2015, 10(2):e0117418. [IF 3.5]

– equally first or corresponding authors*

2. Patents (with licence)

(Ref SATT: DV 749) Patent "Biomarkers and Methods for the prognosis of Glioblastoma", priority deposit the 03/23/2011, PCT extension in 2012 and publication under the number WO2012126542. Now in evaluation phase in the US under the number US14/006023. Déposants: Université de Rennes 1, CNRS, CHU Rennes et Université Pierre et Marie Curie

(Ref SATT: DV 2455) Logiciel "VARIANT DX" version du 10/01/2014, deposit at the Agence pour la Protection des Programme (APP), number IDDN.FR.001.500023.000.S.P.2014.000.31230. Déposants: Université de Rennes 1 and CHU Rennes This logiciel concerns the development of an algorithms pipeline for the analysis of NGS data derived from heterogeneous cancer samples.

(Ref SATT: DV 2457) Enveloppe Soleau, deposit to the INPI le 11/24/2014, number 522286 : attestation of the paternity of the process of library construction before NGS for molecular somatic diagnosis of solid tumors.

3. Conferences (actual team members)

- International

202

2010 Marc AUBRY et al.: 9th Meeting of the European Association of Neurooncology, Maastricht, The Netherlands, September 16-19, 2010. A four-gene signature associated with clinical outcome in high-grade gliomas. Marie de TAYRAC et al.: 9th Meeting of the European Association of Neurooncology, Maastricht, The Netherlands September 16-19, 2010. Integrative genome-wide analysis in GBM. (invitation for oral presentation)

2011 AUBRY M, ETCHEVERRY A, IDBAIH A, MARIE Y, SANSON M, DELATTRE JY, MOSSER J. The 2011 European Multidisciplinary Cancer Congress, Stockholm, 23-27 september 2011. Epigenetic markers identify MGMT-methylated glioblastoma poorly responding to combined radiotherapy-temozolomide (Stupp regimen). (sélection pour présentation orale) BEAU-FALLER, Michele; BLONS, Helene; MAUGUEN, Audrey; PRETET, Jean-Luc; PIARD, Francoise; DEGEORGES, Armelle; PENAULT-LLORCA, Frederique; de CREMOUX, Patricia; BRINGUIER, Pierre Paul; OLSCHWANG, Sylviane et al. Various sensitive molecular detection techniques for EGFR mutations in NSCLC, by 20 french centers-preliminary results on cell lines of ermetic-2/predict. Amm studies, journal of thoracic oncology,6,6,s1070-s1071,2011

2013 AVRIL, Tony; HAMLAT, Abderrahmane; RESTE, Le; MOSSER, Jean; QUILLIEN, Veronique. Tumor migration of human glioblastoma is modulated by the molecule CD90 (Thy-1),Neuro-Oncology,15,12-12,2013,"Oxford univ press inc journals dept, 2001 evans rd, cary, nc 27513 USA" AVRIL, Tony; VAULEON, Elodie; HAMLAT, Abderhamman; MOSSER, Jean; QUILLIEN, Veronique. Flow cytometry analysis of tumor-infiltrating cells in a large series of glioblastoma patients: impact of lymphocyte infiltration on survival,Neuro-Oncology,15,3,65-66,2013, LESPAGNOL, A; NDIAYE, B; MOSSER, ANNICK; CHAPLAIS, C; GOURDET, H; GUENOT, FRÉDÉRIQUE; BOUVET, RÉGIS; MORON, G; MOSSER, J; de TAYRAC, M. NGS panel V1. 1 for the routine deep sequencing-based diagnostic of somatic hotspot theranostic mutations on FFPE tumours: A prospective study of 500 cancer samples, European Journal of Cancer, 49,2013 (poster) BARLESI, F; BLONS, H; BEAU-FALLER, M; ROUQUETTE, I; OUAFIK, LH; MOSSER, J; et al. Biomarkers (BM) France: Results of routine EGFR, HER2, KRAS, BRAF, PI3KCA mutations detection and EML4-ALK gene fusion assessment on the first 10,000 non-small cell lung cancer (NSCLC) patients (pts), J Clin Oncol,31,15S,486s,2013

2014 AUBRY M, de TAYRAC M, ETCHEVERRY A, CLAVREUL A, SAIKALI S, MENEI P and MOSSER J. ‘From the core to beyond the margin’: a genomic picture of glioblastoma intratumor heterogeneity. ANOCEF 2014, 16-17 mai 2014, Lausanne. AUBRY, M; de TAYRAC, M; ETCHEVERRY, A; CLAVREUL, A; SAIKALI, S; MENEI, P; MOSSER, J. O4. 04 ‘From the core to beyond the margin’: a genomic picture of glioblastoma intratumor heterogeneity, Neuro-Oncology,16,suppl2,ii7- ii7,2014. 11th meeting of the european association of NeuroOncology, Turin, Italy, October 6-12, 2014

- National

2010 Annabelle MONNIER et al.: Assises de Génétique, Strasbourg, 28-30 Janvier 2010. Recherche de gènes modificateurs de l’hémochromatose de type 1. Jean MOSSER et al.: Association des Neuro-Oncologues d'Expression Française, Marseille, 18-19 juin 2010. Grand- Ouest Glioma project - Résultats du WP3 : Génomique fonctionnelle intégrée du glioblastome,

2012 M de TAYRAC, R BOUVET, J MORCET, M PERRIN, AM JOUANOLLE, E GÉNIN, Y DEUGNIER, J MOSSER 6e Assises de Génétique humaine et médicale, Marseille, 2-4 février 2012. Recherche pangénomique de gènes modificateurs de la pénétrance de l’hémochromatose-HFE. (sélection pour présentation orale) Jean MOSSER: 6e colloque du Canceropôle PACA, Journée inter-cancéropôles « les glioblastomes », Marseille, 22 février 2012. Méthylome et réponse au traitement. (invitation pour présentation orale).

2014 BARLESI, F; BLONS, H; BEAU-FALLER, M; ROUQUETTE, I; OUAFIK, L; MOSSER, J; MERLIO, JP; BRINGUIER, PP; JONVEAUX, P; LE MARECHAL, C; et al. Biomarqueurs France: résultats de l’analyse en routine de EGFR, HER2, KRAS, BRAF, PI3K, et ALK sur 10 000 patients (pts) atteints de cancer bronchique non à petites cellules (CBNPC)",Revue des Maladies Respiratoires,31,,A13-A14,2014

2015 Jean MOSSER: Place du NGS dans les laboratoires de genomique somatique. ANOCEF 2015, 19-20 juin 2015, Amiens. 203

4. Funding

2007-2013 CPER Volet Génopole – Transfert vers la clinique– Coord. J. MOSSER Equipment 680 k€ Personnel 150 k€ 2008-2010 ANR GENOPAT – Coord. J. MOSSER 500 k€ 2008-2010 Ministère de la santé et des sports 349 K€ 2009-2010 Région Bretagne 30 K€ 2010 IBiSA- Plateforme génomique santé Biogenouest Equipment NGS 280 k€ 2011 Région Bretagne – Biogenouest séquençage massif – Equipment 300 k€ + Personnel: 1 TCN and 1 IE 2011 COREC 2011 – Coord. J. MOSSER 30 k€ 2011 A0 Cancéropôle GO 2011 – Coord. E. GARCION, INSERM U646 Angers 14 k€ 2012 Région Bretagne – Fonds de maturation 80 k€ 2012 COREC, Projet EXOCANCER – Coord. J. MOSSER 37.8 k€ 2013 Ligue – Coord. J. MOSSER 25 k€ 2013-2016 AP INCa – Coord. J. MOSSER Volet 1 Implementation of NGS in laboratories - 18 months 288 k€ Volet 2 Referent teams in bioinformatics) - 18 months 137.7 k€ 2014 Contract ROCHE – Co-coord. J. MOSSER & H. LENA 60 k€ 2014 Ligue – Coord. V. QUILLIEN 35 k€ 2014 AO Emergence Cancéropôle GO, Projet MemiR – Coord. P.-F. CARTRON, INSERM Nantes 8.5 k€ 2014 Réseau REpiCGO 14 équipes Projet DeSED – Coord. P.-F. CARTRON, INSERM Nantes 20 k€ 2015 AP INCa PAOLA-BRCA – Coord. D. VAUR, CLCC Caen 9.2 k€

5. Training Teaching - L3 Coordination: Licence 3: UE Analyse des données biologiques (Annabelle MONNIER) - Master 1 Master Coordination: Master I Biologie Agronomie Santé, spécialité Bioinformatique et Génomique (BIG) (Annabelle MONNIER) UE coordination: Master 1 BIG: - UE Analyse des données de génomique et post-génomique (Annabelle MONNIER) - UE stage (Annabelle MONNIER) Student mentoring: Master Bioinformatics and genomics and Master SCMV (Sciences Cellulaires et Moléculaires du Vivant): 11 students since 2010 Master Santé: 4 students per year Lectures: Master I SCMC and Master santé - Master 2 Coordination: Master II Biologie Agronomie Santé, spécialité Sciences Cellulaires et Moléculaires du Vivant (SCMV) - UE Analyse génétique des caractères multifactoriels (Marie de TAYRAC, Jean MOSSER) Student mentoring: 1 student every 2 years Lectures: - Master II SCMV - Master Génomique et Génétique statistique - M2 Santé Publique Paris XI & Master Sciences du Génome et des Organismes, Université Evry Val d'Essonne - Other Lectures: - Ateliers Inserm - DIU NeuroOncologie Master 2011 - Mailys RUPIN– M1 University Rennes 1 - directed by Jean MOSSER (2 months) 2013 - Jean-François BAUDRON – M2 University Rennes 1 - co-directed by Marie de TAYRAC & Marc AUBRY (6 months) 2014 - Sophie BOISSEAU– M1 University Rennes 1 - directed by Marie de TAYRAC (4 months) - Tania CUPPENS– M1 University Rennes 1 - co-directed by Marc AUBRY (4 months) - Pierre SEMLER-COLLERY - M2 Agrocampus & University Rennes 1 - directed by Marie de TAYRAC (6 months) - Sophie LEMERCIER - M2 UPMC Sorbonne Universités - directed by Marie de TAYRAC (6 months) 2015 - Artem KIM– M1 University Rennes 1 - directed by Marie de TAYRAC (3 months) - Jérôme PIVERT– M1 University Rennes 1 - co-directed by Marc AUBRY (3 months) - Pauline SARAROLS– M1 University Rennes 1 - directed by Marie de TAYRAC (4 months) 204

- Charles VAN GOETHEM – M1 University Rennes 1 - co-directed by Annabelle MONNIER (4 months) - Tania CUPPENS – M2 University Rennes 1 - directed by Marie de TAYRAC - 6 months PhD 04/10/2012: Amandine ETCHEVERRY – Dir. Thèse Jean MOSSER – Currently IR CHU 14/12/2012: Elodie VAULEON – Dir. Thèse Véronique QUILLIEN & Jean MOSSER – Currently PH in CRLCC

205

Team 15 "Gene expression and development" (GED) Leader: Luc PAILLARD

207

2.1. Team presentation

Our team has a long-standing interest in the post-transcriptional (PT) controls of gene expression, which are exerted at the RNA level. We study the contribution of these regulations to the coordinated development of multicellular organisms, essentially mouse and Xenopus. We analysed PT controls in somite segmentation as well as male gametogenesis, and we are now focusing on skin development. Our approaches integrate high-throughput genomics (RNAseq, CLIPseq) with phenotype analyses. Because developmental defects are at the origin of an impressive number of human pathologies, we continuously endeavour to link our findings to human health.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

AUDIC Yann CR1 CNRS LERIVRAY Hubert MCU-HC UR1 DESCHAMPS Stéphane MCU-HC UR1 MEREAU Agnès CR1 CNRS GAUTIER-COURTEILLE Carole MCU UR1 PAILLARD Luc PU2 UR1 HARDY Serge PU1 UR1 VIET Justine TCN UR1 LEGAGNEUX Vincent CR1 INSERM

2.2.2.2. Temporary staff

N/A

2.2.2.3. Permanent staff who left the team during the contract

MOTTIER Stéphanie IE1 CNRS until 08/2014

2.2.3. Achievements

2.2.3.1. Scientific achievements

- Saulière et al., Nature Struct Mol Biol 2012. In the last 4 years, we set up CLIP technologies (CLIP, iCLIP) in the lab, which are aimed at systematically identifying all the RNAs associated with a given 208

RNA-binding protein (RBP) in living cells and to map the binding sites. This technology was transferred to another lab as a collaboration. This yielded a high impact co-publication, which describes the RNAs associated with the exon-junction-complex.

- Noiret et al., in revision (Dev Biol). Beyond CLIP, we have become expert in next-generation- sequencing (NGS), including library preparation and data treatment. Here, we describe a PT network involved in Xenopus skin stability.

- Méreau et al., Mol Cell Biol 2015. This article identifies Esrp1 as a PT regulator of Ptbp1 abundance (an RBP) in Xenopus skin.

- Boulanger et al., in revision (Mol Cell Biol). The male mice disrupted for the gene encoding the RBP CELF1 are sterile due to impaired spermatogenesis. We show here that this phenotype relies on a lack of testosterone due to excessive aromatase activity. We demonstrate that CELF1 directly represses the translation of the mRNA encoding aromatase. Hence, CELF1 represses aromatase to achieve sufficient concentrations of testosterone for spermatogenesis, with implications for reproductive troubles in men.

2.2.3.2. Scientific dissemination and influence

Yann AUDIC: Invited talk in an international conference (joint meeting of the SFBD and EFOR network, 2014). Member of 1 PhD thesis jury, dismissing theses prepared in the research team. Member of 1 HDR jury (jury d'habilitation à diriger les recherches). Member of the French society for bioinformatics and of the French society for developmental biology (SFBD).

Carole GAUTIER-COURTEILLE: Member of 2 PhD thesis juries, dismissing theses prepared in the research team.

Stéphane DESCHAMPS: promotion as a senior assistant professor/Maître de conférences hors classe.

Serge HARDY: Promotion as a first class professor (2013). Invitation by Dr Stefan Hoppler (University of Aberdeen) and Pr Peter Vize (University of Calgary) to write a chapter related to the usage of Xenopus to study Alternative splicing in the new edition of the volume of Methods in Molecular Biology on Xenopus. Member of 1 "Comité de recrutement" (recruitment panels to recruit University professors or assistant professors). Member of 2 PhD thesis juries, dismissing theses prepared in the research team. Member of 3 HDR juries (jury d'habilitation à diriger les recherches).

Agnès MEREAU: Member of a recruitment panel for an INSERM engineer. Member of the board (conseil d'administration) of the French society for biochemistry SFBBM.

Luc PAILLARD: PES/PEDR (2010-2014 and 2014-2018). Invited talk in a non-french laboratories (University of Nottingham UK, 2013, invited by Cornelia de Moor). Invited talk in an international conference (14th International Xenopus conference, 2012). Member of 3 "Comités de recrutement" (recruitment panels to recruit University professors or assistant professors). Member of 3 PhD thesis juries, dismissing theses prepared in the research team. Member of one assessment commitee for AERES. Member of the CNU (Conseil National des Universités, section 65), 2011-2015. Member of the French society for biochemistry SFBBM and of the American society for microbiology.

Justine VIET: Member of the comité régional d'éthique (local ethics committee).

GED Team: We contributed RNA seq data for genome-wide annotation of Xenopus laevis centralised by the Marcotte lab (University of Austin TX, http://www.marcottelab.org/index.php/XENLA_ WorldCup). Members of the team reviewed articles submitted for publication in Development, Nucleic Acids Research, Gene, RNA, Developmental Biology, Scientific reports, BMC Cell Biol, Biology of the Cell, Experimental Cell Research, Molecular Reproduction and Development, Journal of Molecular and Cellular Cardiology, PLoS One.

2.2.3.3. Interaction with the economic, social and cultural environment

Luc PAILLARD (2013-2014), Serge HARDY (2015): "Amphithéâtres des lycéens": Talks about genetics and genomics for high school pupils, 3.

Agnès MEREAU & Stéphane DESCHAMPS: "Forum des lycées" or "Forum des métiers" (presentation of 209

research jobs for high schools pupils): 5.

Agnès MEREAU: "Festival des sciences" (scientific festival for general public): exhibition of scientific photographs.

2.3. Projects, scientific strategies & perspectives (5 years)

Team's background.

With the development of NGS, it has become possible to understand genome-wide how RBPs shape a cell's transcriptome. We used CLIPseq (which relies on RBP immunoprecipitation and deep sequencing of the co-immunoprecipitated RNAs) to identify the repertoire of RNAs associated with the RBP CELF1 and we integrated binding data with changes in RNA abundance following the depletion of this protein. We found that, taken globally, there are only limited relationships between the capacity of an mRNA to bind to an RBP, and changes to its abundance in the absence of the regulatory protein. Yet, we identified specific examples of mRNAs that are regulated by CELF1, allowing us to investigate the biological functions of these regulations.

Technologically, our first series of CLIP have been a success since we are able to run CLIP experiments on virtually any RBP. Our expertise in the field attracted collaborations and the CLIP of the EJC component eIF4AIII, which was published in NSMB (Saulière et al. 2012 Nat Struct Mol Biol. 19:1124- 31), was developed in our lab. Based on our experience, we now aim to use CLIP and transcriptome (RNAseq) data as screens to focus on specific post-transcriptional relationships, in order to answer specific biological questions.

Epidermis development and skin stability defects

A wealth of data demonstrates that PT controls of gene expression play roles in epidermis or epitheliums. For example, the RNA exosome degrades GRHL3 mRNA in epidermal progenitor cells, and repression of the RNA exosome is required for the accumulation of GRHL3 mRNA and differentiation (Mistry et al. 2012 Cell Stem Cell. 11:127-35). The epithelial-to-mesenchymal transition (EMT) requires massive changes to splicing program, suggesting that specific splicing patterns are required to maintain epithelial identity (Warzecha and Carstens. Semin Cancer Biol. 2012 22:417-27). We also observed that knocking-down the mRNAs encoding the RBP Ptbp1 or Esrp1, or the mRNA encoding the RNA exosome component Exosc9, is associated with skin stability defects in Xenopus embryos (Méreau et al. 2015 Mol Cell Biol 35:758-68; Noiret et al., in revision, Dev Biol).

Better understanding epidermis or epithelial development may have significant consequences for human pathologies. EMT is a key event for metastasis, while genodermatoses such as epidermolysis bullosa, cutis laxa or Ehlers-Danlos syndromes are characterized by epithelium and skin stability defects that highly impact the life of the affected individuals (Fine et al. 2014 J Am Acad Dermatol. 70:1103-26; Urban and Davis. 2014 Matrix Biol. 33:16-22). Some of them also affect mucocilliary human airways (Has et al. 2012 New Eng. J. Med. 366: 1508-1514). In these syndromes, some structural genes are identified as being mutated, but the phenotypic variability of the diseases suggests that modifiers genes may affect the expressivity of the phenotype (Kern et al. 2009 Br J Dermatol. 161:1089-97). Our results suggest that genes that control expression networks may be among these modifiers. In addition, the epidermis of Xenopus embryos has been identified recently as an important model to study development of the mucociliary epithelium (Dubaissi and Papalopulu 2011 Dis Model Mech. 4:179-92; Werner and Mitchell 2012 Genesis 50:176-85). We think therefore that post-transcriptional controls in epidermis and epithelium development and disease are largely under-investigated, and we propose to use Xenopus embryos as models to tackle these questions.

Ptbp1 and Esrp1 in Xenopus epidermis

We recently observed that the RBPs Ptbp1 and Esrp1 are mandatory for correct epidermis development in Xenopus embryos, in as much as ptbp1 and esrp1 morphants display blister-like structures in the dorsal fin (Méreau et al. 2015 Mol Cell Biol 35:758-68). The same is true for exosc9 (which encodes a component of the RNA exosome) morphants, but through different routes (Noiret et al., in revision, Dev Biol). In all of these cases, the causes of the observed phenotypes are missing, and we will seek them. In a first approach, the expression of relevant markers will be analysed to more precisely describe the phenotypes.

210

Next, our working hypothesis is that Ptbp1 and Esrp1 bind to and regulate several RNAs in epidermis, and that their deregulation in the respective morphants is responsible for the observed skin instability defects. On the one hand, we propose to systematically identify the RNAs associated with the Ptbp1 and Esrp1 by CLIP. In the absence of immunoprecipitating antibodies, we will use tagged proteins expressed from recombinant bacterial artificial chromosomes (BAC). BAC transgenesis in Xenopus embryos is described as an efficient technique to recapitulate endogenous gene expression (Fish et al. Genesis. 2012 50:307-15). We will prepare libraries for deep sequencing from the co-immunoprecipitated RNAs using CLIP/iCLIP that is a routine approaches in our lab, and treat the deep sequencing data. On the other hand, we propose to systematically identify the differentially expressed genes and differentially matured RNAs by deep RNA sequencing of dissected epidermis. Our past experience indicates that the genes at the overlap of these two repertoires should be relatively few in number. Among them, we will endeavour to identify candidate causal genes in the skin instability phenotype based on the following criteria: relationships to human skin instability disease, ontology, and expression pattern. We will test the candidates by genome editing with CRISPR/Cas9 or using more standard morpholino knockdown. Experiments published by our group in Xenopus (Cibois et al. 2010 RNA 16:10-5) or by other groups (e.g. Varghese et al. 2010 Genes Dev. 24:2748-53) suggest that a limited number of mis-regulated genes may explain the phenotype of mutant or morphants of PT regulators of gene expression.

The output of these experiments will be, (i) a list of RNAs associated with Esrp1 or Ptbp1 in Xenopus epidermis, and the location of the binding sites on pre-mRNA and mRNAs; (ii), a list of RNAs with differential abundance and the identification of altered splicing events in the epidermis of esrp1 or ptbp1 morphants; (iii), the identification of genes and splicing events directly controlled by Esrp1 or Ptbp1, whose mis-regulation in the respective morphants is responsible for the epidermis phenotype.

The read-out of the functional inactivation studies will not be limited to the analysis of the blistering phenotype, but we will develop tools and collaborations to gain access to antibodies pertaining to the study of the stability of the epidermis. For example, antibodies used for clinical diagnosis of genodermatosis will be systematically assessed in collaboration with clinicians. Furthermore, the developing epidermis of Xenopus is highly accessible to confocal imaging for detailed analysis of cellular biology.

Other RNA-binding proteins in Xenopus epidermis

A valuable output of the above experiments is that we can identify the genes encoding RBPs that are expressed in Xenopus epidermis. Based on deep RNA sequencing data and annotated orthologues, we will define a set of RBPs with a high expression in the epidermis. We will confirm their expression by in situ hybridization, and inactivate their genes. For the ones displaying an interesting skin instability phenotype, we will seek the causal gene(s) by approaches similar to those described above.

We also think that skin instability may be associated with defective EMT. ESRP1 is a key regulator of the EMT in human cells (Warzecha and Carstens 2012 Semin Cancer Biol. 22:417-27), and we have observed that inactivating its orthologue in Xenopus makes blisters appear in the dorsal fin (Méreau et al. 2015 Mol Cell Biol 35:758-68). Hence, seeking CRISPR/Cas9 mutants with skin instability may lead to the discovery of novel PT regulators of the EMT. We would then consider analysing the role of these novel regulators in the EMT, possibility with mammalian cultured cells to improve the relevance of our findings for cancer and metastasis.

Together, these experiments will provide the community with a large set of RBPs present in epidermis, important for epidermis biology, with the identification of key regulated genes. This will represent a significant step toward understanding epidermis development and function, with possible strong impacts for human disease.

Collaborative projects

The above described project is the major project of the team. However, our skills in PT regulations, high-throughput approaches, and/or Xenopus biology attracted several requests for collaboration, which led to several co-publications (e.g. Saulière et al, NSMB 2012; Kim et al, HMG 2014). We're still on call for other collaborations. It is the team's policy to select them on the basis of the scientific interest on the one hand, and of our real skills to fulfil the project on the other hand (we reject those demanding a strong technological development). We also establish collaborations when our findings drive us toward questions where we have limited skills, like human diseases. The current striking collaborative projects are with:

211

- Salil LACHKE (Univ. of Delaware USA), dealing with the PT control of gene expression in lens development and disease (cataracts). A collaborative manuscript is in preparation, and the project will continue beyond the publication of this first article. - Takeshi MORIHARA (Univ. of Osaka Japan). Dr MORIHARA recently linked Alzheimer disease with specific RNA splicing patterns (Morihara et al. 2014 Proc Natl Acad Sci U S A. 111:2638-43), and we are investigating the function of the RBP CELF1 in splicing control related to Alzheimer disease.

2.4. Collaboration

2.4.1. Within the IGDR

Collaborations with co-publications: Gilles SALBERT (Quintin et al MCB 2014); Marie-Dominique GALIBERT (Primot et al PCMR 2010) - Strong interactions: Reynald GILLET (RNA biology), Marie-Dominique GALIBERT (skin pathologies).

2.4.2. Other collaborations

Rennes D. TAGU (Legeai et al BMC Gen 2010).

National H. LE HIR, ENS Paris (Saulière et al NSMB 2012) - L. KODJABACHIAN, IBDML Marseille (Cibois et al BiO 2013) - C. BRANLANT, AREMS Nancy (Piazzon et al NAR 2013).

International M. MAHADEVAN, University of Virginia, USA (Kim et al HMG 2014) - S. LACHKE, University of Delaware, USA (ongoing project, PT controls in lens) - T. MORIHARA, University of Osaka, Japan (ongoing project, PT controls in Alzheimer disease).

2.5. SWOT Analysis STRENGTHS A large number of highly experienced researchers with a permanent position. Strong skills in NGS (CLIPseq, RNAseq). Strong skills in developmental biology, including a capacity to carry out experiments with both Xenopus laevis and Xenopus tropicalis. WEAKNESSES A lack of secure fundings. A lack of high-impact publications. A risk of wandering between too many projects. OPPORTUNITIES A high international visibility, resulting in high-level collaborations (USA, Japan). Our will to link molecular mechanisms of PT regulations with physiological, pathological or developmental processes defines a niche with manageable competition. Genomics ressources and tools are available for Xenopus tropicalis. The local environment is favourable, especially regarding imaging facilities. THREATS The scarcity of grants not directly related to human health. A heavier and heavier administrative workload (especially, but not only, for University staff). The scarcity of antibodies for protein work.

212

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Cibois M, Gautier-Courteille C, Legagneux V, Paillard L. Post-transcriptional controls - adding a new layer of regulation to clock gene expression. Trends Cell Biol. 2010 Sep;20(9):533-41. [IF=12.3] Cibois M, Gautier-Courteille C, Vallée A, Paillard L. A strategy to analyze the phenotypic consequences of inhibiting the association of an RNA-binding protein with a specific RNA. RNA. 2010 Jan;16(1):10-5. [IF=4.6] Hardy S, Legagneux V, Audic Y, Paillard L. Reverse genetics in eukaryotes. Biol Cell. 2010 Oct;102(10):561-80. [IF=3.9] Legeai F, Rizk G, Walsh T, Edwards O, Gordon K, Lavenier D, Leterme N, Méreau A, Nicolas J, Tagu D, Jaubert- Possamai S. Bioinformatic prediction, deep sequencing of microRNAs and expression analysis during phenotypic plasticity in the pea aphid, Acyrthosiphon pisum., 2010, BMC Genomics 11(1):281. [IF=4.0] Le Tonquèze O, Gschloessl B, Namanda-Vanderbeken A, Legagneux V, Paillard L, Audic Y. Chromosome wide analysis of CUGBP1 binding sites identifies the tetraspanin CD9 mRNA as a target for CUGBP1-mediated down- regulation. Biochem Biophys Res Commun. 2010 Apr 16;394(4):884-9. [IF=2.3] Massé J, Laurent A, Nicol B, Guerrier D, Pellerin I, Deschamps S. Involvement of ZFPIP/Zfp462 in chromatin integrity and survival of P19 pluripotent cells. Exp Cell Res. 2010 Apr 15;316(7):1190-201. [IF=3.4] Primot A, Mogha A, Corre S, Roberts K, Debbache J, Adamski H, Dreno B, Khammari A, Lesimple T, Mereau A, Goding CR, Galibert MD. ERK-regulated differential expression of the Mitf 6a/b splicing isoforms in melanoma. Pigment Cell Melanoma Res. 2010 Feb;23(1):93-102. [IF=5.8]

2011 Massé J, Piquet-Pellorce C, Viet J, Guerrier D, Pellerin I, Deschamps S. ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate. Exp Cell Res. 2011 Aug 1;317(13):1922-34. [IF=3.4]

2012 Cibois M, Boulanger G, Audic Y, Paillard L, Gautier-Courteille C. Inactivation of the Celf1 gene that encodes an RNA- binding protein delays the first wave of spermatogenesis in mice. PLoS One. 2012;7(10):e46337. [IF=3.5] Mereau A, Hardy S. Investigating alternative RNA splicing in Xenopus. Methods Mol Biol. 2012;917:347-68. [IF=1.3] Morcel K, Watrin T, Jaffre F, Deschamps S, Omilli F, Pellerin I, Levêque J, Guerrier D. Involvement of ITIH5, a candidate gene for congenital uterovaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome), in female genital tract development. Gene Expr. 2012;15(5-6):207-14. [IF=1.4] Noiret M, Audic Y, Hardy S. Expression analysis of the polypyrimidine tract binding protein (PTBP1) and its paralogs PTBP2 and PTBP3 during Xenopus tropicalis embryogenesis. Int J Dev Biol. 2012;56(9):747-53. [IF=2.6], 2 citations Saulière J, Murigneux V, Wang Z, Marquenet E, Barbosa I, Le Tonquèze O, Audic Y, Paillard L, Roest Crollius H, Le Hir H. CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex. Nat Struct Mol Biol. 2012 Nov;19(11):1124-31. [IF=11.6]

2013 Cibois M, Gautier-Courteille C, Kodjabachian L, Paillard L. A gene regulation network controlled by Celf1 protein-rbpj mRNA interaction in Xenopus somite segmentation. Biol Open. 2013 Aug 30;2(10):1078-83. [IF not yet eligible] Piazzon N, Schlotter F, Lefebvre S, Dodré M, Méreau A, Soret J, Besse A, Barkats M, Bordonné R, Branlant C, Massenet S. Implication of the SMN complex in the biogenesis and steady state level of the signal recognition particle. Nucleic Acids Res. 2013 Jan;41(2):1255-72. [IF=8.8]

2014 Kim YK, Mandal M, Yadava RS, Paillard L, Mahadevan MS. Evaluating the effects of CELF1 deficiency in a mouse model of RNA toxicity. Hum Mol Genet. 2014 Jan 15;23(2):293-302. [IF=6.7] Quintin J, Le Péron C, Palierne G, Bizot M, Cunha S, Sérandour AA, Avner S, Henry C, Percevault F, Belaud-Rotureau MA, Huet S, Watrin E, Eeckhoute J, Legagneux V, Salbert G, Métivier R. Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities. Mol Cell Biol. 2014 Jul;34(13):2418-36. [IF=5.0]

2015 Méreau A, Anquetil V, Lerivray H, Viet J, Schirmer C, Audic Y, Legagneux V, Hardy S, Paillard L. A post-

213

transcriptional mechanism that controls Ptbp1 abundance in Xenopus epidermis. Mol Cell Biol 2015 Feb;35(4):758-68. [IF=5.0] Noiret M, Hardy S, Audic Y. zfp36 expression delineates both myeloid cells and cells localized to the fusing neural folds in Xenopus tropicalis. Int J Dev Biol, 2015 in press. [IF=2.6]

2. Patents (with licence)

N/A

3. Conferences (actual team members)

- International

2012 Yann AUDIC: 14th International Xenopus Conference, 2012, (poster) Gaëlla BOULANGER: 17th European Testis Workshop, Djurönäset Sweden (Oral presentation) Agnès MEREAU & Yann AUDIC: Keystone symposia on Molecular and Cellular Biology "Protein−RNA Interactions in Biology and Disease", Santa Fe New Mexico USA, 2012 (2 posters) Luc PAILLARD: 14th International Xenopus conference (Invited speaker)

- National

2010 Yann AUDIC: 8th "rencontre SifrARN", 2010 (Oral présentation)

2013 Agnès MEREAU: Oral presentation at the 9th "rencontre SifrARN", 2013 (Oral présentation)

2014 Yann AUDIC: Joint meeting of the SFBD and EFOR network, 2014 (Invited speaker) Agnès MEREAU: RNA Club IECB-Annual Meeting, 2014 (Oral présentation)

4. Funding

2007-2011 ANR Programme Jeunes chercheuses et jeunes chercheurs 167,2 k€ 2009-2010 Association Française contre les Myopathies 35 k€ 2011 Ligue régionale contre le cancer & région Bretagne, PhD student Géraldine DAVID – L. PAILLARD 90 k€ 2012 Ligue régionale contre le cancer – L. PAILLARD 25 k€ 2013 Retina France – L. PAILLARD 25 k€ 2014 BIOSIT – Y. AUDIC 3 k€ 2015 BIOSIT – Y. AUDIC 6 k€

5. Training Noticeable involvement in training Hubert LERIVRAY has been the dean of the faculty of biology, University of Rennes, since 2010. Serge HARDY has been responsible of the licence (bachelor's degree) in life sciences, University of Rennes (1400 students), since 2012. Luc PAILLARD has been responsible of a master degree in molecular and cell biology, University of Rennes (120 students), since 2008. One third of the graduated students join a research laboratory to prepare a PhD thesis. Carole GAUTIER-COURTEILLE has been responsible of the first year of a master degree in molecular and cell biology for students in medicine, pharmacy and dentistry, since 2008. Agnès MEREAU is PCRP ("personne compétente en radio-protection") for the institute, in charge of the lab equipments, working rules, safety instructions and student training for experiments using radio-labelled compounds. Licence 2011 - Marion BODIN – L3 (6 weeks) 214

2012 - Perrine LAVALOU, L3 (6 weeks) - Julien SALLAIS – L3 (2,5 months) 2013 - Justine HABAULT – L2 (4 weeks) - Gaëlle ANGRAND – L3 (6 weeks) - Marie WARBURTON – L3 (6 weeks) 2014 - Artem KIM – L3 (6 weeks) 2015 - Marvin SYLVESTRE – L3 (6 weeks) BST 2014 - Pierre-Yves BASLE – BTS 2e année (2 months) Erasmus 2012 - Munthar MIAH, school of pharmacy, University of Nottingham, UK - Hannah TAI, school of pharmacy, University of Nottingham, UK Master 2010 - Géraldine DAVID, M1 (2 months) - Etienne GAMON & Clémence LE PAPE, M1 UE11 (2 weeks) - Claire BERTRAND, M2 (5 months) 2011 - Renaud CAOUS, M1 (2 months) - Damien BREZULIER & Faustine SERRAND, M1 UE11 (2 weeks) - Elisa MARIVIN, M2 (5 months) - Claire SCHIRMER, M2 (5 months) 2012 - Lynda LAMRI, M1 (2 months) - Mohammed SAOULI, M1 (2 months) - Arnaud DELEZIRE & Marwane GHEMAME, M1 UE11 (2 weeks) 2013 - Hélène JAGLINE, M1 (2 months) - Charly JEHANNO, M1 (2 months) - Emmanuelle NIVET & Irène NZIGAMASABO, M1 UE11 (2 weeks) 2014 - Florence LORDINOT, M1 (2 months) - Thomas LE GAL, M1 (2 months) - Caroline FOURNIS & Matilde NGUYEN, M1 UE11 (2 weeks) 2015 - Laure LE CALVEZ, M1 (2 months) - Margaux VÉRON, M1 (2 months) - Laurelie BEVIERE & Mathildde BIDEAU, M1 UE11 (2 weeks) - Gaëlle ANGRAND,M2 (5 months) PhD 05/02/2010: Julie MASSÉ – Dir. Thèse Isabelle PELLERIN & Stéphane DESCHAMPS 30/04/2010: Olivier LE TONQUEZE – Dir. Thèse Luc PAILLARD 27/09/2012: Gaëlla BOULANGER – Dir. Thèse Luc PAILLARD 30/11/2012: Maud NOIRET – Dir. Thèse Serge HARDY 15/01/2015: Géraldine DAVID – Dir. Thèse Luc PAILLARD & Yann AUDIC Post-doctorants 2008-2010: Bernhard GSCHLOESSL (ANR), post-doctoral fellow 2 years

215

Team 16 "Reverse engineering cell division" (CeDRE) Leader: Jacques PÉCRÉAUX

217

2.1. Team presentation

The CeDRE team investigates cell division through a multidisciplinary approach, linking molecular details to cell-level events: How does a robust and adaptive cell division emerge from the numerous interactions of involved players? The robustness to perturbations (e.g. chromosomal instability in cancer) or adaptability (e.g. to protein evolution (Riche, S., et al., J. Cell Biol. 2013; 201, 653-662)) emerges from a network of interacting players well approached by statistical physics (Kitano, H., Nat. Rev. Genet. 2004; 5, 826-837) (Kitano, H., Mol. Syst. Biol. 2007; 3, 7-). While the proteins differ between organisms, the network is evolutionarily conserved. Such a change of paradigm is highly promising for future applications in cancer therapy. We focused on in vivo mechanics, i.e. regulation of forces that position the spindle, separate sister chromatids, etc. (Thompson, S.L., et al., Curr. Biol. 2010; 20, R285-295). We investigated not only the dynamics of components but also pseudo-balance in forces i.e. slow drift in position (out- of-equilibrium), which is thought to provide advantageous adaptability (Prost, J., et al., Nat. Phys. 2015; 11, 111-117). We mostly developed microscopy and image processing tools to quantify the dynamics in vivo using Caenorhabditis elegans as a model organism and we modeled it using out-of-equilibrium statistical physics. The team is therefore multidisciplinary; this is a competitive advantage. A strong attention was paid to communication quality (people being trained accordingly). Furthermore, we alternate labmeetings with biologists (MICHAUX Lab) and biophysicists (TRAMIER Lab and group of S. HUET); all lab members speak in both flavors of labmeetings. Recruitment in the lab is project-based to keep the lab dynamic. Quality of recruitment is key and I devoted time to find right and high profile candidates with good track records. We wish to make the lab a nice place to live and work and fostered social activities to this aim. We eventually pay attention to have a multicultural and international team (Z. ALAYAN from Lebanon, R. RODRIGUEZ GARCIA from Cuba, H. BOUVRAIS, PhD in Denmark, X. PINSON post-doc in Canada).

2.2. Assessment from January 1st 2010 to June 30th 2015

The lab was created on 1st of January 2011, supported by an ATIP/Avenir starting grant.

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

PECREAUX Jacques CR1 CNRS Physics, Biology, Math, … BOUVRAIS Hélène CR2 CNRS Bio-/Soft-Matter-Physics LE CUNFF Yann MCU UR1 Math, Statistics, Modeling CHESNEAU Laurent IE2 CNRS Biology – Lab manager PASTEZEUR Sylvain AI UR1 [50 %] Biology

2.2.2.2. Temporary staff

PINSON Xavier Post-doc Biology MERCAT Benjamin PhD Student (2012/2016) Mechanics, Scientific comp.

218

2.2.3. Achievements

2.2.3.1. Scientific achievements

During asymmetric division in the C. elegans one-cell embryo, the mitotic spindle is first centered until anaphase onset, when it is elongated by its internal forces and cortical force generators pulling on astral microtubules (MTs). Cortical forces also move the spindle to the posterior and make it oscillate during anaphase (Ahringer, J., Curr. Opin. Cell Biol. 2003; 15, 73-81) (Kimura, A., and Onami, S., Dev. Cell 2005; 8, 765-775) (Pecreaux, J., et al., Curr. Biol. 2006; 16, 2111-2122).

The lab was created through a starting grant (ATIP), proposing achievements A1-A3.

A1. Mechanism that maintains the spindle in cell center during metaphase Achievements: Our previous experiments (Pecreaux, J., et al., Curr. Biol. 2006; 16, 2111-2122) (Redemann, S., et al., PLoS ONE 2010; 5) suggest that a mechanism maintains the spindle in cell center during metaphase although the details are highly debated (McNally, F.J., J. Cell Biol. 2013; 200, 131-140). (A1a) We finished the tool initiated during my postdoc to measure a mechanical blueprint that recapitulates how the spindle is maintained in the cell center during metaphase. Only embryos with no large spindle motion (no drift) can be analyzed. We could hypothesize that centering maintenance is due to MTs pushing against the cell cortex, with an enhancement of stability due to MT buckling [J. Pecreaux, S. Redemann, Z. Alayan, B. Mercat, S. Pastezeur, C. Garson-Coral, A. A. Hyman, J. Howard, “High positional stability of the one-cell C. elegans mitotic spindle during metaphase accords with centering by a MT-pushing mechanism”, re-submitted]. (A1b) We upgraded this tool to be insensitive to drift and screened for about 40 players putatively involved in stability of centering, in a gene candidate approach. We found in particular that LIN-5/NuMA may contribute to MTs stabilization (1-2 s at the cortex) in the absence of GPR-1/-2/LGN (Du, Q., and Macara, I.G., Cell 2004; 119, 503-516) on top of its role in cortical pulling forces (Nguyen-Ngoc, T., et al., Nat. Cell Biol. 2007; 9, 1294-1302). We also found ZYG-8/DCX, which could rigidify MTs (Bellanger, J.M., et al., J. Cell Sci. 2012; 125, 5417-5427) (Tolomeo, J.A., and Holley, M.C., Biophys. J. 1997; 73, 2241-2247) and account for the discrepancy with model predictions [Main contrib. Z. ALAYAN]. This work continues in project P1b. Significance: Above identifying the centering mechanism, we suggested a direct consequence of regulating MT mechanical properties along cell division. This is pretty original since MTs are usually considered as passive scaffold or mediators of molecular motors action.

A2. Characterizing spindle length and mechanics (blueprint) to understand correction of chromosome mis-attachments. Achievements: (A2a) We deeply improved the approach above (A1a) to have a blueprint of spindle mechanics along time and resolve its assembly, maintenance and elongation (Spindle Spectral Signature, S3 assay)1. We found that the spindle behaved like a spring, a damper and an inertial element in parallel (Kelvin-Voigt with inertia). During metaphase, the spindle slowly elongated, slowed down by viscous drag and mildly by inertia. The very low spring stiffness suggests a weak/lack of mechanism maintaining spindle length. We found similar results, although inertia seems absent, in S. pombe (coll. S. TOURNIER and Y. GACHET, LBCMCP, Toulouse). By laser-spindle-cut, we are calibrating this assay to measure forces [Main contrib. B. MERCAT] [Mercat B., Pinson, X., Fouchard, J., Mary, H., Alayan Z., Pastezeur S., Bouvrais H., Gachet Y., Tournier S., Pecreaux J., “Biophysical tool to characterize the temporal evolution of the mitotic spindle mechanics”, manuscript in preparation]. (A2b) We are currently finalizing a merotely assay in C. elegans using a temperature-sensitive mutant of the AIR-2/Aurora B (Schumacher, J.M., et al., J. Cell Biol. 1998; 143, 1635-1646) with a temperature-controlled microfluidic device to trigger merotely in metaphase, observe its resolution in anaphase and analyze the role of various forces [Main contributor: X. PINSON]. We will apply these tools to spindle mechanics (project P2). Significance: The holocentric C. elegans chromosomes are close to mammalian ones (several MTs per chromosome, chromokinesins), mimicking a magnified human centromeric region (Stear, J.H., and Roth, M.B., Mol. Biol. Cell 2004; 15, 5187-5196), while simple attachments in S. pombe provide a single MT/event view in vivo. On top of offering a fundamental understanding of the spindle and paving the way to modeling robustness and adaptability (see projects), our work will provide a basis to understand the

1 In more technical terms, we used short time Fourier transform by sliding windows and we performed a corrected weighted least square fitting (Norrelykke and Flyvbjerg, Rev. Sci. Instrum. 2010; 81, 075103-) (home developed) to approach the data by a heuristic model and extract mechanical blueprint. 219

major parameters in chromosome alignement and attachment corrections, whose defects can lead to cancer. (Thompson, S.L., et al., Curr. Biol. 2010; 20, R285-295).

A3. Dynamics of cytoplasmic dynein and targeting to the cell cortex Achievements: The unique cytoplasmic C. elegans dynein fulfills many roles in mitosis, including generating cortical pulling forces (Gonczy, P., et al., J. Cell Biol. 1999; 147, 135-150) (Nguyen-Ngoc, T., et al., Nat. Cell Biol. 2007; 9, 1294-1302). However, regulation of its localization is poorly known. We imaged dynein intermediate light chain DYCI-1 expressed under its endogenous promoter (Poser, I., et al., Nat. Methods 2008; 5, 409-415). We denoised (Coupe, P., et al., Med. Image Anal. 2012; 16, 849-864) (Sage, D., et al., IEEE Trans. Image Process. 2005; 14, 1372-1383), tracked (modified utrack, (Jaqaman, K., et al., Nat. Methods 2008; 5, 695-702)) and classified tracks (Huet, S., et al., Biophys. J. 2006; 91, 3542-3559) (Monnier, N., et al., Biophys. J. 2012; 103, 616-626). We identified spots, composed of ~50 dynein molecules each, moving toward cell periphery in a comet like fashion and in a CeEBP-2/HsEB1 dependent manner. We suggested that MT (+)-end is used to target the dynein at the cortex. We observed that dynein/MT tips explore the cortex before being engaged in pulling and reside there for a very short time (> 1 s) [Main contrib: R. RODRIGUEZ GARCIA] [R. Rodriguez Garcia, L. Chesneau, A. Pacquelet, J. Roul, M. Tramier, J. Pecreaux, “Dynamics of dynein intermediate chain (dyci-1) in the C. elegans zygote,” in preparation]. Significance: Dynein was thought to be localized at the cell cortex and reside there on the long run. In contrast, we showed here that it is highly dynamical, residing very briefly at the cortex, thus fostering adaptability. Dynein is enriched at the MT (+)-end, enabling GPR-1/-2 to be the limiting and regulating factor (Park, D.H., and Rose, L.S., Dev. Biol. 2008; 315, 42-54).

A4. Positional control of cortical pulling forces, a robustness mechanism. Achievements: We aimed to investigate the role of astral MTs (Kozlowski, C., et al., Cell 2007; 129, 499-510) in the modeling of anaphase spindle rocking (Pecreaux, J., et al., Curr. Biol. 2006; 16, 2111-2122). We compared spindle oscillations in C. elegans and C. briggsae embryos (coll. M. DELATTRE, LBMC, Lyon), included MTs dynamics in our model to account for the positional switch on cortical pulling forces revealed by the delay in oscillation onset in C. briggsae (Riche, S., et al., J. Cell Biol. 2013; 201, 653-662). We proved that MTs dynamics create this control by analyzing MTs contacts at the cortex (landing assay, labeling MTs not EB1) through advanced image denoising and tracking. We found significantly more contacts in cortical regions closer to centrosomes. We suggest that MTs, through their dynamics, probe cell geometry and control the posterior pulling forces on top of the processivity2 [Main contrib. J. PECREAUX, H. BOUVRAIS] [Bouvrais H., Chesneau L., Riche S., Argoul F., Zouak M., Arneodo A., Delattre M., Pecreaux J., “Space and time regulation of the spindle positioning in one-cell embryo of C. elegans,” manuscript in preparation]. Significance: Combining positional and temporal switches provides robustness in the final spindle position by buffering variations in the initial position, cell shape, final force generators processivity, or compensating in C. elegans vs C. briggsae for evolution/duplication of essential gene(s) gpr-1/2. We demonstrated that MTs create a positional feedback, suggesting that mechanics can create robustness. It also highlights the forefront role of MTs contrasting with their traditional ancillary function.

A5. Spatial and temporal regulation of MTs, towards a prominent role Achievements: A1 and A4 revealed the prominent role of MTs. An advanced statistical analysis of the landing assay above showed two populations of astral MTs with distinct residency times at the cortex: one short, 0.2 - 0.5 s and one long, 1 - 2 s. We observed temporal and spatial regulations of these populations along cell division: (1) 10 - 15% of the MTs (i.e. 0.05 MTs/min/µm²) displayed a short residency time during metaphase, while this proportion increased to about 55-60% in anaphase (i.e. 0.51 MTs/min/µm²). Our observations also suggested a decrease in the MTs dynamics (an increase of residency time) from metaphase to anaphase, consistent with the increase of processivity2 (Pecreaux, J., et al., Curr. Biol. 2006; 16, 2111-2122); (2) Spatial modulation: long-lived MTs showed similar densities in anterior and posterior sides during anaphase (around 0.40 MTs/min/µm²), while short-lived appeared polarized with 1.5 fold more contacts in the posterior (where it is though there is more pulling force generators (Grill, S.W., et al., Science 2003; 301, 518-521) (Redemann, S., et al., PLoS ONE 2010; 5)) vs anterior (0.64 MTs/min/µm² vs 0.41 MTs/min/µm²). We hypothesize that the short-lived MTs might mediate pulling forces while long-lived might push against the cortex [Main contrib. H. BOUVRAIS, Y. LE CUNFF]. We will pursue this in project P1a.

2 i.e. the number of steps a motor runs before detaching from the microtubule. It depends on the load. 220

Significance: The measurement of two populations of MTs displaying distinct dynamical behaviors at the cortex could underline a complex mechanism for the mitotic spindle positioning. E.g. recent work suggested that dynein motors could play a role in bringing the spindle in cell center prior to mitosis, via side-on attachments to the cortex (Gusnowski, E.M., and Srayko, M., J. Cell Biol. 2011; 194, 377-386), and in pulling at MT (+)-ends to create posterior displacement.

A6. Towards faster optical microscopy acquisition with lower phototoxicity Achievements: [Main contributors: J. PECREAUX, Y. LE CUNFF] To support the projects presented above, we performed technical developments: (A6a) Fluorescence microscopy is limited by the destruction, due to the excitation light itself, of living tissue (phototoxicity) and of the fluorophore (photobleaching). In collaboration with TRAMIER Lab (same institute) using C. elegans (Tinevez, J.Y., et al., Methods Enzymol. 2012; 506, 291-309), we investigated commercially available sources and adjustable parameters to determine the best compromise. We found two phototoxicity processes at work, one during cell division and the other during interphase. More surprisingly, we found that a wavelength band close to, but not exactly at, 488 nm (used to excite GFP) could putatively be highly toxic and we noticed differences in phototoxicity on symmetric (AB) and asymmetric divisions (P0, P1). (A6b) Currently, we are revisiting the driving of multidimensional optical microscope (POMM, Pilotage Optimal pour la Microscopie Multidimensionnelle, Optimal Driving for Multidimensional Microscopy) by developing a specific hardware module that will communicate with the devices and tremendously foster the acquisition speed, cutting out the delays due to devices movements. This project is funded through a maturation project (SATT Ouest) shared by TRAMIER- and PECREAUX-lab and the concept was patented. (A6c) We improved image denoising and tracking (coll, C. KERVRANN, INRIA, Rennes, related to A3, A5). We benchmarked various methods published and algorithms developed in the KervrannLab to find the more appropriate one to our application, in particular the measurement of the dynamics of the MTs. This project is still ongoing. Significance: The goal of capturing the dynamics of objects (like the centrosomes) at cell level or, even more challenging, objects at the molecular level (like the (+)-ends of the MT) calls for improving first imaging. We believe that our developments are attractive features beyond the needs of our team and therefore we pay a strong attention to dissemination of this expertise.

2.2.3.2. Scientific dissemination and influence

Although the team is young, its expertise at the interface of soft matter physics and cell biology, as well as the high quality of its quantification are recognized. This appears through recruitments, collaborations and funding: (1) during the term, L. CHESNEAU was recruited as permanent engineer from A. ECHARD Lab (Pasteur Institute, Paris), H. BOUVRAIS as researcher from MEMPHYS (dir. O. MOURITSEN, Denmark), Y. LE CUNFF as assistant professor from K. PAKDAMAN lab (Jacques Monod Institute, Paris); (2) the lab was approached for collaborations requiring advanced quantification in vivo and modeling by M. DELATTRE (LBMC, ENS Lyon) and S. TOURNIER and Y. GACHET (LBCMCP, Toulouse). The high quality data also enabled to collaborate with C. KERVRANN (INRIA Rennes) towards advancing simulations (see project P3); (3) eventually, the lab managed to obtain very competitive funding such as Plan Cancer for fundamental research (~ANR), an EMBO long-term postdoc fellowship (H. BOUVRAIS) or an ATIP starting grant. The CeDRE team is involved in several training actions (see details in annex): 1) Yann LE CUNFF teached in Biology masters, especially statistics and modelling; 2) Through practicals (1-week duration) at master and in PhD level, the lab introduce trainees to quantitative cell biology and modelling; 3) The lab is also strongly involved in professional training in microscopy and image processing and recently statistics; 4) eventually, Jacques PECREAUX lead the PhD Prgram of the institute.

2.2.3.3. Interaction with the economic, social and cultural environment

Disseminating innovations in pedagogy: Through Y. LE CUNFF, the lab is involved in disseminating innovation in pedagogy and learning through research methodologies. Several seminars have been held on these topics, at the initiative of important socio-economic institutions (Chambre de Commerce et de l'Industrie, Bretagne Développement Innovation, Institut de Gestion de Rennes), as well as a paper published in the "Place Publique" magazine (see the list in annex).

Collaborative R&D with private sector: We have know-hows in microscopy (achievement A6a) from which we set up a R&D common program with the company Photonlines, together with the lab of M. TRAMIER (same institute) and entitled “Quantitative study by multivariate statistical analysis of the effects of illumination parameters on photo-toxicity and -bleaching in fluorescence microscopy. Application to development of a light source optimized for fluorescence microscopy”. In fine, we will transfer knowledge on optimized illumination (Application note e.g.) and we also foresee the potential 221

development of advising software to mitigate phototoxicity and photobleaching with other requirements of an experiment (wavelengths used, duration, minimum illumination intensity, etc.).

Maturation (SATT funded), optimal driving for multidimensional microscopy: This project (see achievement A6b) is funded through a maturation project by SATT Ouest. We think that our development could create a product able to upgrade not only any existing microscope used for research in biology but also more broadly, any optical system regardless of its application. We foresee a putative business model, where the upgrading of the hardware could be sold by a company and driving software and customization could be either developed by the user or taken care of by the company as a service together with the support (similar to the “Red Hat” business model).

2.3. Projects, scientific strategies & perspectives (5 years)

Context and significance

A high number of proteins are involved in the mitotic spindle. The network of astral microtubules (MTs) and their regulating proteins both in cytoplasm and cortex constitute a highly complex supra molecular machine. If biochemistry has offered fantastic progress to the field, it has had little successed so far in providing the big molecular picture of the mitotic apparatus at large, in particular because it misses all the mechanical interactions regulating the activity of the components (see e.g. (Ward, J.J., et al., eLife 2014; 4, e03398)). During the next term, we will carry on, with a multidisciplinary approach, investigating the big question: How does a robust and adaptive cell division emerge from the numerous interactions of involved players? Understanding these properties is key to the fundamental understanding of mitosis and also an important challenge in cancer biology. Indeed, such mechanisms designed to ensure that cell division goes on and remains as faithful as possible are ambivalent, essential to the correct development of organisms, allowing mutations and evolution on the one hand, but on the other hand, permitting the survival of cancerous cells despite their accumulated defects (e.g. aberrant number of chromosomes and centrosomes) and fostering resistance to anti-mitotic drugs, especially the one targeting cell division players as microtubules e.g.. The cell division machinery will be approached as a complex, dynamic (in permanent evolution) and out-of-equilibrium (slowly evolving since forces are not balanced) system (see achievements introduction and approach subsection below). During this term, we will put a special effort in developing coarse-grained models 3 and simulations. In such models, the extensive complexity at molecular scale is "summarized" into a lower number of parameters (degrees of freedom) to make simulations and modeling more amenable4. This is, at least in the first steps of simulating/modeling, an established way to deal with molecular redundancy and, to a lower extent, with complexity (Bausch, A.R., and Kroy, K., Nat. Phys. 2006; 2, 231-238). One challenge will lie in backing such modeling and agent-based simulations5 by experimental data (chiefly the ones developed in achievements A2 and A5). To meet this challenge, the lab has established expertise in advanced analysis of data and we wish now to unleash modeling and simulation power to take full advantage of these data.

Objectives

3 Model where molecular components with quite similar properties/roles (e.g. different molecular motors in the spindle contributing to shorten it) are represented into the model by a single “typical motor”, whose parameters average the various represented molecular components. This is a needed step in modeling to capture the essential of a mechanism. 4 For example, a simple “average” force generator can represent forces generated by a diverse group of kinesins, respectively by dynein and MT depolymerization. 5 In such simulations, for sake of simplicity and tractability, one discards the molecular details to recapitulate a full network of proteins/molecular players into a single entity governed by a simple equation. This entity is called an agent. Furthermore, the "big picture" of the simulated process is not solved by equations but emerges of the simulation through agents interactions. 222

We posit that MTs, their regulators and molecular motors form a system, essential to allow cells to adapt to perturbations in such a way that it ensures faithful and robust divisions. The proposed research — systems cell division mechanics: toward a better understanding of dynamics and robustness of cell division — will be developed into three core objectives:

(P1) How do microtubule dynamics and mechanics contribute to cell division robustness? Previous achievements (A4, A5) suggest spatial and temporal regulation of MT dynamics as well as a role of their mechanics (A1). From there, we hypothesize that the short lifetime population of MTs relates to dynein pulling on them from the cortex (see A5, also related to A3), while the long lifetime population relates to the mechanism maintaining the spindle in cell center during metaphase (A1). (P1a) To test this hypothesis, we will adopt a candidate gene approach together with comparing symmetric (AB) and asymmetric (P0 and P1) divisions and investigate the spatiotemporal regulation of long-lived and short- lived populations. We will model the results including the displacement of the MT organizing centers, cell geometry (3D) and regulation of MT dynamics through cell cycle. (P1b) We will measure and model the role of MT mechanics, their rigidity in particular, since we observed that the MTs dynamics alone couldn’t explain the spindle centering (see A1). Altogether, the correlations made between the molecular interactions at the microscopic scale (measurements of the fine regulation of the MT dynamics and mechanics) and the properties at the cell level (robustness, adaptability, reliability, etc.) will feed the biophysical model of cell division.

(P2) How do the dynamics and constant evolution of the spindle (out–of–equilibrium) make it highly efficient and robust? The S3 assay characterizing the mechanical blueprint of the mitotic spindle (see A2) paves the way to explore how “non-balanced forces” making the spindle “slowly moving” contribute to robust and faithful divisions as suggested for other systems (Brugues, J., and Needleman, D., Proc. Natl. Acad. Sci. U. S. A. 2014; 111, 18496-18500) (Prost, J., et al., Nat. Phys. 2015; 11, 111-117). We are currently collaborating with the TOURNIER-GACHET lab (LBCMCP, Toulouse) to investigate how forces contribute to merotely correction during anaphase both in S. pombe, to see single correction events and in C. elegans, whose holocentric chromosomes mimic a magnified mammalian centromeric region and highlight collective effects (Plan Cancer). Carrying on this collaboration, we will investigate: (P2a) the mechanics of the spindle as a dynamic and constantly evolving system (out-of-equilibrium) and its robustness when facing external perturbations (temperature, cell size following successive divisions at constant volume, etc.) and internal ones (chromosomes number, attachment, cohesion, etc.); (P2b) chromosome mis-attachments and their corrections through forces regulated by specific robustness mechanisms, for which dynamics is instrumental to be adaptive; (P2c) how do internal but also external forces, e.g. related to spindle position, alter anaphase onset timing, putatively through the spindle assembly checkpoint (SAC).

(P3) Develop mixed analytical models and agent-based simulations. The wealth of in-depth data produced by our previous work and by P1 and P2 projects calls for progress into modeling. Creating a detailed theoretical model of cell division mechanics (shaped as mathematical stochastic differential equations, Langevin e.g.) is a very valuable but also challenging goal, largely out of the scope of this term. We rather aim to create an analytical model of important aspects of mitosis robustness built from molecular details and modeling collective effects, to understand mechanical regulation at cell scale. The remaining aspects will initially be included by modeling a single molecular player (called an agent) into an agent-based simulation. Such simulations are classically used by biophysicists to explore and gain understanding of complex systems, prior to advancing in the analytical modeling. In collaboration with the teams of C. KERVRANN, E. MEMIM (INRIA Rennes) and J. SALAMERO (Curie Institute, Paris), we aim to build such a mixed analytical-model-based and agent-based simulation to estimate the parameters of the model (cell behavior) by linking the simulation to data (similar to forcing simulation to fit the data to gain insight into the process). To do so, we propose to explore data assimilation methods that couple and synchronize numerical simulation models to microscopy-based measurements with the purpose of predicting the state of the biological system under study6. Such an approach will beautifully recapitulate our data and boost our understanding of the robustness and adaptability of spindle length and positioning.

(PP1) Prospective Project: Modeling cell division mechanics in human cells. In collaboration with the team of M.-D. GALIBERT, and particularly M.-B. TROADEC (same institute), we plan to use human B lymphocytes as a model system because of their lack of adhesion, known to modify the behavior of the spindle, in a healthy or leukemic context. We will measure, analyze and model mitosis and movements of the spindle (building on the work done in C. elegans) using cultured cell lines (immortalized) at first, and

6 Data assimilation is generally recommended to merge various measurements, including image measurements, which are uncertain and potentially irregularly distributed in space and time, given an a priori dynamical model. 223

then aim at validating obtained results using primary cultured cells and at delineating whether cancerous (or even immortalized) cells display modified cell division mechanics. Beyond mechanisms that may differ between healthy and cancerous cells, without prejudice on the molecular players involved, we will identify vulnerabilities that should allow more selective targeting of cancerous cells during therapy.

(PRD1) Project R&D: in fluorescence microscopy to increase acquisition rate. The project POMM (optimal driving for multidimensional microscopy) will be pursued with TRAMIER Lab (see A6b).

Strategy / Approach

Biological and microscopy experiments: We will use molecular genetics associated with live cell video-microscopy, advanced image/signal processing, quantification, statistics and biophysical modeling to challenge the proposed model in-depth. In particular we will: quantify forces through laser cut experiments; use the S3 assay to obtain the cell-level mechanical blueprints; measure inter-sister kinetochore distances to assess tension; use trajectories of chromosomes and spindle poles and use the merotely assay (see A2b) to test chromosomes attachment mechanisms. To challenge the robustness, we will use non-genetic perturbations to minimize the risk of being out of physiological conditions, e.g. cell size changes (using P lineage) and symmetric/asymmetric divisions, changes in chromosomes number or in dynamics of the components (through temperature e.g.).

Models: The details of cell division mechanism and forces cannot be simply drawn from assembling a few components, each with a distinct function but ignoring their interactions. Instead, we will: (1) focus on developing a system approach, changing the paradigm from "one molecule – one cell function" to “one network of players – one function”; this can be viewed as a generalized signaling pathway; (2) account for the players dynamics (MTs, motors binding/unbinding e.g.) and their dependence on forces, position, shape, etc. of the spindle; (3) consider the system, in particular the spindle in vivo, as unlikely to be at/reach a "balanced forces state" (equilibrium); e.g. forces elongating the spindle do not perfectly balance the shortening forces leading to its slow elongation over time in prometaphase/metaphase. This situation is particularly interesting to create adaptability to internal or external perturbations. However, the robustness arising from an out-of-equilibrium state is different from the one resulting from the dynamics7. Proving that this phenomenon is relevant in vivo is also an expected result of this term.

Simulations will be developed using Cytosim, an agent-based engine able to run variability and robustness studies (Ward, J.J., et al., eLife 2014; 4, e03398). We will develop a spindle module (we are in contact with F. NEDELEC, who authored Cytosim) and connect it with TOURNIER-GACHET simulation (P2) to visualize simultaneously spindle dynamics and specific events at the kinetochores. Running a batch of simulations will provide the first sensitivity analysis and highlight the key parameters of the model.

Data analysis: Optimized wide field and spinning disk microscopes combined with image denoising and restoration workflow (P1 and P3, coll. C. KERVRANN, INRIA Rennes) will allow imaging a full division at a high frame rate with minimal phototoxicity/photobleaching. Advanced statistical analyses are performed and are keys to high quality data (P1, P2 especially). We will control our conclusions with in silico fabricated data to challenge the robustness of statistical and image analysis tools.

Perspective and Significance

Our approach will enable us to pinpoint the networks and collective effects at the heart of robustness and adaptability properties of cell division. Our work will also contribute to establish C. elegans embryo (and S. pombe through collaboration) as an in vivo model organism to understand biophysical networks; our conclusions, expressed in the universal language of physics, will be relevant to other organisms. Beyond mitosis, this project will also contribute to a better understanding of the role of mechanisms underlying plasticity and robustness of asymmetric divisions, which appear important also in stem cells and Cancer Stem Cells (CSC)8. Indeed, targeting them could limit tumor growth, prevent relapse and tumor dissemination (metastasis). Interfering with robustness mechanisms would only weakly

7 Imagine you are taking the start of a race with your dog. The dog is in front of you and you attempt to keep a stick touching it. In one case (at equilibrium, regime 1), your bike is stopped in a shallow hole to ensure it stays at its position: if the dog moves slightly forward (corresponding to the kinetochore that detaches and is pulled towards the opposing pole, the stick being a MT), you will need a substantial effort to move forward your bike and to reestablish contact with your dog. Similarly, the start of the race (spindle elongation in anaphase) will require a strong effort. Now, imagine you are not stopped (not at-equilibrium; regime 2) but that you are braking in a slope and slowly moving forward: reestablishing contact with the dog will simply consist in releasing the brake. Similarly, the start of the race will be considerably easier. Observing the slow increase of spindle length over time and a mechanics dominated by viscous drag (a brake) suggests that the spindle rather performs in regime 2 of our analogy. 8 Initially identified in leukemia, and later in many cancer types, CSC are rare, highly tumorigenic and share common characteristics with stem cells (self-renewal e.g.). 224

penalize healthy cells (under satisfactory environment), while cancer cells would be severely hindered (as they critically need robustness) enabling high selectivity.

2.4. Collaboration

2.4.1. Within the IGDR

M. TRAMIER (A6, PRD1) - M.-B. TROADEC and M.-D. GALIBERT (PP1).

2.4.2. Other collaborations

Rennes C. KERVRANN (INRIA Rennes) (A5, P1, image processing, and P3) - E. MÉMIN (INRIA, Rennes) (P3).

National J. SALAMERO (Inst. Curie, Paris) (P3) - Y. GACHET and S. TOURNIER (LBCMCP, Toulouse) (A2b, P2ab) - M. DELATTRE (LBMC, Lyon) (A4).

2.5. SWOT Analysis STRENGTHS Interdisciplinarity within the lab A permanent researcher in each major field WEAKNESSES Delay in publication of the lab; longer delay to establish a story (multiple fields) Lack of technical support (computer e.g.) No international collaboration OPPORTUNITIES Hot topics, thus fundable Team attractive for collaborations THREATS Competitive fields More complicated communications (several fields) Difficult to find (and fund) talented PhD students and to a lower extend postdocs

225

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Redemann, S., Pecreaux, J., Goehring, N.W., Khairy, K., Stelzer, E.H., Hyman, A.A., and Howard, J. (2010). Membrane invaginations reveal cortical sites that pull on mitotic spindles in one-cell C. elegans embryos. PLoS ONE 5. (IF=3.5)

2013 Riche, S., Zouak, M., Argoul, F., Arneodo, A., Pecreaux, J., and Delattre, M. (2013). Evolutionary comparisons reveal a positional switch for spindle pole oscillations in Caenorhabditis embryos. J Cell Biol 201, 653-662. (IF=9.8)

2014 Bajard, L., Morelli, L.G., Ares, S., Pecreaux, J., Julicher, F., and Oates, A.C. (2014). Wnt-regulated dynamics of positional information in zebrafish somitogenesis. Development 141, 1381-1391. (IF=6.3)

2015 Gillard, G., Shafaq-Zadah, M., Nicolle, O., Damaj, R., Pecreaux, J., and Michaux, G. (2015). Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells. Development. (IF=6.3) Mary H., Fouchard J., Gay G., Reyes C., Gauthier T., Gruget C., Pecreaux J., Tournier S., and Gachet Y. (2015). Fission yeast Kinesin-8 controls chromosome congression J. Cell Sci. accepted (IF=5.4).

2. Patents (with licence) / Computer software

ROUL, J., PECREAUX, J., and TRAMIER, M. (2012). Flimager for realtime fluorescence lifetime imaging computation (Agence pour la Protection des Programmes (AAP) Nr IDDN.FR.001.140028.000.S.P.2012.000.31235).

ROUL, J., PECREAUX, J., and TRAMIER M. Procédé de pilotage multi-modules fonctionnels incluant un dispositif d'imagerie multi-longueur d'onde, et système de pilotage correspondant. Patent pending, 2014.

3. Conferences (actual team members)

- International

2012 Riche, S., Argoul, F., Zouak, M., Arneodo, A., Pecreaux, J., and Delattre, M. (2012). Evolution of GPR regulation in the control of spindle positioning for two Caenorhabditis species embryos. Poster presented at: Annual meeting of American Society of Cell Biology (December 14-19 2012, San Francisco, CA, USA). Riche, S., Argoul, F., Zouak, M., Arneodo, A., Pecreaux, J., and Delattre, M. (2012). What did a comparative modeling of spindle oscillations in C. elegans and C. briggsae embryos teach us about time and positional control of spindle positioning? Poster presented at: Microtubules: Structure, Regulation and Functions (EMBO series) (EMBL Heidelberg, Germany).

2013 Rodriguez Garcia, R., Chesneau, L., Roul, J., Tramier, M., and Pecreaux, J. (2013). Characterizing dynein dynamics by live cell imaging suggests transport to the cell cortex by microtubule (+)-end. Poster presented at: Seeing is Believing – Imaging the Processes of Life (EMBO series) (Heidelberg, Germany). Rodriguez Garcia, R., Chesneau, L., Roul, J., Tramier, M., and Pecreaux, J. (2013). Dynamics of dynein intermediate light chain (dyci-1) in C. elegans zygote. Poster presented at: Physical Biology of Cancer (EMBO series) (Candiolo, Italy).

2014 Rodriguez Garcia, R., Chesneau, L., Roul, J., Tramier, M., and Pecreaux, J. (2014). Dynein intermediate chain tracks microtubule plus end in an EBP-2 dependent manner in C. elegans one cell embryo. Poster presented at: Building the cell (Paris, France). Bouvrais, H., Le Cunff, Y., Roul, J., Alayan, Z., Rodriguez Garcia, R., Tramier, M., and Pecreaux, J. (2014). Astral microtubules displaying two different dynamical behaviours may perform different functions during the division of C. elegans one-cell embryo. Poster presented at: Building the cell (Paris, France). 226

Bouvrais, H., Le Cunff, Y., Roul, J., Alayan, Z., Rodriguez-Garcia, R., Tramier, M., and Pecreaux, J. (2014). Two populations of astral microtubules during the division of C. elegans one-cell embryo may perform different functions. Poster presented at: EMBO conference: Microtubules: Structure, regulation and functions (Heidelberg, Germany). Rodriguez Garcia, R., Chesneau, L., Roul, J., Tramier, M., and Pecreaux, J. (2014). Dynein intermediate chain tracks microtubule plus end in an EBP-2 dependent manner in C. elegans one cell embryo. Poster presented at: EMBO conference: Microtubules: Structure, regulation and functions (Heidelberg, Germany).

- National

2011 Pecreaux, J., and Oates, A.C. (2011). Biophysical active contours and level sets segmenting tissues brightfield- microscopy images. Talk presented at: journées GdR ISIS/GdR 2588/GIS Europia (Rennes, France). Pecreaux, J., Redemann, S., Hyman, A.A., and Howard, J. (2011). Mitotic spindle centering in C. elegans one-cell embryo. Paper presented at: Cell cycle cancer and development (St Malo, France). Pecreaux, J., Redemann, S., Hyman, A.A., and Howard, J. (2011). Mitotic spindle positioning in C. elegans one-- cell embryo. Talk presented at: 14th workshop “Ver midi” (Feb -18 2011, Paris, France).

2012 Rodriguez Garcia, R., and Pecreaux, J. (2012). Comment la modélisation peut aider à planifier des expériences? Talk presented at: Mifobio 2012: Microscopie fonctionnelle en biologie (Talmont Saint-Hilaire (Vendée)). Roul, J., Tramier, M., and Pecreaux, J. (2012). Etude de la phototoxicité cellulaire en microscopie de fluorescence en fonction du mode d'excitation: continu, modulé, pulsé. Talk presented at: Mifobio 2012: Microscopie fonctionnelle en biologie (Talmont Saint-Hilaire (Vendée)). Riche, S., Argoul, F., Zouak, M., Arneodo, A., Pecreaux, J., and Delattre, M. (2012). What did a comparative modeling of spindle oscillations in C. elegans and C. briggsae embryos teach us about time and positional control of spindle positioning? Poster presented at: Ver midi XV (Paris, France).

2013 Alayan, Z., Bouvrais, H., Abdouni, A., Pastezeur, S., Chesneau, L., and Pecreaux, J. (2013). How is the mitotic spindle kept in cell center during the metaphase of C. elegans one-cell embryo division? Poster presented at: French microtubule network (Marseille). Rodriguez Garcia, R., Chesneau, L., Roul, J., Tramier, M., and Pecreaux, J. (2013). Dynamics of dynein intermediated light chain (dyci-1) in C. elegans zygote. Poster presented at: French microtubule network (Marseille). Bouvrais, H., Roul, J., Tramier, M., Alayan, Z., Rodriguez Garcia, R., and Pecreaux, J. (2013). Central role of astral microtubules to control spindle position and orientation. Poster presented at: French microtubule network (Marseille). Bouvrais, H., Chesneau, L., Riche, S., Argoul, F., Zouak, M., Arneodo, A., Delattre, M., and Pecreaux, J. (2013). Space and time regulation of the spindle positioning in one-cell embryo of C. elegans. Talk presented at: French microtubule network (Marseille). Rodriguez-Garcia, R., Chesneau, L., Roul, J., Tramier, M. and Pecreaux, J. (2013). Dynamics of dynein light chain in C. elegans zygote. Poster presented at: Ver midi XVI (Lyon, France). Alayan, Z., Pastezeur, S., Abdouni, A., Chesneau, L. and Pecreaux, J. (2013). How is the mitotic spindle kept in cell center during the metaphase of C. elegans one-cell embryo division? Poster presented at: Ver midi XVI (Lyon, France). Riche, S., Argoul, F., Zouak, M., Arneodo, A., Chesneau L., Bouvrais H., Delattre, M. and Pecreaux, J., (2013). Space and time regulation of the spindle positioning in one-cell embryo of C. elegans. Poster presented at: Ver midi XVI (Lyon, France).

2014 Pecreaux, J. (2014). Positional fluctuations of the mitotic spindle poles reveal cell division mechanics: a study by image processing and Fourier analysis. Invited talk presented at: Traject 2014: Random Trajectories in Cells and Tissues: extraction, modeling, analysis (Villeneuve d'Ascq, France). Bouvrais, H., Roul, J., Tramier, M., Alayan, Z., Rodriguez Garcia, R., Le Cunff, Y., and Pecreaux, J. (2014). Central role of astral microtubule to control spindle position and orientation. Poster presented at: Ver midi XVII (Illkich, France). Rodriguez Garcia, R., Chesneau, L., Roul, J., Tramier, M., and Pecreaux, J. (2014). Dynamics of dynein intermediate chain (dyci-1) in the C. elegans zygote. Poster presented at: Ver midi XVII (Illkich, France). Alayan, Z., Bouvrais, H., Abdouni, A., Pastezeur, S., Chesneau, L., and Pecreaux, J. (2014). How is the mitotic spindle kept in cell center during the metaphase of C. elegans one-cell embryo division? Poster presented at: Ver midi XVII (Illkich, France).

227

2015 Rodriguez Garcia, R., Chesneau, L., Roul, J., Tramier, M., and Pecreaux, J. (2015). Dynein intermediate chain tracks microtubule plus end in an EBP-2 dependent manner in C. elegans one cell embryo. Talk presented at: Ver midi XVIII (Paris, France). Bouvrais, H., Le Cunff, Y., Roul, J., Alayan, Z., Rodriguez Garcia, R., Tramier, M., and Pecreaux, J. (2015). Astral microtubules display two distinct dynamical behaviors, which may perform different functions during the division of C.elegans one-cell embryo. Poster presented at: Ver midi XVIII (Paris, France). Mercat, B., Pinson, X., Alayan, Z., Le Cunff, Y., Bouvrais, H., and Pecreaux, J. (2015). A biophysical tool to characterize the temporal evolution of the mitotic spindle mechanics during the cell division. Poster presented at: CompSysBio, Advanced lecture course on computational systems biology (Aussois, France). Le Cunff, Y., Bouvrais, H., and Pecreaux, J. (2015). A robust statistical and simulation approach to identify subpopulations of microtubules in C. elegans dividing embryo. Poster presented at: CompSysBio, Advanced lecture course on computational systems biology (Aussois, France). Bouvrais, H., Le Cunff, Y., Roul, J., Alayan, Z., Rodriguez Garcia, R., Tramier, M., and Pecreaux, J. (2015). Astral microtubules display two distinct dynamical behaviors, which may perform different functions during the division of C. elegans one-cell embryo. Talk and poster presented at: CompSysBio, Advanced lecture course on computational systems biology (Aussois, France).

- Selected list of contributions in pedagogy for the public

"L’open innovation, clé de renouveau économique pour la Bretagne", January 2014, Seminar IGR-IAE: "Illustrations de pratiques d'innovation ouverte".

"Et si l’open innovation devenait une pratique ordinaire de nos organisations pour stimuler l'innovation?", March 2014, technical seminar organized by the Chambre de Commerce et de l'Industrie de Rennes: "Prérequis pour aider à mettre en œuvre une démarche d’open innovation".

"360 possibles", October 2014, organised by Bretagne Développement Innovation (http://www.360possibles.fr/): "Le rôle de l’approche pluridisciplinaire dans l’innovation".

"L’économie collaborative, nouveau pilier de développement pour la Bretagne?", IGR seminar - IAE, February 2015: Apports méthodologiques.

"La ville, écosystème d’innovations" Yann LE CUNFF, 2015. In Place Publique (Rennes).

4. Funding

2011-2013 ATIP CNRS / LNCC 277.4 k€ 2011-2013 SAD Région Bretagne 100 k€ 2013 RTSisComm (Coll. D. CHRETIEN, IGDR, C. KERVRANN, INRIA Rennes) 4 k€ 2013-2014 EMBO Long Term postdoctoral fellowship (Hélène BOUVRAIS) interrompu à 15/24 mois 80 k€ 2013-2016 Plan Cancer Biologie des Systèmes (Coll. S. TOURNIER/ Y. GACHET, LBCMCP Toulouse) 230 k€ 2013-2016 Bourse de thèse LNCC (Benjamin MERCAT) 88.5 k€ 2014-2015 SATT Ouest, Maturation Projet POMM (Coll. M. TRAMIER, IGDR) (montant total 2 équipes) 42 k€ 2014-2015 Photonlines, contrat R&D (Coll. M. TRAMIER, IGDR) 25 k€ 2014-2015 ATIP+ 60 k€ 2014-2015 AIS Rennes Métropole (Yann LE CUNFF) 40 k€

5. Training 5.1. Training actions

Members of the CeDRE team teach in the master "mention Biologie-Agronomie-Santé (BAS)" (Biology-Agronomy-Health), "spécialité SCMV: Sciences Cellulaires et Moléculaires du Vivant" (option Cell and Molecular Life Sciences) and "Spécialité BIG: Bio-informatique et génomique" (option Bio-informatics and Genomics). The team contributes to "Ecole Doctorale VAS, Vie Agro Santé" (Graduate School Life, Agronomy, Health) as member of the "Commission de Suivi des Thèses" (PhD Thesis Monitoring Committee) and as member of several thesis advisory committees. Teachings and practicals The CeDRE team is strongly committed into developing innovative "learning through research" programs for undergraduate and graduate biology students. Over the past few years, Y. LE CUNFF has taken the lead on designing and carrying out interdisciplinary teaching. As early as the second year at the university (Licence 2), biology students are now introduced to dynamic systems and bifurcations in biology. This teaching is then complemented by two advanced modules at the Master level: first, an advanced statistics course has been built and is directly inspired by ongoing research projects in the lab and, for example, this year, it has been focusing on optimal experimental design and model selection, with datasets directly extracted from lab data; second, he designed, together with S. HUET (same institute), a course to initiate students to conducting projects ranging from biological experiments (wet-lab) 228

to mathematical modeling and numerical simulations of the acquired data (one week cumulated duration). The team hosted one of these practicals. Validation of this course was composed of two complementary parts: a peer- review (each group of students reviewing another) and a lab meeting-like presentation of the results. The dynamics was such that students themselves were actually asking most of the questions at the end of the presentation. J. PECREAUX also provided "Research conference" to master students to make them discover physical biology in master BAS/SCMV9 and once in master SPI/MCSA10. Professional training / continuing éducation "microscopie photonique en biologie: principes et applications" (photonic microscopy in biology: principles and applications) Duration and dates: 5 days, given 3 times: 16-20th May 2011 ; 12-16 November 2012 ; 24-28 March 2014. Goals: this training aims at providing to the trainees all the theoretical bases and first-hand practicals covering all commonly used microscopy techniques in biology (wide-field with or without time-lapse in DIC and phase contrast and fluorescence, confocal and spinning disk microscopy, two photons, deconvolved wide field, introduction to F- techniques, etc.) as well as side methods as options. It also introduces trainees to sample preparation, images manipulation and archiving. This training was supported by CNRS, INSERM, INRA and Univ. Rennes 1. Participation: members of the lab contribute through theoretical courses teaching and organizing of practicals "analyse d'images de microscopie optique" (processing of optical microscopy images) Duration and dates: 3 days, given 3 times: 12-14 December 2011 and 9 January 2012 ; 15-17 April 2013 and 15th May 2013 ; 10-12 December 2014 and January 12th 2015. Goals: this training aims at introducing the trainee to all the major image processing techniques (intensity quantification and co-localization, filtering, segmentation, tracking, automation through macro development) to enable them to autonomously use established algorithms (using Fiji/ImageJ software) and have a common language with image analysts. This training was supported by CNRS, INSERM, INRA and Univ. Rennes 1. Participation: members of the lab contribute through theoretical course teaching and organizing of practicals Internal training to statistics Introductory sessions to a proper use of statistics in biology were offered by Y. LE CUNFF at the IGDR. These sessions were opened to anyone willing to (re)discover the standard techniques of biostatistics as well as to discuss the key limitations of the traditional frequentist approaches. More than 40 persons attended such 4 hour-long sessions. The key ideas were to introduce a bit of the theory behind statistical testing, provide minimal working examples of the widespread tests (t-test, Mann-Whitney, Chi-2, Anova...) and to stress potential misconducts in the field. In addition to technical skills, this was also the opportunity to gather students, permanent researchers and technicians in the same interactive framework. PhD Program Jacques PECREAUX initiated and was invited by the direction of the IGDR to lead a PhD program, teaming up with Grégoire MICHAUX, Sébastien HUET and Guillaume HALLET. We aim at supplementing the training offered by the "École doctorale VAS" (Graduate school Life, Agronomy, Health). The PhD program is a research oriented training, which aims at (1) opening perspectives to student by making them discover other research topics explored at IGDR, (2) fostering their vision of the various fields in biology, (3) providing them with practical training in soft skills. This program is further described in section "1.3.3 Oragnisation of life in the Institute" and in the Annex 3. 5.2. Trainees in the lab Master / Engineer (Bac +4 & +5) 2011 - Ahmed ADDOUNI, M1 SCMV (Biology), U. Rennes 1 (4 months) - Supervisor J. PECREAUX - Reza SOUFIAN, M2 MCSA (Physics), U. Rennes 1 (6 months) - Supervisor J. PECREAUX 2012 - Benjamin MERCAT, M2 MCSA (Physics), U. Rennes 1 (6 months) - Supervisor J. PECREAUX - Walid BEDHIAFI, M2 BIG (Bio-info.), U. Rennes 1 (6 months) - Supervisor J. PECREAUX 2013 - Mo ZHANG, Eng. 4th y. engineering in mathematics, INSA, U. Rouen (2 months) - Supervisors J. PECREAUX, C. KERVRAN (INRIA, Rennes) & D. CHRÉTIEN (IGDR) 2014 - Geoffrey DIEFFENBACH, M2 BIBS, U. Paris-Sud Orsay (computer science, applied math., biology) (8 months) - Supervisors J. PECREAUX & C. KERVRAN (INRIA, Rennes) 2015 - Gary de NEIDHARDT, M1 SCMV (Biology), U. Rennes 1 (2 months) - Supervisor L. CHESNEAU - Ludovic LEBLANC, M1 SCMV (Biology), U. Rennes 1 (2 months) - Supervisors H. BOUVRAIS & X. PINSON - Céline DELETTRE, Eng. 5th y. ESIEE Paris, (electronics, computer science) (6 months) - Supervisors J. PECREAUX, M. TRAMIER (IGDR) PhD 10/2011-10/2014: Zahraa ALAYAN, Biology - Dir. Thèse J. PECREAUX, interrupted thesis Since 10/2012: Benjamin MERCAT, Mechanics, Scientific comp. – Dir. Thèse J. PECREAUX Post-doctorants 2011-2014 : Ruddi RODRIGUEZ GARCIA, Bio-/Soft-Matter-Physics, post-doctoral fellow 2 years & 9,5 months 2014-2016 : Xavier PINSON, Biology, post-doctoral fellow 1 year & 11,5 months

9 “mention Biologie-Agronomie-Santé (BAS)” (Biology Agronomy Health), “spécialité SCMV : Sciences Cellulaires et Moléculaires du Vivant” (option cell and molecular sciences of the living) 10 “mention Sciences pour l’ingénieur (SPI)” (Sciences for engineer), “spécialité Modélisation, Calcul scientifique et Applications” (option modeling, scientific computing and applications) 229

Team 17 "Cell cycle" Leader: Claude PRIGENT

231

2.1. Team presentation

One of the most fascinating events during cell cycle is when the cell distributes its genetic material to its two daughter cells and divides. This stage, called mitosis, is a tightly controlled and highly regulated process, and any defect can trigger chromosome instability and lead to cancer. During these events the cell co-ordinately prepares itself for the most critical stage of its life, cytokinesis, the physical separation of the two daughter cells. In the group we study the mechanisms that regulate mitosis. We focus on how bipolar spindles assemble, how chromosomes condense and separate and how cells divides. In parallel, we also study mitosis quality controls mechanisms such as cell cycle checkpoints. We are currently focusing on late mitotic events, post metaphase stages. The approaches we use mainly focus on post-translational modifications of proteins such as phosphorylation. We are also deeply engaged in medical research focusing on two aspects (1) genetic diseases related to chromatid cohesion and (2) cancer research. Not only do we search for inhibitors of cell cycle controls but also do we try to understand how misregulation of these control mechanisms participates to carcinogenesis.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

BENAUD Christelle CR1 INSERM TASSAN Jean-Pierre CR1 CNRS CHARTRAIN Isabelle MCU UR1 WATRIN Erwan CR1 CNRS KUBIAK Jacek DR2 CNRS REBOUTIER David IR UR1 OMILLI Francis MCU UR1 LE DEZ Gaëlle AI CNRS PRIGENT Claude DR1 CNRS MIRONOV Svetlana AJT2 CNRS RABUT Gwénaël* CR1 INSERM TIFFOCHE Christophe IR1 CNRS

*Gwénaël RABUT wishes to set up his own group in Jan 2017.

2.2.2.2. Temporary staff

BARON Yorann Post-doc HATTE Guillaume PhD student (2013-2016) BROSSARD Audrey IE LE BOULCH Marie* PhD student (2015-2018) WATRIN Tanguy IR DAMORAN Arun Prasath PhD student (2015-2018) BLASZCZAK Ewa PhD student (2011-2015) VAUFREY Lucie PhD student (2013-2016) GAVARD Olivia PhD student (2011-2015)

232

2.2.2.3. Permanent staff who left the team during the contract

CREMET Jean-Yves IE1 CNRS until 11/2013

2.2.3. Achievements

2.2.3.1. Scientific achievements

Mol Cell. 2011 Aug 5;43(3):488-95. (G. RABUT). Cullin-RING ligases constitute a large family of ubiquitin ligases that regulate numerous cellular functions. Their catalytic activity is controlled through modification of the cullin subunit in a process termed neddylation. Here we show that Tfb3, a conserved subunit of the transcription factor TFIIH, is required for efficient neddylation of the yeast cullins Rtt101 and Cul3, and that it directly interacts with the neddylation enzyme Ubc12. These results suggest that Tfb3 controls the activation of a subset of cullin-RING ligases.

Nature. 2012 Sep 13;489(7415):313-7 (E. WATRIN). Cornelia de Lange syndrome (CdLS) is a developmental disorder caused by mutations in genes encoding components of cohesin and its regulators. SMC3 is acetylated during S-phase to establish cohesiveness of cohesin. We discovered and characterised HDAC8 as the vertebrate SMC3 deacetylase. Furthermore, we identified loss-of-function HDAC8 mutations in six CdLS probands, where loss of HDAC8 activity results in increased SMC3 acetylation and decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

J Cell Biol. 2012 Apr 2;197(1):19-26 (D. REBOUTIER/C. PRIGENT). Nucleophosmin/B23 (NPM) and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. NPM induces AurA auto-phosphorylation on serine 89 stimulating AurA activity. Depletion of NPM by siRNA eliminated phosphorylation of CDC25B on S353 at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA.

J Cell Biol. 2013 Apr 1;201(1):65-79. + biobytes (D. REBOUTIER/C. PRIGENT). We designed a human as-Aurora-A engineered by chemical genetics techniques. This mutant is fully functional and is rapidly and specifically inhibited by 1-NaPP1 in vivo. We discovered that Aurora A is required for central spindle assembly in anaphase through phosphorylation of Ser 19 of P150Glued. This paper thus describes a new Aurora A function that takes place after the metaphase-to-anaphase transition and a new powerful tool to search for and study new Aurora A functions.

EMBO Rep. 2014 Sep;15(9):948-55. + Cover Image (E. WATRIN/C. PRIGENT). Timely establishment and termination of sister-chromatid cohesion are required for proper chromosome segregation and mitotic progression. Cohesion establishment occurs in S phase, and requires binding of sororin to cohesin, which antagonizes the cohesin releasing factor Wapl to stabilize cohesin on chromatin. Here we showed that perturbation of spliceosome caused cohesion defects due to the selective impairment of sororin pre-mRNA splicing. We propose that the splicing machinery contribute to the establishment of cohesion by promoting Sororin accumulation during S phase, and are, therefore, essential to the maintenance of genome stability.

Nature. 2014 Dec 18;516(7531):410-3 (G. RABUT). In this manuscript, we describe a new protein degradation pathway at the inner nuclear membrane (INM). We show that the INM-localized Asi complex (consisting of the RING-domain proteins Asi1 and Asi3) is a ubiquitin ligase that functions together with the ubiquitin conjugating enzymes Ubc6 and Ubc7. Our data indicate that this ubiquitin ligase primarily targets trans-membrane proteins from the cellular endomembrane system, suggesting that it acts to maintain the integrity of the INM.

EMBO Rep. 2015 Apr;16(4):481-9 (C. BENAUD/C. PRIGENT). We demonstrate that annexin A2 is necessary in the early phase of cytokinesis to maintain the association between the equatorial cortex and the central spindle. We propose that annexin A2 participates in central spindle-equatorial plasma membrane communication.

2.2.3.2. Scientific dissemination and influence

Claude PRIGENT: - Member of CNRS Comité National section 28 : Developmental Biology 2010-2012 - 233

President of ANR committee SVSE2 from 2011 to 2013 - Member of National Scientific Council LNCC from 2011 to 2015 - Representative of the DG CNRS within the Scientific Council and the Board of Director of the Cancéropôle Grand Ouest since 2013 - Representative of the President of University of Rennes 1 at the Board of Director Interne School Biological Station of Roscoff (since 2008) - Scientific director of the MRic core facility (Microscopy Rennes Imaging Center) - Associate Professor at the University Laval of Quebec 2014-2015-2016.

Gwénaël RABUT: Member of the Scientific Council de l’UR1.

David REBOUTIER: Member of the "commission consultative paritaire" (CCP) of the University of Rennes 1.

Erwan WATRIN: Since 2013, member of the médical Council of the French association Cornelia de Lange syndrome (AFSCDL).

2.2.3.3. Interaction with the economic, social and cultural environment

Claude PRIGENT: Member of the Scientific Council of the "Espace des Sciences" in Rennes since 2007.

2.3. Projects, scientific strategies & perspectives (5 years)

Since its creation the Cell Cycle Team is working to understand the function of the mitotic protein kinase Aurora-A and its role in tumorigenesis. Although this is the main project of the lab we are also engaged is other mitotic related projects. Below are the details of our projects.

Aurora-A (Claude PRIGENT, this is the main project of the team in which everybody is participating) Aurora-A is a key cell cycle kinase regulating many events during mitotic progression as well as being involved in carcinogenesis. By taking advantage of an ATP-analogue sensitive isoform of Aurora-A that we prepared, we are in a position to discover new functions of the kinase and to study them. 1 - We found that Aurora-A kinases activity is required to maintain the Spindle Assembly Checkpoint (SAC) active in prometaphase (a manuscript is under revision in JCS). This data was unexpected since many publications already report that overexpression of Aurora-A leads to SAC override (Anand et al., Cancer Cell 2003 Jan;3(1):51-62) (Katayama et al., Cancer Cell 2012 Feb 14;21(2):196-211). Here we found that without Aurora-A activity the checkpoint proteins Mad2 and BubR1 do not stay at kinetochores even in the presence of nocodazol or paclitaxel. How Aurora-A regulates this even remains to be elucidated. As Aurora-A kinase activity is required, its substrates remain to be searched. 2 - We found that Aurora-A kinase activity is required to nucleate MT in the midzone during anaphase (Reboutier et al., J Cell Biol 2013 Apr 1;201(1):65-79). Inhibition of Aurora-A just after metaphase/anaphase transition clearly leads to anaphase B inhibition. The reason for this inhibition is the absence of microtubule nucleation. We have search for substrates to explain the role of Aurora-A in this even and we identified the dynactin subunit p150Glued and the gTuRC anchoring protein NEDD1 (a manuscript is under revision in JCB). Investigation of Aurora-A function and search for substrates during anaphase are still ongoing. David REBOUTIER, researcher in the lab, is in charge of this project. 3 – Because Aurora-A was identified as a tumour suppressor in Drosophila (Wang et al., Genes & Dev 2006 Dec 15;20(24):3453-63) (Lee et al., Genes & Dev 2006 Dec 15;20(24):3464-74) we have decided to investigate its function during asymmetric cell division in collaboration with the team of Roland LE BORGNE. We first identified Numb as a substrate of Aurora-A and report a function of this phosphorylation in cell fate during asymmetric cell division (a manuscript is under revision in Development). To clarify further Aurora-A functions during asymmetric cell division in Drosophila we took advantage of CRISPR Cas9 method to create Aurora-A mutant KI (without kinase activity) and KO (without Aurora-A protein). Its role as tumour suppressor is investigated during neuroblast cell division. Lucie VAUFREY PhD in the lab is in charge of this project. We also took advantage of some of the tools we developed during the set up of the methods to use the ATP-analogue sensitive isoform of Aurora-A. We first built an interactome of Aurora-A that also revealed new functions. We have decided to start two projects based on it. 4 - Several mitochondria proteins have been found in Aurora-A interactome. This was interesting because few years ago, using immuno-EM, we did observe Aurora-A within mitochondrias in Xenopus XL2 cells. Also recently it was reported that Aurora-A phosphorylates the small GTPase RalA to regulate mitochondrial fission (Kashatus et al., Nat Cell Biol 2011 Aug 7;13(9):1108-15). Various reports also 234

indicate that several Aurora-A inhibitors induce apoptosis and autophagy (Drug Des Devel Ther 2015 Jan 14;9:487-508 & Jan 9;9:425-64 & Mar 12;9:1555-84 & Mar 16;9:1627-52). We decided to investigate the link between Aurora-A and the mitochondria in collaboration with Marc TRAMIER and Roland LE BORGNE teams. Giulia BERTOLIN post-doc in Tramier’s lab is in charge of this project. 5 - Several proteins involved in mRNA splicing have been found in Aurora-A interactome. A splice response (defect in RNA splicing) has been observed in cells treated with drugs inducing mitotic arrest in particular drugs or siRNA targeting Aurora-A (Moore et al., Cell 2010 Aug 20;142(4):625-36). We have controlled that the presence of proteins that participate to the splicesome in Aurora-A interactome was not due to the presence of RNA molecules. A function of Aurora-A in the regulation of splicing of specific population of RNA is likely to occur. Together with Dr Juan VALCARCEL from Barcelona we have decided to tackle this function of Aurora-A using a method recently developed in his lab and described in two recent publications in Mol Cell (Papasalkas et al., Mol Cell 2015 Jan 8;57(1):7-22) (Teledor et al., Mol Cell 2015 Jan 8;57(1):23-38). A PhD student financed by the LNCC/Region Bretagne will be recruited soon to work between Rennes and Barcelona. 6 – In order to follow Aurora-A activity in vivo we have developed two different FRET biosensors in collaboration with Olivier GAVET (IGR Paris) and Marc TRAMIER (IGDR). The first one is based on a substrate of the kinase, and phosphorylation of this substrate-biosensor induces a FRET signal that we detect by FLIM, using a Fast-FLIM microscope developed by Marc TRAMIER. The second is a conformational biosensor that is the kinase itself. Activation of the kinase induces a FRET signal. Moreover this last biosensor is an active Aurora-A kinase in vivo, it rescues endogenous Aurora-A depletion. We wish to introduce the ATP-analogue sensitive mutation in this biosensor to be able to manipulate it in vivo. These biosensors will be used to visualise Aurora-A activity in time and space vivo.

MELK (Jean-Pierre TASSAN & Jacek KUBIAK) We have previously shown that the Maternal Embryonic Leucine zipper Kinase (MELK) is involved in cytokinesis of Xenopus embryo epithelial cells. At the anaphase onset, the kinase is massively re-localized at the cell cortex (Le Page et al., J Cell Sci, 2011 Mar 15;124(Pt 6):958-68) (Chartrain et al., Biol Open 2013 Aug 21;2(10):1037-48). The aim of our project is to understand the function of MELK in cytokinesis. Jean-Pierre TASSAN is in charge of this project, he is focusing on the mechanism of MELK re-localization during mitosis and on a search for partners involved in this redistribution. He has also observed that during epithelial cell division in Xenopus embryo ingression progresses asymmetrically, and that this phenomenon depends on the size of the cells. He is studying this mechanism to understand how it is regulated.

Annexin 2 (Christelle BENAUD) Our recent study has highlighted the role of annexin A2 in the central spindle-equatorial plasma membrane communication during early cytokinesis (Benaud et al., EMBO Rep 2015 Apr;16(4):481-9). In the lab Christelle BENAUD is in charge of the project and she is currently addressing the potential role of annexin A2 in anchoring the central spindle and the nascent equatorial cortex and/or in the actin dependent vesicular trafficking between the two.

Chromatid cohesion and cohesinopathies (Erwan WATRIN) In all Eukaryotes cohesin complex mediates sister chromatid cohesion and is therefore essential for faithful transmission of genetic information from one cell or organism to the next. In addition cohesin also acts in DNA damage response, chromatin architecture and gene regulation. Reflecting its involvement in virtually all aspects of chromosome biology, mutations in genes encoding cohesin subunits or regulators are responsible for human diseases: cancers and developmental pathologies, Cornelia de Lange and Roberts syndromes (Deardorff et al., Nature 2012 Sep 13;489(7415):313-7). In the lab, Erwan WATRIN is in charge of this project and he is focusing on understanding at the molecular level how cohesion apparatus functions in normal cells, and how its defects trigger human pathologies.

2.4. Collaboration

2.4.1. Within the IGDR

Marc TRAMIER (Aurora-A biosensors - Aurora-A and mitochondria - Melk biosensors) - Roland LE BORGNE (Aurora-A and Numb - Aurora-A and asymmetric cell division - Aurora-A and mitochondria) - Christian JAULIN (Aurora-A and kinetochore - Chromosome cohesion – CENPA Ubi) - Gilles SALBERT (Genome plasticity) – Raphaël MÉTIVIER (Cohesin & Genome organisation).

235

2.4.2. Other collaborations

Rennes Oliver LAVASTRE, University of Rennes (chemistry) - Sophie JAILLARD, CHU Rennes (Cohesin & Cornelia De Lange).

France Olivier GAVET, IGR, Paris, France (Aurora-A biosensors) - Gaëlle LEGUBE, LBCMCP, Toulouse, France (Cohesins & DNA repair).

Europe Frank KAISER, IHG, Lubeck, Germany (Cohesin & Cornelia De Lange) - Roman KÖRNER, Max Planck, Martinsried, Germany (proteomics) - Michael KNOP, DKFZ-ZMBH Alliance, Heidelberg, Germany (Ubiquitination) - Sebastian LEIDEL Lab, Max Planck, Münster, Germany (Ubiquitination) – Kerstin S. WENDT, Erasmus Medical Center, Rotterdam, Netherland (Cohesin & Cornelia De Lange) - Ewa BORSUK, Warsaw University, Warsaw, Poland – Jacek M. SZYMURA, Jagiellonian University, Cracow, Poland - Z. POLANSKI, Jagiellonian University, Cracow, Poland - Bill EARNSHAW, Welcome Trust, Edinbourg, Scotland, UK (DT40 cells) - David FITZPATRICK, IGMM, Edinbourg, Scotland, UK (Cohesin & Cornelia De Lange) - Thimo KURZ, SCILLS, Dundee, Scotland, UK (Ubiquitination) - Mark PETRONCZKI, CRUK, London Research Institute, Londres, UK (Cohesin regulation) - Marcos MALUMBRES, CNIO, Madrid, Spain (transgenic mice) - Juan VALCARCEL, CRG, Barcelona, Spain (RNA splicing) - Isabelle VERNOS, CRG, Barcelona, Spain (anaphase MT nucleation) - Per LJUNGDAHL, The Wenner-Gren Institute, Stockholm, Sweden (Ubiquitination) - Matthias PETER, IBC, ETH Zürich, Switzerland (Neddylation).

USA Heidi K. BAUMGARTNER Wilson, Univ Colorado, Denver, USA - Raymond DESHAIES, HHMI, Caltech, USA (Protein degradation) - Kendji FUKASAWA, Tampa, Florida, USA (Nuclephosmin) - Malgorzata KLOC, Houston Methodist Research Institute, Houston, Tx, USA - Ichiro NAKANO, The Ohio State University, Columbus, USA - Kevan SHOKAT, UCSF, USA (ATP analog sensitive kinase).

Canada Jean-Philippe GAGNÉ, CHUL, Quebec, Canada (Cohesin & DNA damage) - Arnaud DROIT, CHUL, Quebec, Canada (bioinformatics) - Guy POIRIER, CHUL, Quebec, Canada (proteomics).

Japan Satoshi YOSHITOME, Tottori Univ, Yonago, Japan (cyclin B2 & spindle) - Katsuhiko SHIRAHIGE, Tokyo Univ, Japan (Cohesin régulation).

2.5. SWOT Analysis STRENGTHS Expertise focused on mitosis Several projects insuring various sources of financial support Cell Biology and imaging (microscopy) Original tools to study Aurora-A kinase (ATP analogue sensitive, interactome, biosensors) Multiple model organisms (Yeast, Xenopus, Drosophila, tissue culture) Numerous collaborations that bring expertise (asymmetric cell division, biosensors, RNA splicing, mitochondria, genetics) WEAKNESSES Too many projects? Time consuming administrative charges: direction of the IGDR, direction of MRic OPPORTUNITIES Novelties: - New function of Aurora-A kinase in Splicing, anaphase, mitochondria, asymmetric division - New tools ATP-analogue sensitive Aurora-A, Aurora-A Biosensors - New approaches: Cell biology in an organism : Drosophila

236

THREATS Competition Financial support

237

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

Within the team Division and Cell Polarity (Jean-Pierre TASSAN) until Jan 1st 2012

2010 Badouel C, Chartrain I, Blot J, Tassan JP. Maternal embryonic leucine zipper kinase is stabilized in mitosis by phosphorylation and is partially degraded upon mitotic exit. Exp Cell Res. 2010 Aug 1;316(13):2166-73. (IF 3.372)

2011 Le Page Y, Chartrain I, Badouel C, Tassan JP. A functional analysis of MELK in cell division reveals a transition in the mode of cytokinesis during Xenopus development. J Cell Sci. 2011 Mar 15;124(Pt 6):958-68. (IF 5.325) Tassan JP. Cortical localization of maternal embryonic leucine zipper kinase (MELK) implicated in cytokinesis in early xenopus embryos. Commun Integr Biol. 2011 Jul;4(4):483-5.

Within the team Cell Cycle

2010 Baldini E, Arlot-Bonnemains Y, Mottolese M, Sentinelli S, Antoniani B, Sorrenti S, Salducci M, Comini E, Ulisse S, D'Armiento M Deregulation of Aurora kinase gene expression in human testicular germ cell tumours. Andrologia. 2010 Aug;42(4):260-7. (IF 1.17) Bouter A, Buisine N, Le Grand A, Mouchel N, Chesnel F, Le Goff C, Le Tilly V, Wolff J, Sire O Control of vitellogenin genes expression by sequences derived from transposable elements in rainbow trout. Biochim Biophys Acta. 2010 Aug;1799(8):546-54. (IF 4.6) Courapied S, Cherier J, Vigneron A, Troadec MB, Giraud S, Valo I, Prigent C, Gamelin E, Coqueret O, Barré B. Regulation of the Aurora-A gene following topoisomerase I inhibition: implication of the Myc transcription factor. Mol Cancer. 2010 Aug 3;9:205. (IF 5.397) D'Inca R, Marteil G, Bazile F, Pascal A, Guitton N, Lavigne R, Richard-Parpaillon L, Kubiak JZ. Proteomic screen for potential regulators of M-phase entry and quality of meiotic resumption in Xenopus laevis oocytes. J Proteomics. 2010 Jun 16;73(8):1542-50. (IF 3.929) Ferchichi I, Stambouli N, Marrackchi R, Arlot Y, Prigent C, Fadiel A, Odunsi K, Ben Ammar Elgaaied A, Hamza A. Experimental and computational studies indicate specific binding of pVHL protein to Aurora-A kinase. J Phys Chem B. 2010 Jan 28;114(3):1486-97. (IF 3.377) Gohin M, Bobe J, Chesnel F Comparative transcriptomic analysis of follicle-enclosed oocyte maturational and developmental competence acquisition in two non-mammalian vertebrates. BMC Genomics. 2010 Jan 8;11:18. (IF 3.9) Kubiak JZ. Yes Fyn can: individual or team player in SFKs? Cell Cycle. 2010 Apr 15;9(8):1460-1. (IF 5.006) Marteil G, D'Inca R, Pascal A, Guitton N, Midtun T, Goksøyr A, Richard-Parpaillon L, Kubiak JZ. EP45 accumulates in growing Xenopus laevis oocytes and has oocyte-maturation-enhancing activity involved in oocyte quality. J Cell Sci. 2010 May 15;123(Pt 10):1805-13. (IF 5.325) Romé, P., Montembault, E., Franck, N., Pascal, A., Glover, D. M., and Giet, R. (2010). Aurora A participates to dynactin phoshorylation and function during mitosis. Journal of Cell Biology, 189(4):651-9. (IF 9.7) Romé P, Prigent C, Giet R. [Centrosomes, mitotic spindle and cancer: find the odd one out!]. Med Sci (Paris). 2010 Apr;26(4):377-83. (no IF) Schneider M, Hsiao HH, Will CL , Giet R, Urlaub H & Lührmann R. Human Prp4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation (2010). Nature Structural Molecular Biology, 17(2):216-221. (IF 11.6) Teperek-Tkacz M, Meglicki M, Pasternak M, Kubiak JZ, Borsuk E. Phosphorylation of histone H3 serine 10 in early mouse embryos: active phosphorylation at late S phase and differential effects of ZM447439 on first two embryonic mitoses. Cell Cycle. 2010 Dec 1;9(23):4674-87. (IF 5.006) Ulisse S, Arlot-Bonnemains Y, Baldini E, Morrone S, Carocci S, Di Luigi L, D'Armiento M. Inhibition of the aurora kinases suppresses in vitro NT2-D1 cell growth and tumorigenicity. J Endocrinol. 2010 Feb;204(2):135-42. (IF 3.7)

2011 Amson R, Kubiak JZ, Van Montagu M, Telerman A. Could TCTP contribute to Armin Braun's paradigm of tumor reversion in plants? Cell Cycle. 2011 Jan 1;10(1):1 (IF 5.006)

238

Baldini E, Arlot-Bonnemains Y, Sorrenti S, Mian C, Pelizzo MR, De Antoni E, Palermo S, Morrone S, Barollo S, Nesca A, Moretti CG, D'Armiento M, Ulisse S.Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT. BMC Cancer. 2011 Sep 26;11:411. (IF 3.3) Burschowsky D, Rudolf F, Rabut G, Herrmann T, Peter M, Wider G. Structural analysis of the conserved ubiquitin- binding motifs (UBMs) of the translesion polymerase iota in complex with ubiquitin. J Biol Chem. 2011 Jan 14;286(2):1364-73. (IF 4.6) Diallo A, Prigent C. [The serine/threonine kinases that control cell cycle progression as therapeutic targets]. Bull Cancer. 2011 Nov;98(11):1335-45. (IF 0.635) Dobrzyński M, Bernatowicz P, Kloc M, Kubiak JZ. Evolution of bet-hedging mechanisms in cell cycle and embryo development stimulated by weak linkage of stochastic processes. Results Probl Cell Differ. 2011;53:11-30. (no IF) Franck, N., Montembault, E., Romé, P., Pascal, A., Cremet, J. Y. and Giet, R. (2011). CDK11 is required for centriole duplication and Plk4 targeting to the mitotic centrosome. PLoS ONE, 6(1):e14600. (IF 3.5) Gabillard JC, Ulisse S, Baldini E, Sorrenti S, Cremet JY, Coccaro C, Prigent C, D'Armiento M, Arlot-Bonnemains Y. Aurora-C interacts with and phosphorylates the transforming acidic coiled-coil 1 protein. Biochem Biophys Res Commun. 2011 May 20;408(4):647-53. (IF 2.281) Gohin M, Bodinier P, Fostier A, Chesnel F, Bobe J. Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation. Mol Reprod Dev. 2011 Jul;78(7):510-8. (IF 2.7) Gohin M, Bodinier P, Fostier A, Bobe J, Chesnel F. Aromatase expression in Xenopus oocytes: a three cell-type model for the ovarian estradiol synthesis. J Mol Endocrinol. 2011 Sep 7;47(2):241-50. (IF 3.7) Hoffmann S, Maro B, Kubiak JZ, Polanski Z. A single bivalent efficiently inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I. PLoS One. 2011;6(11):e27143. (IF 3.534) Khan J, Ezan F, Crémet JY, Fautrel A, Gilot D, Lambert M, Benaud C, Troadec MB, Prigent C. Overexpression of active Aurora-C kinase results in cell transformation and tumour formation. PLoS One. 2011;6(10):e26512. (IF 3.534) Kubiak JZ. Proteomics of M-phase entry: 'Omen' vs. 'Omre', the battle for oocyte quality and beyond. Folia Histochem Cytobiol. 2011;49(1):1-7. (IF 1) Kubiak JZ, El Dika M. Canonical and Alternative Pathways in Cyclin-Dependent Kinase 1/Cyclin B Inactivation upon M- Phase Exit in Xenopus laevis Cell-Free Extracts. Enzyme Res. 2011;2011:523420. (no IF) Maciejewska Z, Pascal A, Kubiak JZ, Ciemerych MA. Phosphorylated ERK5/BMK1 transiently accumulates within division spindles in mouse oocytes and preimplantation embryos. Folia Histochem Cytobiol. 2011;49(3):528-34. (IF 1) Moh C, Kubiak JZ, Bajic VP, Zhu X, Smith MA, Lee HG. Cell cycle deregulation in the neurons of Alzheimer's disease. Results Probl Cell Differ. 2011;53:565-76. (no IF) Rabut G, Le Dez G, Verma R, Makhnevych T, Knebel A, Kurz T, Boone C, Deshaies RJ, Peter M. The TFIIH subunit Tfb3 regulates cullin neddylation. Mol Cell. 2011 Aug 5;43(3):488-95. (IF 14.464)

2012 Deardorff MA*, Bando M*, Nakato R*, Watrin E*, Itoh T, Minamino M, Saitoh K, Komata M, Katou Y, Clark D, Cole KE, De Baere E, Decroos C, Di Donato N, Ernst S, Francey LJ, Gyftodimou Y, Hirashima K, Hullings M, Ishikawa Y, Jaulin C, Kaur M, Kiyono T, Lombardi PM, Magnaghi-Jaulin L, Mortier GR, Nozaki N, Petersen MB, Seimiya H, Siu VM, Suzuki Y, Takagaki K, Wilde JJ, Willems PJ, Prigent C, Gillessen-Kaesbach G, Christianson DW, Kaiser FJ, Jackson LG, Hirota T, Krantz ID, Shirahige K. HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle. Nature. 2012 Sep 13;489(7415):313-7. *Equal contribution ((IF 42.351) Eot-Houllier G, Giet R, Fisher D, Kubiak JZ, Prigent C. Le ciblage des kinases cycline-dépendantes (CDK) et des kinases mitotiques Aurora et Polo-like. In Jacques Robert, Eric Raymond, (eds), 2012, Les cibles nucléaires en oncologie., Montrouge, John Libbey Eurotext, 7:50-66. (no IF) Ferchichi I, Arlot Y, Cremet JY, Prigent C and Elgaaied AB. Aurora A kinase interacts with and phosphorylates VHL protein. Biologia 2012:67/5 : 1026-1030. (IF 0.696) Ferchichi I, Kourda N, Sassi S, Romdhane KB, Balatgi S, Cremet JY, Prigent C, Elgaaied AB. Aurora A overexpression and pVHL reduced expression are correlated with a bad kidney cancer prognosis. Dis Markers. 2012;33(6):333-40. (IF 2.174) Jaglarz MK, Bazile F, Laskowska K, Polanski Z, Chesnel F, Borsuk E, Kloc M, Kubiak JZ. Association of TCTP with centrosome and microtubules. Biochem Res Int. 2012;2012:541906. (no IF) Kloc M, Ghobrial RM, Borsuk E, Kubiak JZ. Polarity and asymmetry during mouse oogenesis and oocyte maturation. Results Probl Cell Differ. 2012;55:23-44. (no IF) Kloc M, Kubiak JZ, Ghobrial RM. Translationally controlled tumor-associated protein. Biochem Res Int. 2012;2012:432590. (no IF) Kloc M, Tejpal N, Sidhu J, Ganachari M, Flores-Villanueva P, Jennings NB, Sood AK, Kubiak JZ, Ghobrial RM. Inverse

239

relationship between TCTP/RhoA and p53 /cyclin A/actin expression in ovarian cancer cells. Folia Histochem Cytobiol. 2012 Oct 8;50(3):358-67. (IF 1) Kubiak JZ, Prigent C The centrosome life story in Xenopus laevis. In: Heide SCHATTEN, (ed.), 2012, The centrosome: cell and molecular mechanisms of functions and dysfunctions in disease, Humana Press, 347-364. (no IF) Marteil G, Gagné JP, Borsuk E, Richard-Parpaillon L, Poirier GG, Kubiak JZ. Proteomics reveals a switch in CDK1- associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition. Int J Biochem Cell Biol. 2012 Jan;44(1):53-64. (IF 4.24) Piprek RP, Pecio A, Kubiak JZ, Szymura JM. Differential effects of testosterone and 17β-estradiol on gonadal development in five anuran species. Reproduction. 2012 Aug;144(2):257-67. (IF 3.262) Piprek RP, Pecio A, Kubiak JZ, Szymura JM. Differential effects of busulfan on gonadal development in five divergent anuran species. Reprod Toxicol. 2012 Nov;34(3):393-401. (IF 2.771) Polański Z, Homer H, Kubiak JZ. Cyclin B in mouse oocytes and embryos: importance for human reproduction and aneuploidy. Results Probl Cell Differ. 2012;55:69-91. (no IF) Rabut G. Introduction to the pervasive role of ubiquitin-dependent protein degradation in cell regulation. Semin Cell Dev Biol. 2012 Jul;23(5):481. (IF 5.971) Rannou Y, Salaun P, Benaud C, Khan J, Dutertre S, Giet R, Prigent C. MNK1 kinase activity is required for abscission. J Cell Sci. 2012 Jun 15;125(Pt12):2844-52. + Journal cover image. (IF 5.325) Reboutier D, Prigent C. Aurora kinases. Sangdun Choi. Encyclopedia of Signaling Molecules, 2012:10.1007/978-1- 4419-0461-4_81 (no IF) Reboutier D*, Troadec MB*, Cremet JY, Fukasawa K, Prigent C. Nucleophosmin/B23 activates Aurora A at the centrosome through phosphorylation of serine 89. J Cell Biol. 2012 Apr 2;197(1):19-26. *Equal contribution (IF 9.688) Skelton TS, Tejpal N, Gong Y, Kubiak JZ, Kloc M, Ghobrial RM. Allochimeric molecules and mechanisms in abrogation of cardiac allograft rejection. J Heart Lung Transplant. 2012 Jan;31(1):73-84. (IF 5.611) Yoshitome S, Furuno N, Prigent C, Hashimoto E. The subcellular localization of cyclin B2 is required for bipolar spindle formation during Xenopus oocyte maturation. Biochem Biophys Res Commun. 2012 Jun 15;422(4):770-5. (IF 2.281) Watrin E, Prigent C Sister chromatid cohesion and aneuploidy. In: Suzana Storchova, (ed.), 2012, Aneuploidy in health and disease, Rijeka, InTech, 41-58. (no IF)

2013 Chartrain I, Le Page Y, Hatte G, Körner R, Kubiak JZ, Tassan JP. Cell-cycle dependent localization of MELK and its new partner RACK1 in epithelial versus mesenchyme-like cells in Xenopus embryo. Biol Open. 2013 Aug 21;2(10):1037- 48. (no IF) Ferchichi I, Sassi Hannachi S, Baccar A, Marrakchi Triki R, Cremet JY, Ben Romdhane K, Prigent C, Ben Ammar El Gaaied A. Assessment of Aurora A kinase expression in breast cancer: a tool for early diagnosis? Dis Markers. 2013;34(2):63-9. (IF 2.174) Kubiak JZ. Protein kinase assays for measuring MPF and MAPK activities in mouse and rat oocytes and early embryos. Methods Mol Biol. 2013;957:77-89. (no IF) Kubiak JZ, Ciemerych MA. [From Gurdon to Yamanaka--a brief history of cell reprogramming]. Postepy Biochem. 2013;59(2):124-30. (no IF) Kubiak JZ, Chartrain I, Tassan JP (2013). The Xenopus embryo as a model system to study asymmetric furrowing in vertebrate epithelial cells. In Xenopus Development 2013 (Eds Kloc and Kubiak). John Wiley & Sons, Inc, Oxford. pp 103−111. (no IF) Piprek RP, Pecio A, Laskowska-Kaszub K, Kloc M, Kubiak JZ, Szymura JM. Retinoic acid homeostasis regulates meiotic entry in developing anuran gonads and in Bidder's organ through Raldh2 and Cyp26b1 proteins. Mech Dev. 2013 Nov- Dec;130(11-12):613-27. (IF 2.238) Piprek RP, Pecio A, Laskowska-Kaszub K, Kubiak JZ, Szymura JM. Sexual dimorphism of AMH, DMRT1 and RSPO1 localization in the developing gonads of six anuran species. Int J Dev Biol. 2013;57(11-12):891-5 (IF 2.567). Polanski Z, Kubiak JZ. Free-hand bisection of mouse oocytes and embryos. Methods Mol Biol. 2013;957:255-65. (no IF) Reboutier D*, Troadec MB*, Cremet JY, Chauvin L, Guen V, Salaun P, Prigent C. Aurora A is involved in central spindle assembly through phosphorylation of Ser 19 in P150Glued. J Cell Biol. 2013 Apr 1;201(1):65-79. *Equal contribution + biobytes. (IF 9.688) Zemla A, Thomas Y, Kedziora S, Knebel A, Wood NT, Rabut G, Kurz T. CSNand CAND1-dependent remodelling of the budding yeast SCF complex. Nat Commun. 2013;4:1641. (IF 10.742)

240

2014 El Dika M, Dudka D, Prigent C, Tassan JP, Kloc M & Kubiak JZ. (2015). Control of timing of embryonic M-phase entry and exit is differentially sensitive to CDK1 and PP2A balance. Intl. J. Dev. Biol. 2014;58(10-11-12):767-774 (IF 2.567). El Dika M, Laskowska-Kaszub K, Koryto M, Dudka D, Prigent C, Tassan JP, Kloc M, Polanski Z, Borsuk E, Kubiak JZ. CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition. Dev Biol. 2014 Sep 30. pii: S0012-1606(14)00480-1. (IF 3.637) Khmelinskii A*, Blaszczak E*, Pantazopoulou M, Fischer B, Omnus DJ, Le Dez G, Brossard A, Gunnarsson A, Barry JD, Meurer M, Kirrmaier D, Boone C, Huber W, Rabut G#, Ljungdahl PO#, Knop M#. Protein quality control at the inner nuclear membrane. Nature. 2014 Dec 18;516(7531):410-3. *Equal contribution #Corresponding Authors. (IF 42.351) Kloc M, Kubiak JZ, Li XC, Ghobrial RM. The newly found functions of MTOC in immunological response. J Leukoc Biol. 2014 Mar;95(3):417-30. (IF 4.304) Quintin J, Le Péron C, Palierne G, Bizot M, Cunha S, Sérandour AA, Avner S, Henry C, Percevault F, Belaud-Rotureau MA, Huet S, Watrin E, Eeckhoute J, Legagneux V, Salbert G, Métivier R. Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities. Mol Cell Biol. 2014 Jul;34(13):2418-36. (IF 5.036) Watrin E, Demidova M, Watrin T, Hu Z, Prigent C. Sororin pre-mRNA splicing is required for proper sister chromatid cohesion in human cells. EMBO Rep. 2014 Sep;15(9):948-55. + Journal cover image. (IF 7.858)

2015 Benaud C, Le Dez G, Mironov S, Galli F, Reboutier D, Prigent C. Annexin A2 is required for the early steps of cytokinesis. EMBO Rep. 2015 Apr;16(4):481-9. (IF 7.858) Braunholz D, Obieglo C, Parenti I, Pozojevic J, Eckhold J, Reiz B, Braenne I, Wendt KS, Watrin E, Vodopiutz J, Rieder H, Gillessen-Kaesbach G, Kaiser FJ. Hidden mutations in cornelia de lange syndrome limitations of sanger sequencing in molecular diagnostics. Hum Mutat. 2015 Jan;36(1):26-9. (IF 5.05) Kloc M, Kubiak JZ. (2015). Are Morpholino technology dilemmas an affidavit of structural function of mRNA? Trends in Dev. Biol. [in press] (no IF). Kloc M, Kubiak JZ, Li XC, Ghobrial RM. (2015). Pericytes, microvasular dysfunction and chronic rejection. Transplantation 2015 Apr;99(4):658-67 (IF 3.828). Pacquelet A, Uhart P, Tassan JP, Michaux G (2015). PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division. J Cell Biol, accepted (IF 9.8). Parenti I, Gervasini C, Pozojevic J, Graul-Neumann L, Azzollini J, Braunholz D, Watrin E, Wendt KS, Cereda A, Cittaro D, Gillessen-Kaesbach G, Lazarevic D, Mariani M, Russo S, Werner R, Krawitz P, Larizza L, Selicorni A, Kaiser FJ. Broadening of cohesinopathies: exome sequencing identifies mutations in ANKRD11 in two patients with Cornelia de Lange-overlapping phenotype. Clin Genet. 2015 Feb 4. [Epub ahead of print]. (IF 3.652) Piprek RP, Kloc M, Kubiak JZ (2015). The Bidder’s organ – structure, development and function. Intl. J. Dev. Biol. [in press] (IF 2.567). Piprek RP, Pecio A, Kloc M, Kubiak JZ, Szymura JM. (2015). Evolutionary trend for metamery reduction and gonad shortening in Anurans revealed by comparison of gonad development. Int. J. Dev. Biol., 2015, [ in press ] (IF 2.567).

Edited books 2011 - Cell Cycle in Development, ed. by J. Z. Kubiak, Springer Verlag, Germany, 2011; 500 p., Hardcover. 2012 - Mouse Development: From Oocyte to Stem Cells, ed. by J. Z. Kubiak, Springer Verlag, Germany, 2012 ; 429 p. Hardcover. 2014 - Xenopus Development, ed. by M. Kloc & J. Z. Kubiak, Wiley’s, USA, 2014, 413 p. Hardcover. 2015 - Frontiers Oncology - Molecular and Cellular Oncology - Research Topic: Aurora kinases: classical mitotic roles, non-canonical functions and translational views. Edited by Ignacio Pérez de Castro Insúa, David Glover, Claude Prigent & Mar Carmena, (Chef Editor Giuseppe Giaccone, Georgetown University, USA) EPFL Innovation Park, Building I - CH – 1015 Lausanne - Switzerland

2. Patents (with licence)

Anticorps monoclonal anti-aurora-a, son procédé d’obtention, et ses utilisations dans le diagnostic et le traitement des cancerss – CA2489214A1 – CA2489214C – DE60325725D1 – EP1511771A1 – EP1511771B1 – US7514231 – US20070117163 - 7 licences : SEROTEC, ABCAM, EMD BIOSCIENCE, HYCULT BIOTECHNOLOGY, SIGMA, ZYMED, CELL SIGNALING TECHNOLOGY – Claude PRIGENT & Anne MARTIN.

Protéine Aurora A mutée sensible à un inibiteur - Brevet d’invention # 13 52932 (# de publication 3 003 871) Claude PRIGENT, David REBOUTIER, Marie-Bérengère TROADEC & Patrick SALAUN.

241

3. Conferences (actual team members)

- International 2010 Jacek KUBIAK: Lights and shadows of Xenopus laevis proteomics: Proteomics of M-phase entry and exit. Conference Advanced Bioinformatics Tools, Warsaw, Poland, May 14-16. (invited speaker) Jacek KUBIAK: Novel ubiquitinated mitotic proteins in cell-free extract of Xenopus laevis. International Conference on Analytical Proteomics, Caparica, Portugal, September 7-9 (invited speaker) Claude PRIGENT: Control of mitosis by unexpected protein kinases. PepCon – BIT's 3rd Annual World Protein and Peptide Conference, Beijing International Convention Center, Beijing, China, March 21-23. (invited speaker) 2011 Jacek KUBIAK: Canonical and alternative pathways in Cyclin-Dependent Kinase 1/cyclin B inactivation upon M-phase exit in Xenopus laevis oocytes and embryos. I Symposium: Chosen aspects of gametogenesis and early embryo development, Warsaw, Poland, April 9. (invited speaker) Claude PRIGENT: Aurora-A kinase : Multiple ways for being degraded - Chairman session Proteomics of Protein Degradation & Ubiquitin - BIT Life Science 2ndWorld DNA and Genome Day – World Expo Center, Dalian, China, 24-29 April. (invited speaker) Claude PRIGENT: Co-organisator with B Ducommun of the The “Cell Cycle Cancer & Development“ Conference CCCD 2011. 34th SBCF meeting “French Society of Cellular Biology”. Palais des congrès de Saint Malo (France) in May 25-28 Erwan WATRIN: Cancer Research-UK, Clare Hall Laboratories, London, UK, September (invited by Mark Petronczki) 2012 Claude PRIGENT: Nucleophosmin/B23 activates Aurora-A at the centrosome through phosphorylation of Serine 89. Cell Division: from single molecule mechanics to multicellular organisms. Jacques Monod Conference, Roscoff, France, September 5-9. (selected speaker) Erwan WATRIN: Fondation Rennes I, Rennes France (invited speaker) Erwan WATRIN: Association Française du syndrome de Cornelia de Lange, Paris, septembre (invited speaker) 2013 Jacek KUBIAK: Regulation of M-phase of the cell cycle in Xenopus embryo cell-free extract. Puzzles of Development: from Cell Divisions to Brain Wiring, Zakopane, Poland, May 17-19 (invited speaker). Jacek KUBIAK: Xenopus in laboratory practice. III polish conference PolLasa Animals in laboratory practice, Warsaw, Poland, September 9-11 (invited speaker). Jacek KUBIAK: Xenopus laevis and Xenopus tropicalis as experimental models in biomedical research. Warsaw University, Poland, April 2013 (invited by M. Maleszewski) Jacek KUBIAK: Cell cycle regulation in oocytes and embryos: from cell fusion to cell-free extracts. Mammalian Embryology Conference, Celebrating Pioneering Work of Professor Andrzej K. Tarkowski, Warsaw, Poland, October 25- 26 (invited speaker). Jacek KUBIAK: How frog and mouse embryos may help to understand cancer? Summer School Developmental Biology for Biomedical Research, Warsaw, Poland October 23 (invited speaker). Jacek KUBIAK: Regulation of M-phase of the cell cycle in Xenopus embryo cell-free extract, Jagiellonian University, Cracow, Poland, May 2013, (invited by J. Szymura) Claude PRIGENT: Aurora-A is involved in central spindle assembly through phosphorylation of Ser 19 in p150Glued – 3rd Cell Cycle and Cancer meeting (SBCF) – Montepellier, France, April 2-5. (selected speaker) Claude PRIGENT: A chemical genetic approach to identify new functions of Aurora-A late in mitosis / Une approche chimie génétique pour identifier de nouvelles fonctions de Aurora-A dans les phases tardives de la mitose. Amphithéâtre du CRCEO, L'Hôtel-Dieu de Québec, Quebec city, Quebec, Canada. April 26. (invited by Guy Poirier) 2014 Jacek KUBIAK: Proteomics in cell cycle research. XXth polish conference on Mathematics in Biology and Medicine, Lochow, Poland, September 24 (invited speaker). Jacek KUBIAK: Proteomics in cell cycle research. Computational Molecular Biology, Warsaw, Poland, September 25-27 (invited speaker). Jacek KUBIAK: Mitotic activation of CDK1: from frog development to prostate cancer. 1st conference of the Polish Society for Biomedical Research, Jurata, Poland, May 26-30 (invited speaker) Jacek KUBIAK: The role of CDC6 in mitotic entry, University of Manchester, September, (invited by B. Plusa) Jacek KUBIAK: The role of CDC6 in mitotic entry, EMBL, Heidelberg, Germany, November, (invited by T. Hiiragi). 242

Jacek KUBIAK: Progress in biomedical research, Polska Akademia Umiejetnosci, Cracow, Poland, December 15, (invited by A. Bialas). Claude PRIGENT: Aurora-A kinase in Cancer: detection & inhibition. Chairman session Protein Biomarkers & Cancer Biomarkers. 173rd OMICS Group Conference, 5th International Conference on Biomarkers & Clinical Research. St Hilda’s College – University of Oxford, UK, April 15-17. (invited speaker). Claude PRIGENT: to be or not to be an oncogène or a tumor suppressor. 173rd OMICS Group Conference, 5th International Conference on Biomarkers & Clinical Research. St Hilda’s College – University of Oxford, UK, April 15-17. (Keynote speaker). Claude PRIGENT: Aurora-A kinase activity and cancer. Chairman Molecular Medicine /Oncology. 19th World Congress on Advances in Oncology and 17th International Symposium on Molecular Medicine. Metropolitan Hotel, Athens, Greece, October 9-11 (invited speaker). Gwénaël RABUT: The physiological network of ubiquitin conjugating and ligating enzymes probed by fluorescence complementation in yeast (ID 99). The 1st Proteostasis Meeting, the Centro de Investigación Principe Felipe, Valencia, Spain, November 5-7. (selected speaker). Erwan WATRIN: Institut Curie, UMR3348, Paris, France (invited by Dr Sarah Lambert) Erwan WATRIN: Association Française du syndrome de Cornelia de Lange. Nouan Le Fuzelier, septembre (invited speaker) 2015 Jacek KUBIAK: The role of CDC6 in mitotic entry, University of Birmingham, UK, February, (invited by A. Gambus). Jacek KUBIAK: The role of CDC6 in mitotic entry, University of Southampton, UK, February (invited by K. Jones). Jacek KUBIAK: Xenopus laevis and Xenopus tropicalis as experimental models in biomedical research, Warsaw University, Poland, April, (invited by M. Maleszewski) Jacek KUBIAK: Regulation of embryonic mitoses in Xenopus embryo: role of CDC6 in mitotic entry, Warsaw University, Poland, April (invited by P. Borsuk) Jacek KUBIAK: The role of CDC6 in CDK1 activation and mitotic entry, University of Tel Aviv, Israel, March, (invited by R. Shalgi). Claude PRIGENT: Spindle assembly checkpoint and Aurora-A kinase activity. BIT’s 5th Annual World Congress of Molecular & Cell Biology. Nanjing International Youth Conference Hotel (NIYCH), Nanjing, China, April 25-28 (invited speaker). Claude PRIGENT: Aurora-A kinase activity and cancer : inhibition & detection. Chairman session Protein dynamics, Stability and Signalling Transduction. BIT's 8th Annual World Protein and Peptide Conference. Nanjing International Youth Conference Hotel (NIYCH), Nanjing, China, April 25-28 (invited speaker). Gwénaël RABUT: Microscale Thermophoresis (MST) interactions and beyond. Theoretical and Practical Workshop, Institut Pasteur, Paris, March 3-6 (invited speaker).

- National 2011 Claude PRIGENT: Les inhibiteurs des kinases du cycle cellulaire – Meeting of the Société Française du Cancer. Paris 9 juin. (invited speaker) 2012 Claude PRIGENT: The protein kinase Aurora-C is an oncogene – 14ème Colloque de la Recherche à la Ligue Nationale Contre le Cancer. Centre des Salorges, Nantes, January 26-27. (invited speaker) Claude PRIGENT: Polymorphisms of Aurora-A kinases & cancer – Forum de la Société Française de Toxicologie Génétique. Variabilité Génétique, Génotoxicité et Cancer. Diapason, Rennes. 25 Mai. (Invited speaker) Claude PRIGENT: Nucleophosmin/B23 activates Aurora-A at the centrosome through phosphorylation of Serine 89. CRCNA Nantes 7 juin. (Invited by Dr Sophie Barillé-Nion) Gwénaël RABUT: The TFIIH subunit Tfb3 regulates cullin neddylation - 10eme rencontre Levures Modeles Outils, Toulouse, France April 2-4 (selected speaker) 2013 Claude PRIGENT: Les inhibiteurs des kinases du cycle cellulaire – Meeting of the Société Française du Cancer. Paris, March 22. (Invited speaker) Claude PRIGENT: Aurora-A and anaphase – Institut Curie, CNRS UMR 144. Paris Dec 3rd. (Invidet by Anne Paoletti) Gwénaël RABUT: Institute of Biochemistry, ETH Zurich, Switzerland (invited by Matthias Peter)

243

Gwénaël RABUT: Use of Bimolecular Fluorescence Complementation to probe the physiological network of ubiquitylating enzymes in yeast - International Centre for Genetic Engineering and Biotechnology. Trieste, Italie, November 8th. 2014 Claude PRIGENT: New function of Aurora-A kinase in anaphase – Institut Gustave Roussy, CNRS UMR 8200. March 20. (Invited by Said Aoufouchi) Gwénaël RABUT: Institut Jacques Monod, CNRS UMR 7592, Paris 2015 Gwénaël RABUT: LIOAD UMRS 791 - Nantes

4. Funding

2007-2010 Ligue Nationale Contre le Cancer (label) 251,2 k€ 2008-2011 INCA 90 k€ 2009-2014 Région Nord-Pas de Calais 126,5 k€ 2010 ANR Aurora-A 2010-2014 406 k€ 2010 ANR Kinbiofret 230 k€ 2010 SAD Région Bretagne ProtNEDD (Gwénaël RABUT) 50 k€ 2010 LNCC 35 31 k€ 2010 PEPS CNRS 10 k€ 2010 UEB 31 k€ 2010 UR1 action incitative 30 k€ 2010 ARED Region Bretagne (Emmanuel GALLAUD) 90 k€ 2010 ARC libre (Régis GIET) 144 k€ 2010-2011 Région Bretagne + FEDER 30 k€ 2010-2013 Région Bretagne + FEDER 150 k€ 2010 UR1 defis émergents (Régis GIET) 25 k€ 2011 LNCC 30 k€ 2011 Rennes Metropole – Equipement & fonctionnement (Gwénaël RABUT) 75 k€ 2011 BIOSIT microscopy (Régis GIET) 13 k€ 2011 Institut Fédératif de Recherche 140 - Génétique fonctionnelle, Agronomie et Santé 12,5 k€ 2011-2013 ARC 480 K€ 2011 LNCC (Regis GIET) 25 k€ 2012 INCa, PLBIO-2012 156 k€ 2012 Conseil Régional 1.5 k€ 2013 ARED LNCC/Region Bretagne (Lucie VAUFREY) 90 k€ 2013 ANR JCJC Gwénaël RABUT 2013-2015 200 k€ 2013 AVISAN maladies rares 15 k€ 2013 Equipment Rennes metropole 65 k€ 2013 BIOSIT microscopy (Gwénaël RABUT) 3.7 k€ 2013 ERA-net Maladies Rares (UE/ANR) 239 k€ 2013 UR1 Co-tutelle 1 k€ 2013 UR1 défits émergents 11 k€ 2013 Ligue Nationale Contre le Cancer 25 K€ 2014 Université de Rennes 1 1 k€ 2014 LNCC 4th year PhD Ewa BLASZCZAK (Gwénaël RABUT) 14.7 k€ 2014 AP UR1 (Gwénaël RABUT) 1.5 k€ 2014 CRITT Santé 30 k€ 2014 BIOSIT / Protim Central Spindle proteomics 13 k€ 2014 LNCC label 2014-216 300 k€ 2015 ARED LNCC/Région Bretagne 90 k€ 2015 FRM 4th year PhD Olivia GAVARD 30 k€ 2015 BIOSIT thermophoresis (Gwénaël RABUT) 4 k€ 2015 Université de Rennes 1 1 k€

5. Training Licence 2011 - Madleen CANEAU - L3 University Rennes 1 (6 weeks) 2012 - Thibault VAZEILLE – Licence Pro (9 months) 2013 - Ivan REYES TORRES – L3 Erasmus (Spain) (5 weeks) - Marie LE BOULCH - L3 University Rennes 1 (6 weeks) - Nathan GUIET – - L3 University Rennes 1 (6 weeks) 2014 - Eva LE RUN – - L3 University Rennes 1 (6 weeks) 2015 - Emilie EVEN - L3 University Rennes 1 (6 weeks) - Thomas BLÉVIN - L3 University Rennes 1 (6 weeks) 244

- Audrey LÉJART - L3 University Rennes 1 (6 weeks) - Bénédicte LEFÈVRE - L3 University Rennes 1 (6 weeks) IUT/BTS 2012 - Justine COTURLA - BTS (4 months) 2013 - Julie LEGROS – BTS (4 months) 2014 - Amandine LAVASTRE – IUT (1 month) Master 2010 - Carole DONGMO- M1 University Rennes 1 (2 months) collaboration with Grégoire MICHAUX - Marie-Charlotte DUMARGNE - M1 University Rennes 1 (2 months) - Zheng HU - M2 University Rennes 1 (6 months) 2011 - Ambre-Aurore JOSSELIN - M1 University Rennes 1 (2 months) - Sacha ROBERT - M1 University Rennes 1 (2 months) - Erwan CADIC - M2 University Rennes 1 (6 months) - Thibaud RICHARD - M2 University Rennes 1 (6 months) co-directed by Marc TRAMIER 2012 - Julien COUTIER - M1 University Rennes 1 (2 months) - Camille DABRAWSKI - M1 University Rennes 1 (2 months) - Guillaume HATTE - M1 University Rennes 1 (2 months) - Guillaume CORDIER - M2 University Rennes 1 (6 months) - co-directed by Marc TRAMIER - Julien FOURET - M2 University Rennes 1-ENSSA Toulouse (6 months) - Razan JAMMAL - M2 University Rennes 1 (6 months) - Claudia PADRONE - M2 Erasmus Université Federico di Napoli (It) (3 months) - Emeline PASQUIER - M2 University Rennes 1 (5 months) 2013 - Jordan AUGER - M1 University Rennes 1 (3 months) - Safiétou SANKHE - M1 University Rennes 1 (2 months) - Lisa-Maria WECHSELBERGER – M1 Erasmus Université de Graz (Autriche) (2 months) - Bilal DJAGHOUT - M2 Université 8 mai 1945, Guelma (Algérie) (1 month) - Rossella MIGLIACCIO - M2 Erasmus Université Federico di Napoli (It) (3 months) 2014 - Aodrena LE GALL - M1 University Rennes 1 (2 months) - Aaron BROOKS – M2 Erasmus (8 months) - Federico GALLI – M2 Erasmus Université di Modena e Reggio Emilia (Italy) (6 months) - Genco GIANLUCCA – M2 Erasmus Université di Modena e Reggio Emilia (Italy) (6 months) - Théo LEBEAUPIN - M2 University Rennes 1 (5 months) 2015 - Tristan PERRODIN - M1 University Angers (2 months) - Manuel VLACH - M1 University Rennes 1 (2 months) - Marika FRIANO - M2 Erasmus Université Federico di Napoli (It) (6 months) - Stephan NICOLE - M2 University Rennes 1 (6 months) PhD 17/10/2011: Jabbar KHAN – Dir. Thèse Claude PRIGENT & Marie-Bérengère TROADEC - Currently assistant Professor in Pakistan 30/09/2013: Mohammed EL DIKA – Dir. Thèse Jacek KUBIAK – Currently post-doc in Canada 16/12/2013: Jean-Luc VORNG – Dir. Thèse David REBOUTIER & Olivier LASVASTRE 18/12/2013: Alghassimou DIALLO - Dir. Thèse Claude PRIGENT - Currently assistant Professor in Guinée 26/06/2015: Ewa BLASZCZAK (bourse ministérielle + LNCC) – Dir. Thèse Gwénaël RABUT Since 11/2011: Olivia GAVARD (FRM) – Cotutelle University of Rennes 1 and University Laval Quebec et Co-direction Thèse Claude PRIGENT (UR1), Arnaud DROIT et Guy POIRIER (ULQ) Since 10/2013: - Guillaume HATTE (bourse ministérielle) - Dir. Thèse Jean-Pierre TASSAN - Lucie VAUFREY (LNCC/Région Bretagne) – Dir. Thèse Claude PRIGENT & Roland LE BORGNE Starting 10/2015: - Marie LE BOULCH (bourse ministérielle) – Dir. Thèse Gwénaël RABUT - Arun Prasath DAMORAN (LNCC/Région Bretagne) – Dir. Thèse Claude PRIGENT Post-doctorants 2007-2013: David REBOUTIER, post-doctoral fellow 6 years - Direction Claude PRIGENT - now researcher Univ Rennes 1. 2010-2012: Gaëtan PASCREAU, post-doctoral fellow 2 years 2 months - Direction Claude PRIGENT – now post-doc Univ Rennes 1. 2012-2013: Sergio PADILLA-PARRA, post-doctoral fellow 10 months – Direction Jean-Pierre TASSAN & Marc TRAMIER - now group leader, Oxford University, UK 2013-2014: Maria DEMIDOVA, post-doctoral fellow 1 year & 4,5 months – Direction Erwan WATRIN 2013-2014: Agata CENA, post-doctoral fellow 1 year – Direction Gwénaël RABUT 2014-2016: Yorann BARON, post-doctoral fellow 1 year & 4 months – Direction Erwan WATRIN 2013-2015: Gaëtan HERBOMEL, post-doctoral fellow 2 years & 5 months – Direction Jean-Pierre TASSAN & Marc TRAMIER CDD 2013-2015: Tanguy WATRIN - Engineer CNRS, contract 2 years - Direction Erwan WATRIN 2014-2015: Audrey BROSSARD –Engineer assistant CNRS, contract 1 year & 7 months - Direction Gwénaël RABUT

245

Team 18 "Spatio-temporal regulation of transcription in eukaryotes" Leader: Gilles SALBERT

247

2.1. Team presentation

Our genome is used to build a complex organism made of various tissues, each harbouring unique properties. The phenotypic specialization of our 10,000 billions of cells is partly due to the cell-type specific expression of repertoires of genes which can adapt to specific events during life. Within the nucleus, the genome is organized as chromatin, a nucleoproteic fiber in which DNA is wrapped around histone proteins. This typical organization allows for efficient three dimensional folding as well as genome indexation through specific marks added to histones and DNA. Each cell type is endowed with its own index driving the expression of a characteristic gene repertoire. In order to understand how the transcriptional activity of the genome is regulated, we study the action of specialized proteins called transcription factors which can impact chromatin structure dynamics and indexation. We develop a multi- scale approach, involving microscopy, molecular biology, biochemistry and bioinformatics, to analyse the spatio-temporal dynamics of chromatin architecture.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

BARLOY-HUBLER Frédérique CR1 CNRS AVNER Stéphane IR2 CNRS HUET Sébastien MCU UR1 BIZOT Maud ADT UR1 LE PERON Christine MCU UR1 CHAPUIS Catherine TCN CNRS METIVIER Raphaël CR1 CNRS MADIGOU Thierry IR2 CNRS SALBERT Gilles PU1 UR1 PALIERNE Gaëlle TCN CNRS

2.2.2.2. Temporary staff

LEBEAUPIN Théo PhD student (2014-2017) SELLOU Hafida PhD student (2013-2016) MAHE Elise PhD student (2012-2016)

2.2.3. Achievements

248

2.2.3.1. Scientific achievements

The team focuses on chromatin dynamics at transcriptional enhancers. Enhancers are essential non-coding elements of the genome involved in long-distance cell-specific regulation of gene expression and whose mutation can impact on disease development. These small genomic regions are responsible for the fine tuning of cell-specific gene expression and as such participate in cell type determination and maintenance. Their selection and priming for activation depend on the initial binding of sequence-specific transcription factors often called "pioneer factors" as they prepare enhancers for binding additional transcription factors and subsequent activation of target genes. As an example of one such pioneer factor, FOXA1 binds condensed chromatin and is required for the differentiation of pluripotent cells into neural progenitors as well as for the correct establishment of the estrogen receptor (ER) cistrome in breast cancer cells. To understand the relationship between pioneer factor binding and enhancer priming events, chromatin dynamics can be studied through biochemical analysis (chromatin and/or DNA immunoprecipitation - ChIP and DIP) of cell populations, although single-cell analyses would be required for an in depth comprehension of ongoing events. However, single-cell studies can however be carried out with advanced microscopy techniques, and the development of high resolution microscopy by a physicist from the team allows us to investigate chromatin dynamics at the level of individual nucleosomes.

Pioneer factors are believed to stimulate DNA demethylation and mono- and di-methylation of lysine 4 of histone H3 (H3K4) at enhancers. Active DNA demethylation requires oxidation of 5mC to 5hmC and then to 5fC and 5caC by Ten-eleven translocation (TET) dioxygenases, 5fC and 5caC being cleaved by the Thymine DNA glycosylase (TDG) and replaced by unmodified cytosines by the base excision repair machinery. We have shown that the cell-specific activity of FOXA1 enhancers is inversely correlated to their level of 5mC and demontrated that these enhancers get demethylated upon pioneer factor binding (Sérandour et al., Genome Research 2011). We further showed that enhancers selected during mouse neural progenitor differentiation as well as adipocyte specification gain 5hmC (Sérandour et al., Nucleic Acids Res 2012). This was also observed during human plasmablast differentiation from primary naïve B cells (Caron et al., Cell Reports in revision). Among activated enhancers, a fraction was rapidly demethylated with no trace of 5hmC detectable at steady state, whereas a significant amount of enhancers remained associated with high amounts of 5hmC over time, suggesting a specific function of this mark at these genomic sites (Caron et al., Cell Reports in revision). We further discovered that 5hmC apposition favors monomethylation of H3K4, a mark of enhancer priming (Mahé et al., manuscript in preparation) and chromatin opening. These data point to a possible role of DNA hydroxymethylation in the early events accompanying enhancer selection and possibly in subsequent nucleosome dynamics.

Although 5hmC deposition can also be induced at FOXA1 binding sites in breast cancer cells like MCF7, the extremely low levels of 5mC oxidation in these cells preclude a thorough assessment of its role in the process of ER enhancer priming. In parallel, we have conducted an in depth study of the spatio- temporal events regulating the coordinated response of a cluster of genes targeted by the estrogen receptor in response to estradiol (Quintin et al., Mol Cell Biol 2014). This work demonstrated that a coordinated response is set by the engagement of a number of ER-targeted enhancers distributed along the 1 Mb cluster and which cooperate to activate the genes included in this cluster. In addition, by exploring the impact of disrupting the activity of individual enhancers through the use of triplex forming oligonucleotides, we evidenced both non-redundant and redundant actions of these enhancers coupled with the establishment of transient inter-enhancer contacts (Quintin et al., Mol Cell Biol 2014). In addition to establishing a pioneer function for the forkhead protein FOXA2, we also found that GATA2 can exert such function in the absence of FOXA1 (Quintin et al., manuscript in preparation).

With the aim of acquiring a better description of the chromatin architecture in eukaryotic cells, we developed tools to directly assess the chromatin organization and its dynamics at different spatio- temporal scales. First, we used the incorporation of fluorescent nucleotides into DNA during its replication to visualize chromatin movements in living cells over a broad range of timescales, from tens of milliseconds to minutes. With this approach, we analyzed the impact of molecular crowding for the establishment and maintenance of the heterochromatin foci (Walter et al., J Struct Biol, 2013). In addition, in collaboration with the laboratory of J.M Peters (IMP, Vienna), we also used this tool to demonstrate the crucial role played by the cohesin-associated protein Wapl in the regulation of the chromatin architecture (Tedeschi et al., Nature 2013).

In order to monitor the chromatin dynamics at scales lower than those accessible with the nucleotide labeling, we have established a custom imaging system to track single fluorescently labeled core histones by sptPALM (single particle tracking Photoactivation Localization Microscopy). Using two- photon temporal focusing photoactivation, we could image with an optimal spatio-temporal resolution

249

single fluorophores located several micron deep within the cells. With this setup, we analyzed the subdiffusive motion displayed by nucleosomes in living mammalian cells (Cesbron et al., in preparation).

Using our expertise in the analysis of dynamic processes in living cells, we also collaborated to different projects inside and outside the institute. In particular, we contributed to the study of the role of the protein Ensconsin/Map7 in microtubule dynamics (Gallaud, J Cell Biol, 2014) and we were also involved in the analysis of protein diffusion in casein microgels (Peixoto et al., Langmuir, accepted).

2.2.3.2. Scientific dissemination and influence

Sébastien HUET: member of the executive committee of the national research network GDR3070 "CellTiss".

Raphaël METIVIER : invited speaker Keystone symposium on nuclear receptors (March 2010) - invited speaker 27th Conference of european comparative endocrinologists (August 2014).

Gilles SALBERT : founding member of the “Epigenetic” network of Cancéropole Grand Ouest - founding member of the Fédération Hospitalo-Universitaire (FHU) CaMin (Rennes) - invited speaker Jacques Monod Conference on DNA methylation (September 2013 - Roscoff) - invited speaker Meeting EpiNantes (October 2013) - invited speaker Centre de Recherche en Cancérologie de Toulouse (octobre 2013) - invited speaker Institut du Fer à Moulin, Paris (September 2014).

2.2.3.3. Interaction with the economic, social and cultural environment

Members of the team are regularly engaged in meetings with high school students to explain the many facets of biological research and what are the different associated professions.

2.3. Projects, scientific strategies & perspectives (5 years)

The team will focus on chromatin dynamics at transcriptional enhancers. In addition to “traditional” biochemical approaches to study chromatin dynamics, we plan to develop original methods based on super-resolution microscopy. This will allow us to integrate both biochemical and microscopy data to unravel chromatin organization and dynamics at subsets of transcriptional regulatory regions bound by pioneer factors such as FoxA1 and Pbx1, or the estrogen receptor α (ERα). Specifically, we will focus on (a) the relationship between cytosine hydroxymethylation and nucleosome dynamics at pioneer factor bound enhancers, and (b) the relationship between regulatory events at ERα-bound enhancers and DNA damage and repair.

(a) Nucleosome dynamics and DNA modifications

During neural progenitor cell (NPC) commitment, priming of enhancers associates with cytosine hydroxymethylation (5hmC) in correlation with the engagement of pioneer factors like FoxA1 and the TALE-HD proteins Meis1 and Pbx1. Accordingly, we identified Pbx1-bound chromatin as being highly enriched in 5hmC and, by immunoprecipitation coupled to mass spectrometry (ChIP-MS), to contain components of the chromatin remodeling complexes NuRD (including the histone H3K4 demethylase LSD1) and FACT. NuRD, in conjunction with LSD1, is believed to occupy active enhancers in ES cells to regulate subsequent decommissioning during differentiation. However, LSD1 can also demethylate H3K9 in certain circumstances, although it is not known if it associates with NuRD in this case. Whereas NuRD has the capacity to facilitate nucleosome sliding by lowering histone/DNA interactions, both nucleosome eviction and deposition have been associated with FACT engagement. In order to explore the functional relationship between 5hmC, chromatin remodeling and pioneer factor binding, we will first map genome- wide 5mC/5hmC, nucleosomes and NuRD/FACT distribution at different time points of NPC commitment. Next, we will focus on a number of enhancers selected from the set of hydroxymethylated regions that are bound by pioneer factors and which show chromatin remodeling in association with NuRD/FACT engagement and run individual gene silencing to understand their contribution to enhancer priming. In addition, we will run single-cell analysis of the relationship between 5hmC and nucleosome dynamics through super resolution microscopy. Collectively, this will enable us to address the following specific questions: (i) is NuRD and FACT engagement associated with different chromatin organizations? (ii) is DNA hydroxymethylation important for chromatin remodeling at pioneer factor-bound enhancers? (iii) are chromatin remodelers required for enhancer priming and activation?

250

Work program:

1. Kinetic analysis of chromatin dynamics at pioneer factor-bound enhancers. Here, we will follow chromatin changes at enhancers mobilized during differentiation of P19 embryonal carcinoma cells (ECCs) into neural progenitor cells (NPCs) upon induction by retinoic acid (RA). To this aim, samples of cross-linked cells will be taken at 0, 6, 12, 18, 24 and 48 hrs post-treatment with RA and subjected to ChIP-seq with antibodies against proteins identified by Pbx1 ChIP-MS (i.e. SSRP1 and SPT16 for FACT, HDAC1 and CHD4/Mi2-β for NuRD). Nucleosomes will be mapped by MNase-seq and the obtained data will be confronted to the kinetics of chromatin opening already assessed during differentiation by FAIRE-seq. In addition, we will run single-base resolution mapping of 5mC and 5hmC in undifferentiated cells as well as in cells treated for 48 hrs with RA by Bisulfite (BS)-seq and oxidative-BS (oxBS)-seq, either on whole genomic DNA or on genomic DNA enriched by chromatin immunoprecipitation with an anti-Pbx1 antibody. This part will be done in collaboration with Dr Michaël Weber (Strasbourg) who is mastering techniques for the analysis of DNA methylation at single-base resolution. This will allow to precisely define the dynamics of chromatin remodeling at pioneer factor-bound enhancers and to define their DNA modifications at single-base resolution thus identifying their putative spatio-temporal association with nucleosome (re)positioning/eviction at pioneer factor-bound sites. This set of data will allow to (i) identify the nucleosomal organization of pioneer factor bound enhancers in conjunction with NuRD and FACT engagement, and (ii) analyse the DNA modifications at pioneer factor-bound enhancers at single-base resolution thus identifying a putative association with nucleosome dynamics.

2. Functional analysis of the impact of DNA hydroxymethylation and chromatin remodelers on pioneer factor-bound enhancer activity. In this part, we will focus on a number of enhancers selected from the set of hydroxymethylated regions that are bound by pioneer factors and which show chromatin remodeling in association with NuRD/FACT engagement. Using this subset of regions, we will be able to address the following specific questions: (i) is DNA hydroxymethylation important for chromatin remodeling at these enhancers ? (ii) are chromatin remodelers required for enhancer priming and activation ? To answer these questions, we will analyse chromatin dynamics by ChIP-qPCR for NuRD/FACT subunits, H3K4me1, H3K27ac, as well as MNase- qPCR, NOMe-seq (Nucleosome Occupancy and Methylome-sequencing), FAIRE-qPCR and hMeDIP-qPCR at selected enhancers in cells in which TET expression has been silenced or in cells in which selected subunits of NuRD and FACT have been knocked-down. In addition, we will analyse the presence of ubiquitinated H2BK120 at the selected enhancers since it is known to enhance FACT engagement. Finally, we will analyze the chromatin of selected enhancers in cells either depleted or overexpressing FoxA1, Meis1 and Pbx1. These last experiments will allow us to understand the respective contribution of each pioneer factor to the priming of the selected enhancers. If variations in FACT recruitment correlate with changes in cytosine hydroxymethylation levels in TET siRNA experiments, we will then investigate a putative requirement of 5hmC itself by inhibiting TET catalytic domain by the dioxygenase inhibitor DMOG. Collectively, the obtained data will shed light on the functional relationship between pioneer factor binding, DNA hydroxymethylation and nucleosome (re)positioning/eviction by NuRD and FACT.

3. Single-cell analysis of nucleosome dynamics at hydroxymethylated genomic regions. We will observe, at single-cell resolution, the distribution of nucleosomes with respect to hydroxymethylated cytosines by super-resolution microscopy of fixed samples of cells expressing tagged histones and labeled with 5hmC antibodies. Through double antibody-labeling experiments in the context of cells expressing H2B fused to the red photoactivatable protein PATagRFP, we will be able to focus on pioneer factor bound enhancers regions which are enriched in 5hmC (whereas pioneer factor-bound promoters are not enriched in 5hmC). Since enhancers are known to be associated with H2A.Z (a histone variant giving instability to nucleosomes when combined with the other variant H3.3), we will also run this analysis in cells expressing H2A.Z::PATagRFP. The requirement for NuRD and FACT to the observed nucleosome organization at 5hmC-enriched pioneer factor-bound enhancers will be evaluated through knock-down experiments targeting subunits of these chromatin remodelers. Finally, through the development of a fluorescent 5hmC sensor, we expect to extend this analysis to live samples in order to quantify nucleosome dynamics in hydroxymethylated regions bound by pioneer factors like FoxA1 or Pbx1. To this aim, we will generate a fluorescently-tagged catalytic domain of the 5hmC-glucosyltransferase, fused to a nuclear localization signal. This construct will be transiently expressed in cells expressing H2B::PATagRFP or H2A.Z::PATagRFP as well as tagged pioneer factors.

(b) Relationship between regulatory events at enhancers and DNA damage and repair

Like many other transcription factors (TFs), ERα mostly binds to genomic sites that are distant

251

from its target genes, and promotes enhancer/promoter contacts through the controlled formation of chromatin loops. This three-dimensional organization of ERα cistrome and consequent gene regulations require the cohesin complex. Interestingly this protein complex includes RAD21 which is a partner for proteins involved in DNA repair. RAD21 ChIP-MS analyzes performed in both MCF-7 and MDA::ER cellular models confirmed that RAD21 associates with ERα, and the repair proteins XRCC1, XRCC5, XRCC6 and Topoisomerases. In addition to RAD21 and XRCC1 dynamic association with ERα target gene promoters, histone H2A.X is phosphorylated upon ERα binding and transcription activation by estradiol. Phosphorylation of H2A.X (a major mark of DNA damage signaling) in response to hormone treatment reinforces the notion that E2 induced transcription activation is accompanied by DNA damage, at least locally. To further investigate this, we ran a genome wide characterization of double strand break occurrence (biotin-ChIP assay) in response to estradiol in breast cancer cells and found that DNA breaks peak at ERα binding sites, indicating a strong relationship between ERα engagement at enhancers and DNA damage. By using a fluorescently tagged ERα, we will target ERα-bound genomic sites and study the dynamics of DNA repair proteins by super-resolution microscopy in correlation with nucleosome occupancy. This will allow to gain insight into a possible functional link between transcription factor binding, DNA damage and repair.

Work program:

1. Cytosine modifications, DNA breaks and estrogen signaling in breast cancer cells Cytosine modifications are likely to affect the rate of DNA breaking by modulating DNA repair protein binding. In favor of this hypothesis, DNA break-enriched regions are CpG rich in breast cancer cells and cytosine modifications can be directly targeted by DNA repair proteins in vitro. In this part, we will evaluate the impact of altered levels of cytosine modifications on DNA break occurrence. MCF-7 cells show low levels of 5hmC, reduced Tet1 and Tet2 expression levels compared to normal breast tissue, and hence are ideally suited to study the influence of artificially elevated levels of 5hmC on ER cistrome and ERα- dependent DNA breaks. MCF-7 clones expressing the catalytic domains of Tet1, Tet2 and Tet3 and their catalytically defective mutants Tet1-CD, Tet2-CD and Tet3-CD as controls (vectors obtained from Yi Zhang; Harvard Medical School, Boston) will be used for these analyses. We will also establish MCF-7 clones overexpressing DNMT3a or expressing shRNAs targeting DNMT3a. Clones with altered levels of 5mC or 5hmC will be selected by Dot blot analysis of their genomic DNA. Cytosine modifications will then be mapped genome- wide by MeDIP-seq and hMeDIP-seq in the different MCF-7 cell lines. In order to investigate the relationship between cytosine modifications and the occurrence of DNA breaks, biotin-ChIP assays will next be performed in control and clones of MCF-7 cells treated for 50 min with vehicle or E2. The genome- wide maps of DNA breaks will be confronted to the distribution of ERα and its pioneer factors FOXA1 and PBX1 in order to identify DNA breaks directly correlated with ERα binding events. In parallel, we will run RNA-seq experiments to define the transcriptome of each generated MCF-7 clone. This will allow to test whether the prevalence of DNA breaks specifically associated with exons is correlated to transcription levels. The kinetics of recruitment of actors of DNA repair pathways (TDG, Topoisomerase, PARP1, γH2A.X...) will next be investigated at selected loci by ChIP-qPCR in MCF-7 clones with altered levels of 5mC and 5hmC. This should allow to determine whether cytosine modifications can impact DNA repair processes. In order to assess the relevance of the identified mechanisms in primary cancer cells, we will examine the expression and co-localization of ERα with candidate proteins (defined from the ChIP kinetics) in tissue samples from ERα positive breast tumors. This will be tested by immuno-fluorescence under normal or high-resolution imaging. High resolution imaging will also be used to evaluate the colocalization of these proteins with modified cytosines using specific antibodies.

2. Spatio-temporal dynamics of ERα-dependent DNA repair events. The aim of this part is to precisely characterize the molecular and structural remodeling events affecting chromatin at ERα-dependent DNA breaks in relation to the onset of transcription. These remodeling events involve both the dynamic recruitment of the repair proteins identified in part 1 and the spatial reorganization of chromatin which is often observed in relation to DNA repair processes as well as the modulation of transcription. In order to reach an optimal spatio-temporal resolution, we plan to characterize these processes in single living cells using advanced quantitative microscopy-based approaches.

To gain information about the recruitment of proteins involved in DNA repair at ERα-dependent breaks, we will fuse these proteins with fluorophores (GFP, mCherry, turquoise) and follow their localization by confocal microscopy in transfected MCF-7 cells treated or not by E2 and with normal or altered levels of 5mC and 5hmC. The dynamics of binding of these repair proteins to chromatin will be 252

assessed by FRAP and FCS, microscopy methods for which members of the team have a proven expertise. In order to analyze specifically the behavior of the DNA repair proteins at ERα-dependent breaks, it will be necessary to identify the position of these breaks in the nucleus of imaged cells. One possible approach is to perform this analysis at sites of accumulation of fluorescently tagged ERα proteins, which mark the positions of estrogen responsive elements in chromatin. Our preliminary FCS experiments performed on MCF-7 cells expressing a GFP fusion with NBS1, a protein involved in DNA repair, indeed suggest that NBS1 binds a subset of ERα-positive sites. In order to directly monitor the binding kinetics of the repair proteins at sites of ERα accumulation on chromatin, we will also use our custom-made single molecule imaging system to track the movements of single molecules in living cells. Our preliminary data show that our setup is suitable to follow the motion of both bound and unbound proteins in the nuclei of cultured cells, thus allowing to image the dynamic binding of proteins to chromatin.

2.4. Collaboration

2.4.1. Within the IGDR

Marie-Dominique GALIBERT - Marie-Bérengère TROADEC - Catherine ANDRÉ - Jenny WU - Erwan WATRIN - Vincent LEGAGNEUX - Marc TRAMIER - Régis GIET.

2.4.2. Other collaborations

Rennes Thierry FEST (INSERM, Rennes).

National Jerôme EECKHOUTE (Pasteur, Lille) - Philippe LEFEBVRE (Pasteur, Lille) - Jean-François ARNAL (CNRS, Toulouse) - Kerstin BYSTRICKY (CNRS, Toulouse) - Jérôme MOREAUX (INSERM, Montpellier).

International Inaki MARTIN (CEK, Barcelona) - Jason CARROLL (CRUK, Cambridge) - Gyula TIMINSZKY (Munich) - Karsten WEIS (ETH, Zurich) - Jan ELLENBERG (EMBL, Heidelberg).

2.5. SWOT Analysis STRENGTHS Pluridisciplinary team Diversified approaches Perennial know-how WEAKNESSES Large-scale project Project limited to cell lines OPPORTUNITIES Unique tools Original questions THREATS Insufficient funding Highly competitive field

253

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2010 Oger F, Lecorgne A, Sala E, Nardese V, Demay F, Chevance S, Desravines DC, Aleksandrova N, Le Guével R, Lorenzi S, Beccari AR, Barath P, Hart DJ, Bondon A, Carettoni D, Simonneaux G, Salbert G. Biological and biophysical properties of the histone deacetylase inhibitor suberoylanilide hydroxamic acid are affected by the presence of short alkyl groups on the phenyl ring. J Med Chem. 2010 Mar 11;53(5):1937-50 (IF: 5.48)

2011 Lucchetti-Miganeh C, Goudenège D, Thybert D, Salbert G, Barloy-Hubler F. SORGOdb: Superoxide Reductase curated DataBase. BMC Microbiol. 2011 May 16;11:105 (IF: 2.98) Métivier R. Are 'omics of estrogen receptors defining potential targets for breast cancer treatment? Epigenomics. 2011 Oct;3(5):525-8 (IF: 5.21) Salbert G, Weber M. Tracking genomic hydroxymethylation by the base. Nat Methods. 2011 Dec 28;9(1):45-6 (IF: 25.95) Sérandour AA, Avner S, Percevault F, Demay F, Bizot M, Lucchetti-Miganeh C, Barloy-Hubler F, Brown M, Lupien M, Métivier R, Salbert G*, Eeckhoute J*. Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers. Genome Res. 2011 Apr;21(4):555-65 (*corresponding authors) (IF: 13.85)

2012 Bancaud A., C. Lavelle, S. Huet and J. Ellenberg. 2012 A fractal model for nuclear organization current evidence and biological implications, Nucleic Acids Res. 40(18):8783-92 (IF: 8.81) Berthier A, Oger F, Gheeraert C, Boulahtouf A, Le Guével R, Balaguer P, Staels B, Salbert G, Lefebvre P. The novel antibacterial compound walrycin A induces human PXR transcriptional activity. Toxicol Sci. 2012 May;127(1):225-35 (IF: 4.48) Huet, S.*, I. Fanget*, O. Jouannot, P. Meireles, T. Zeiske, N. Larochette, F. Darchen, Desnos C. 2012. Myrip couples the capture of secretory granules by the actin-rich cell cortex and their attachment to the plasma membrane, J Neurosci. 32:2564-77 (*co-premier auteur) (IF: 6.48) Le Bars H, Bousarghin L, Bonnaure-Mallet M, Jolivet-Gougeon A, Barloy-Hubler F. Complete genome sequence of the strong mutator Salmonella enterica subsp. enterica serotype Heidelberg strain B182. J Bacteriol. 2012 Jul;194(13):3537-8 (IF: 2.69) Nicolas A, Lucchetti-Miganeh C, Yaou RB, Kaplan JC, Chelly J, Leturcq F, Barloy-Hubler F, Le Rumeur E. Assessment of the structural and functional impact of in-frame mutations of the DMD gene, using the tools included in the eDystrophin online database. Orphanet J Rare Dis. 2012 Jul 9;7:45 (IF: 3.96) Sérandour AA, Avner S, Oger F, Bizot M, Percevault F, Lucchetti-Miganeh C, Palierne G, Gheeraert C, Barloy-Hubler F, Péron CL, Madigou T, Durand E, Froguel P, Staels B, Lefebvre P, Métivier R, Eeckhoute J, Salbert G. Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers. Nucleic Acids Res. 2012 Sep 1;40(17):8255-65 (IF: 8.81)

2013 Flajollet S, Rachez C, Ploton M, Schulz C, Gallais R, Métivier R, Pawlak M, Leray A, Issulahi AA, Héliot L, Staels B, Salbert G, Lefebvre P. The elongation complex components BRD4 and MLLT3/AF9 are transcriptional coactivators of nuclear retinoid receptors. PLoS One. 2013 Jun 10;8(6):e64880 (IF: 3.53) Meuric V, Martin B, Guyodo H, Rouillon A, Tamanai-Shacoori Z, Barloy-Hubler F, Bonnaure-Mallet M. Treponema denticola improves adhesive capacities of Porphyromonas gingivalis. Mol Oral Microbiol. 2013 Feb;28(1):40-53 (IF: 2.84) Ratié L, Ware M, Barloy-Hubler F, Romé H, Gicquel I, Dubourg C, David V, Dupé V. Novel genes upregulated when NOTCH signalling is disrupted during hypothalamic development. Neural Dev. 2013 Dec 23;8:25 (IF: 3.37) Tedeschi A, G. Wutz, S. Huet, M. Jaritz, A. Wuensche, E. Schirghuber, I.F. Davidson, W. Tang, D.A. Cisneros, V. Bhaskara, T. Nishiyama, A. Vaziri, A. Wutz, J. Ellenberg, J.M. Peters. 2013 Wapl is an essential regulator of chromatin structure and chromosome segregation. Nature. 501(7468):564-8 (IF: 42.35) Walter, A., C. Chapuis, S. Huet*, J. Ellenberg*. 2013. Nuclear crowding is not sufficient for heterochromatin formation and not required for its maintenance. J Struct. Biol., 184:445-53 (*corresponding authors) (IF: 3.37)

2014 Azzouzi N, Barloy-Hubler F, Galibert F. Inventory of the cichlid olfactory receptor gene repertoires: identification of 254

olfactory genes with more than one coding exon. BMC Genomics. 2014 Jul 11;15:586 (IF: 4.04) Brawand D, Wagner CE, Li YI, Malinsky M, Keller I, Fan S, Simakov O, Ng AY, Lim ZW, Bezault E, Turner-Maier J, Johnson J, Alcazar R, Noh HJ, Russell P, Aken B, Alföldi J, Amemiya C, Azzouzi N, Baroiller JF, Barloy-Hubler F, Berlin A, Bloomquist R, Carleton KL, Conte MA, D'Cotta H, Eshel O, Gaffney L, Galibert F, Gante HF, Gnerre S, Greuter L, Guyon R, Haddad NS, Haerty W, Harris RM, Hofmann HA, Hourlier T, Hulata G, Jaffe DB, Lara M, Lee AP, MacCallum I, Mwaiko S, Nikaido M, Nishihara H, Ozouf-Costaz C, Penman DJ, Przybylski D, Rakotomanga M, Renn SC, Ribeiro FJ, Ron M, Salzburger W, Sanchez-Pulido L, Santos ME, Searle S, Sharpe T, Swofford R, Tan FJ, Williams L, Young S, Yin S, Okada N, Kocher TD, Miska EA, Lander ES, Venkatesh B, Fernald RD, Meyer A, Ponting CP, Streelman JT, Lindblad-Toh K, Seehausen O, Di Palma F. The genomic substrate for adaptive radiation in African cichlid fish. Nature. 2014 Sep 18;513(7518):375-81 (IF: 42.35) Dupin C, Tamanai-Shacoori Z, Ehrmann E, Dupont A, Barloy-Hubler F, Bousarghin L, Bonnaure-Mallet M, Jolivet- Gougeon A. Oral Gram-negative anaerobic bacilli as a reservoir of β-lactam resistance genes facilitating infections with multiresistant bacteria. Int J Antimicrob Agents. 2014 Nov 3. pii: S0924-8579(14)00309-4 (IF: 4.26) Falentin H, Naquin D, Loux V, Barloy-Hubler F, Loubière P, Nouaille S, Lavenier D, Le Bourgeois P, François P, Schrenzel J, Hernandez D, Even S, Le Loir Y. Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis LD61. Genome Announc. 2014 Jan 16;2(1). pii: e01176-13 Gallaud, E., R. Caous, A. Pascal, F. Bazile, J. P. Gagné, S. Huet, G. G. Poirier, D. Chrétien, L. Richard-Parpaillon, R. Giet. 2014 Ensconsin/Map7 promotes microtubule growth during mitosis and is required with Kinesin-1 for centrosome separation during interphase in Drosophila neural stem cells. J. Cell Biol. 204(7):1111-21 (IF: 9.79) Huet, S., C. Lavelle, H. Ranchon, P. Carrivain, J.M. Victor, A. Bancaud. 2014. Relevance and Limitations of Crowding, Fractal, and Polymer Models to Describe Nuclear Architecture, In International Review of Cell and Molecular Biology Book Series. Vol 307, Chap 13, R. Hancock and K. Jeon editors, Elsevier Academic Press. Le Bars H, Bonnaure-Mallet M, Barloy-Hubler F, Jolivet-Gougeon A, Bousarghin L. Strong mutator phenotype drives faster adaptation from growth on glucose to growth on acetate in Salmonella. Microbiology. 2014 Oct;160(Pt 10):2264- 71 (IF: 0.71) Oger F, Dubois-Chevalier J, Gheeraert C, Avner S, Durand E, Froguel P, Salbert G, Staels B, Lefebvre P, Eeckhoute J. Peroxisome proliferator-activated receptor γ regulates genes involved in insulin/insulin-like growth factor signaling and lipid metabolism during adipogenesis through functionally distinct enhancer classes. J Biol Chem. 2014 Jan 10;289(2):708-22 (IF: 4.60) Quintin J, Le Péron C, Palierne G, Bizot M, Cunha S, Sérandour AA, Avner S, Henry C, Percevault F, Belaud-Rotureau MA, Huet S, Watrin E, Eeckhoute J, Legagneux V, Salbert G, Métivier R. Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities. Mol Cell Biol. 2014 Jul;34(13):2418-36 (IF: 5.04)

2015 Peixoto PD, Bouchoux A, Huet S, Madec MN, Thomas D, Floury J, Gésan-Guiziou G. Diffusion and partitioning of macromolecules in casein microgels: evidence for size-dependent attractive interactions in a dense protein system. Langmuir. 2015 Feb 10;31(5):1755-65. (IF : 4.38)

2. Patents (with licence)

N/A

3. Conferences (actual team members)

- International

2010 MÉTIVIER Raphaël: Estrogen Receptor Transcriptional Activity: A Dynamic Integration of Multi-Leveled Information. Keystone Symposia on Nuclear Receptors: Signaling, Gene Regulation and Cancer. Keystone, Colorado, USA, March 2010 (Invited speakers) SÉRANDOUR A, AVNER S, PERCEVAULT F, DEMAY F, BIZOT M, LUCHETTI-MIGANEH C, BARLOY-HUBLER F, BROWN M, LUPIEN M, MÉTIVIER R, SALBERT G, EECKHOUTE J. Epigenetic switch involved in activation of pioneer factor FOXA1- dependent enhancers. 9th EMBL conference « Transcription and Chromatin », Heidelberg, August 2010 (Poster) QUINTIN J, CUNHA S, SÉRANDOUR A, LEGAGNEUX V, PALIERNE G, PASTEZEUR S, LE PERON C, BIZOT M, AVNER S, DEMAY F, EECKHOUTE J, SALBERT G, MÉTIVIER R. Spatio-temporal controls of the estrogen-responsive TFF gene cluster activity. 9th EMBL conference « Transcription and Chromatin », Heidelberg, August 2010 (Poster)

2011 HUET Sébastien: International Workshop on “Exploration and Search”, Cargèse, France, April 2011 (Invited speakers)

255

SÉRANDOUR A, AVNER S, PERCEVAULT F, DEMAY F, BIZOT M, LUCHETTI-MIGANEH C, BARLOY-HUBLER F, BROWN M, LUPIEN M, MÉTIVIER R, SALBERT G, EECKHOUTE J. Epigenetic switch involved in activation of pioneer factor FOXA1- dependent enhancers. EMBO conference « Chromatin and Epigenetics », Heidelberg, June 2011 (Poster) QUINTIN J, CUNHA S, SÉRANDOUR A, LEGAGNEUX V, PALIERNE G, LE PÉRON C, DEMAY F, BIZOT M, WATRIN E, EECKOUTE G, SALBERT G, METIVIER R. Spatio−temporal regulations of an estrogen−dependent gene cluster. EMBO Conference on nuclear receptor. Sitges/Barcelona, Spain, September 2011 (Poster)

2012 SÉRANDOUR A, AVNER S, OGER F, BIZOT M, PERCEVAULT F, PALIERNE G, GHEERAERT C, LE PÉRON C, MADIGOU T, LEFEBVRE P, MÉTIVIER R, EECKHOUTE J, SALBERT G. Dynamic hyrdoxymethylation of DNA marks differentiation- associated enhancers. 10th EMBL conference « Transcription and Chromatin ». Heidelberg, August 25th-28th 2012 (Poster)

2013 SALBERT Gilles: Hydroxymethylation of enhancers: what for? Jacques Monod Conference on DNA methylation and demethylation, Roscoff, September 14th-18th 2013 (Invited speakers) Mahé E, Sérandour A, Bizot M, Avner S, Le Péron C, Palierne G, Madigou T, Métivier R, Salbert G. Influence of cytosine state on binding of homeodomain-containing proteins to DNA. Jacques Monod Conference on DNA methylation and demethylation, Roscoff, September 14th-18th 2013 (Poster)

2014 MÉTIVIER R. Genomic organization of estrogen receptor signalling. 27th Conference of european comparative endocrinologists. Rennes, France, August 2014 (Invited speakers) MAHÉ E, SÉRANDOUR A, BIZOT M, AVNER S, LE PÉRON C, PALIERNE G, MADIGOU T, MÉTIVIER R, SALBERT G. Understanding the role of 5-hydroxymethylcytosine at active enhancers during cell differentiation. 11th EMBL conference « Transcription and Chromatin », Heidelberg, August 2014 (Poster)

2015 MAHÉ E, SÉRANDOUR A, BIZOT M, AVNER S, LE PÉRON C, PALIERNE G, MADIGOU T, MÉTIVIER R, SALBERT G. Understanding the role of 5-hydroxymethylcytosine at active enhancers during cell differentiation. EMBO conference « Chromatin and Epigenetics », Heidelberg, May 2015 (Poster)

- National

2012 HUET Sébastien: Annual meeting of the national research group on functional imaging in biology GDR 2588 “MIFOBIO”, Talmont Saint Hilaire, September 2012 (Invited speakers)

2013 SALBERT Gilles: DNA hydroxymethylation of transcriptional enhancers during cell differentiation. EpiNantes, Nantes, October 15th-16th 2013 (Invited speakers)

4. Funding

2009-2011 Label “Equipe Ligue Nationale Contre le Cancer” (coordinator G. SALBERT) 207 k€ 2009-2012 Région Bretagne 186 K€ 2010-2012 - ANR Blanc - Project “Gorgones” (coordinator G. SALBERT) 333 k€ 2010-2015 Université de Rennes 1 50 K€ 2011 Institut Fédératif de Recherche 140 - Génétique fonctionnelle, Agronomie et Santé 5 K€ 2012 Rennes Métropole (coordinator S. HUET) 40 k€ 2012-2013 Région Bretagne (coordinator S. HUET) 50 k€ 2012-2015 ANR Young Investigators (coordinator S. HUET) 234 k€ 2012-2015 EU FP7 Carrier Integration Grant (coordinator S. HUET) 100 k€ 2013 GIS IBISA (coordinator S. HUET) 150 k€ 2013 BIOSIT - Structure fédérative de recherche en biologie santé 15 K€ 2013-2014 Project ARC (coordinateur R. MÉTIVIER) 50 k€ 2013-2014 Campus France/Partenariat Hubert Curien (coordinator S. HUET) 17 k€ 2014 Université Rennes 1 (coordinator S. HUET) 10 k€ 2014-2017 Région Bretagne - ARED IGDR 90 K€ 2015 Project Ligue Contre le Cancer région Grand Ouest (coordinator G. SALBERT) 35 k€ 2015 Cancéropole Grand Ouest (coordinator G. SALBERT) 25 k€ 2015 Université de Rennes 1 1,5 K€

256

5. Training Teaching Three researchers of the team have teaching duties (192 hours per year) at the University. Licence 2012 - Pierre LE GARS - L2 University Rennes 1 (1 week) - Morgane BOURDOULOUS - L3 University Rennes 1 (6 weeks) 2013 - Delphine DIARD - L3 University Rennes 1 (6 weeks) 2014 - Marine QUEFFEULOU – L2 University Rennes 1 (3 weeks) IUT/BTS 2012 - Floriane ALMIRE- DUT (1 week) Master 2011 - Lyndia LAMRI - M1 University Rennes 1 (5,5 months) - Kasimir LERAY - M1 University Rennes 1 (2 months) - Lydia KASDI - M2 (3 months) 2012 - Lucille CHASSE - M1 University Rennes 1 (2 months) - Julien ERABIT - M1 University Rennes 1 (2 months) - Martha BAYDOUN - M2 University Rennes 1 (8 months) - Elise MAHE- M2 University Rennes 1 (5 months) 2013 - Quentin AMOSSE - M1 University Rennes 1 (2 months) - Solenne FARELLY - M1 University Rennes 1 (2 months) 2014 - Erwan BODIOU - M1 University Rennes 1 (2 months) - Pauline SCOTTO DI VETTIMO - M1 University Rennes 1 (2 months) 2015 - François DESCAVES - M1 University Rennes 1 (2 months) - Salifou LAM - M1 University Rennes 1 (2 months) PhD 15/02/2011: David GOUDENEGE – Dir. Thèse Frédérique HUBLER 14/10/2011: Aurélien SERANDOUR – Dir. Thèse Gilles SALBERT 23/10/2012: Aurelie NICOLAS – Dir. Thèse Elisabeth LE RUMEUR & Frédérique HUBLER 06/03/2013: Justine QUINTIN – Dir. Thèse Raphaël METIVIER Since 10/2012: Elise MAHE – Dir. Thèse Gilles SALBERT & Raphaël METIVIER Since 10/2013: Hafida SELLOU – Dir. Thèse Sébastien HUET Since 10/2014: Théo LEBEAUPIN – Dir. Thèse Sébastien HUET Post-doctorant Since 10/2013 2012-2014: Yann CESBRON, post-doctoral fellow 2 years & 6 months CDD 2012-2013: – Martha BAYDOUN, Engineer CNRS, contract 6 months

257

Team 19 "Quantitative fluorescence microscopy" Leader: Marc TRAMIER

259

2.1. Team presentation

Directly connected to imaging facilities MRic-photonics (https://microscopie.univ- rennes1.fr/) and in close collaboration with biologists of Rennes, the team "Quantitative Fluorescence Microscopy" aims at developing techniques and methodologies in fluorescence microscopy to study dynamics of protein-protein interactions and biochemical activities in live sample. Our approach is mainly driven by methodological and technological development, its transfer and its applicability in biology to answer relevant new questions.

Fluorescence based methods encompass different disciplines and a vast number of technical approaches. Advances in different photonic imaging techniques and the development of fluorescent probes, and particularly fluorescent proteins, have raised fluorescence microscopy to the level of in the field of biology.

The project is built following three axes: (i) technological development in fluorescence microscopy, (ii) methodological development using fluorescence microscopy, and (iii) biological applications of fluorescence original methods. The objective is double: (i) the transfer of these methodologies to imaging facility MRic (IBiSA Plateform) and eventually to valorize the development by industrial transfer, and (ii) the use of these approaches in collaboration with other teams which would lead to new results of high quality in cellular and developmental biology.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

TRAMIER Marc IRHC CNRS

2.2.2.2. Temporary staff

BERTOLIN Giulia Post-doc DÉMÉAUTIS Claire PhD student (2013/2016) HERBOMEL Gaetan Post-doc SIZAINE Florian PhD student (2015/2018) BOUCHAREB Otmane IR SATT Ouest Valo.

260

2.2.3. Achievements

2.2.3.1. Scientific achievements

fastFLIM prototype - with Julien ROUL (in collaboration with Jacques PECREAUX, IGDR) IBiSA 2011 250 k€ - Rennes Métropole 50 k€ - Région Bretagne 50 k€ This microscope is based on a super-continuum laser, a spinning disk microscope and a time gated intensifier and permits to acquire several FLIM images/s. The work resulted in a software depot (IDDN.FR.001.140028.000.SP 2012.000.31235), a patent (14/52766) and a publication (Leray et al., PlosOne, 2013). The continuation of this project is the maturation POMM project (see research plane) which is supported by SATT valorization office. The prototype is now transferred to MRic imaging facilities and directly available to any users of the platform.

Temporal focusing - with Julien ROUL (in collaboration with Sébastien HUET, IGDR) IBiSA 2014 150 k€ On the PALM system of Sébastien HUET, we have implemented the temporal focusing method with an IR femtosecond laser to be able to acquire PALM images in thick samples. The system is now fully operational and experiments are carried out by Sébastien HUET to decipher the chromatin dynamics in the nucleus of human cancer cells.

Two colour FLCS - with Sergio PADILLA-PARRA (in collaboration with Maïté COPPEY, IJM) Dual colour FCS is a powerful method to monitor protein-protein interactions in living cells. But, when performing these experiments, the spectral bleed-through produces an artefact that corrupts the cross-correlation data. Fluorescence Lifetime Correlation Spectroscopy (FLCS) applied to dual colour cross-correlation allows the determination of protein-protein interactions in living cells without the spectral bleed through artefact. We have implemented this methodology on a commercial setup (SP8 SMD from Leica) of the imaging facilities and recently a protocol was published (Padilla-Parra et al., Methods Mol Biol, 2014). The method is now available through MRic imaging facilities.

Multiplex FRET - with Claire DEMEAUTIS and Sergio PADILLA-PARRA (in collaboration with Franck RIQUET, ) ANR KinBioFRET 410 k€ - ARED PhD 45 k€ A very exciting challenge is to follow several FRET biosensors in the same time on the same sample. But the multiplex approach suffers two limitations: (i) a spectral bleed through of the first acceptor in the second donor emission band and (ii) the multiple excitation wavelengths which necessitates sequential acquisition. We have developed a method dealing with these different limitations. Taking advantage of the long stoke shift of LSSmOrange, we have used 440 nm single excitation wavelength of the two donor mTFP1 and LSSmOrange and a dual color FLIM to measure simultaneously two genetically encoded FRET biosensors. Moreover, taking advantage of the non-fluorescent acceptor sREACh for mTFP1 and of red-shifted mKate2 for LSSmOrange, we were able to neglect any spectral bleed trough. These acquisitions were carried out on the fastFLIM prototype allowing to measure sequences of biosensor measurements at frequency up to 1Hz. We have applied this methodology to follow the simultaneous activation of ERK and PKA by using modified EKAR2G and AKAR4 biosensors with mTFP1/sREACh and LSSmOrange/mKate2 fluorescent protein pairs. A manuscript is about to be submitted.

Tension sensor measurements - with Sergio PADILLA-PARRA and Gaëtan HERBOMEL (in collaboration with Jean-Pierre TASSAN, IGDR) ANR KinBioFRET 410 k€ Organization and maintenance of tissues are regulated by mechanical forces inside and between cells, but only few quantitative data are available in-vivo. Following spatiotemporal variation of tension at cell-cell junction during xenopus embryo development is a real challenge to understand the role of mechanical forces in formation and homeostasis of tissues. During the last few years, new FRET biosensors have been developed that allow to directly measure mechanichal forces in living cells, using a tension sensor module inserted between two fluorescent proteins (Grashoff, et al., Nature, 2010). We focused on the Cadherin FRET biosensor developed by Nicolas BORGHI (Borghi, et al., PNAS, 2012). Cadherin is localized at the apical pole of the cell and make a direct link between neighboring cells and the actin cytoskeleton via protein partner such α and β Catenin, making it the best candidate to study mechanical tension at the cell-cell junction. Using the fastFLIM prototype developed in the lab with Cadherin tension sensor, we are able to measure mechanical forces, with a picoNewton sensitivity, at cell-cell junctions in developing xenopus embryo. Our results suggest that at the end of blastula stage, forces applied to cadherin are quite stable over time with a tension of approximately 3pN, and do not changes during cell division. A manuscript is in preparation.

261

2.2.3.2. Scientific dissemination and influence

Local dissemination through internal collaboration (J. PECREAUX, S. HUET, R. LE BORGNE, Y. AUDIC, C. PRIGENT, G. RABUT) and through MRic Imaging facilities (technological and methodological transfer).

Regional dissemination through the participation of BioGenOuest (Marc TRAMIER, coordinator axe BioImagerie).

National dissemination through the participation of RTmfm (Marc TRAMIER, member of steering committee), of GDR MIV, and through the active participation of MiFoBio CNRS thematic school (Marc TRAMIER, organization committee and organization of practicals).

National and international recognition for the expertise of the team in F-techniques (FRET-FLIM- FCS).

2.2.3.3. Interaction with the economic, social and cultural environment

Industrial collaboration with Photonlines "Quantitative study by multivariate statistical analysis of the effects of toxicity and bleaching parameters of fluorescence microscopy illumination. Application to development of a light source optimized for fluorescence microscopy". The project has not produced the expected results.

Several contacts through NDA with Photonlines, Lambert Instrument, Leukos for valorization and transfer (supercontinuum laser wavelength selector and fastFLIM prototype).

Maturation project POMM "Pilotage Optimal en Microscopie Multidimensionnelle" (SATT Ouest Valorisation maturation project 120 k€). This project is a continuation of the fastFLIM development and aims at pushing industrial transfer of both: (i) wavelength selector for supercontinuum laser (discussion with Leukos), (ii) fastFLIM prototype (discussion with Photonlines), and (iii) a new electronic device to optimize the acquisition speed of fluorescence microscopes (in development).

2.3. Projects, scientific strategies & perspectives (5 years)

Our research plan is a continuation of projects initiated by the team the last 4 years. After a period of setting up of the team, the next step will be clearly dedicated to the transfer of advanced methods through valorization and collaboration with biology teams. The success of this period will be measured by original biological results coming from the use of these techniques and methods and by the finalization of industrial transfer projects. In the same time, new technological and methodological projects will be initiated.

Technological developments:

1- POMM project (SATT maturation project funded 120 k€) - Otmane BOUCHAREB in collaboration with Jacques PECREAUX (IGDR)

This project is a continuation of the fastFLIM development and aims at pushing industrial transfer of both: (i) wavelength selector for supercontinuum laser (advanced contact with Leukos), (ii) fastFLIM prototype (discussion with Photonlines), and (iii) a new electronic device to optimize the acquisition speed of fluorescence microscopes. For the last development, the project is on-going. Through its minimally invasive nature, the development of endogenous fluorescent probes, light microscopy has become an indispensable tool in biological research. In particular, to observe the living, a major issue of microscopy is to quickly follow the observed dynamic events. It is therefore to optimize the speed of acquisition of multidimensional pictures (time, z, color, position of the microscope plate...). The project proposes to develop a multidirectional communication module device. It rests on three pillars: (i) the filing of a patent from an upstream academic research, (ii) knowledge of the microscopy market and advanced contact with a potential partner, and (iii) the definition of a product that would fit on any type of microscopy systems to optimize its steering speed. After this project maturation, we will be able to accurately assess the potential of the product for the transfer to the industrial world.

262

2- stedFLIM (ANR OHRisque, asked grant) – Marc TRAMIER

Different methods are available for FLIM. Until now, different systems are commercially available such as the TCSPC (Time Correlated Single Photon Counting) under confocal microscope (Becker and Hickl, PicoQuant), the single photon counting in wide field mode (Europhoton), the registration of single photons in confocal (LIMO Nikon), the streak camera (Hamamatsu StreakFLIM), gated intensifiers for time domain (Kentech instrument, Lavision), modulated intensifiers for frequency domain (LIFA Lambert Instrument). The team also developed recently a fastFLIM prototype based on a super-continuum laser, a spinning disk microscope and a time gated intensifier (Leray et al., PlosOne, 2013). But for all of these approaches, a time correlated detection is required which constitutes some difficulties in the dissemination of the FLIM method because of the cost or the implementation of these methods.

We propose here a completely new method using the process of stimulated emission to get rid of time correlated detection. In the literature, there are two examples where the stimulated emission is used to measure the FLIM. In 1995 C.Y. DONG in the team of E. GRATTON developed a FLIM system based on modulated excitation and asynchronous modulated stimulated emission, retrieving lifetime information from the cross correlation signal (Dong et al., Biophys J, 1995). But in this case, the detection has to be time correlated to measure and quantify the signal. In 2012, the team of F.J. KAO uses pulsed excitation and also a pulsed stimulated emission where the intensity of fluorescence was measured in the stimulated emission signal rebuilding the fluorescence decay sequentially (Dellwig et al., J Biomed Opt, 2012). In this case again, this technique requires a synchronous detection since the part of the fluorescence signal in the transmission signal at the stimulated emission wavelength is very low (about 10-6).

Our proposed new method is to resume the use of stimulated emission to quench the fluorescence in a temporally controlled manner so as to measure the fluorescence lifetime. And in our case, we design the method to get rid of time correlated detection. The diagram of principle is shown in Figure 1. Fluorescence is produced by a pulsed excitation and subsequently quenched by a pulsed stimulated emission with a controlled delay. If we measure the conventional fluorescence emission without any time correlation, it comes only from the portion of the fluorescence decay between the two pulses of excitation and stimulated emission. By sequentially acquiring the fluorescence intensity for different delays, we find the fluorescence decay profile by direct subtraction. The proposed scheme thus allows characterizing the fluorescence lifetime only by measuring steady state fluorescence intensities. This approach permits to use a conventional detection without temporal correlation.

Figure 1: Schematic diagram of the measurement of the fluorescence decay by pulsed excitation and delayed stimulated emission.

Methodological developments:

1- New modalities to measure FRET (ANR KinBioFRET 410k€ - ARED PhD 45k€) – Claire DEMEAUTIS in collaboration with Sébastien HUET (IGDR)

FRET measurements necessitate a sufficient level of expression for quantitative acquisition of fluorescent signals whatever the methodology used (ratio or FLIM). This can be a problem since genetically encoded FRET biosensor studies would be carried out at the physiological level of expression. In this case, we propose to use two colour FCS to be able to measure FRET. In the case of co-diffusion as it is the case for intramolecular FRET biosensors, the level of cross-correlation amplitude from the two colour signals depends directly from the level of FRET between the two colour fluorescent proteins. Then we will be able to monitor FRET by 2c-FCS using the SP8 SMD microscope available through MRic facilities. We will test our methodology by using tandem models with different levels of FRET and we will try to follow a biosensor activation such as AKAR or EKAR biosensors directly in cells where the signal is too low for conventional FRET measurement and adapted for FCS measurement. This methodology will be used to reconsider FRET activation of biosensors at the physiological level (where the sensed biochemical activity is not saturated by overexpression of biosensors).

263

2- Development of a two FRET biosensing (ANR MEEN asked grant) – Marc TRAMIER in collaboration with Claude PRIGENT (IGDR) and Olivier GAVET (IGR)

Several numbers of AurkA regulatory partners have been identified, what is the physiological function associate to each activator is not well understood in the frame of spatial and temporal functions of the kinase activity. We propose to investigate this question by using simultaneously our AurkA conformational-based FRET biosensor (see biological application for preliminary results) with a conventional study of protein-protein interaction by FRET. Using this method we will be able to specifically follow the activity of AurkA associated to a specific activator. Taking advantage that the change in the FRET signal from GFP to mCherry of the conformational biosensor corresponds to its activation, we should be able to follow in the same time another FRET signal from mCherry (acceptor of the first FRET fused to AurkA) to far-red fluorescent proteins fused to the different regulatory partners of AurkA (TPX2, Abuja, CEP192, nucleophosmin and others). New far-red fluorescent proteins are now available such as IFP 2.0 (Yu et al., Nat Commun. 2014) or IRFP670 (Shcherbakova et al Nat Methods. 2013). By sequentially measuring FLIM images of GFP and mCherry, GFP donor lifetime will monitor the conformational change of AurkA and mCherry donor lifetime will monitor AurkA-regulator interaction (Figure 2).

Figure 2: Scheme of the two FRET biosensor. By using sequentially GFP/mCherry FRET pair and mCherry/far- red FP FRET pair, we should be able to follow conformational change and protein interaction with one of the AuroraA partner described as co-activator.

3- AutoNET project (European project FET-Open, asked grant) – Marc TRAMIER coordinator in collaboration with Paul FRENCH (Imperial College), Sergio PADILLA-PARRA (Oxford University), Juan LLOPIS (Albacete University), Franck RIQUET (Ghent University), Chiara ZURZOLO (Pasteur Institute) and two companies Leukos (FR) and Kentech Instrument (UK).

The AutoNET project aims to establish a new radically different approach to interrogate cell signalling networks in a physiologically realistic context to enable screening of complex signalling networks for the systematic study of perturbations on a genomic scale. This will be realised by building a high content analysis platform for automated “single-shot” assays of multiple cell signalling readouts in 3- D cell cultures. Multiplexed fluorescence-based readouts of biomolecular processes will be realised using Förster resonant energy transfer (FRET) based biosensors. We will exploit the power of automated imaging technology and develop analysis tools to probe a specific set of signalling pathways acquiring 100’s of parallel image-based readouts from a single multi-well plate. Cells will be labelled in each well with different subsets of the designed biosensors, thus generating a panel of stable cell lines expressing the various combinations of biosensors. We will design labelling strategies to optimise multiplexed readouts using spectrally efficient FRET probes with constructs utilising homo- and hetero-FRET with dark acceptors. FRET readouts will be complemented by 2-colour correlation imaging readouts to provide a robust method for protein interactions. Arrays will be prepared in 3-D cell cultures to provide a more physiologically realistic context than conventional cell-based assays. To evaluate the efficacy of this approach and refine the technology, we will use the Epithelial-Mesenchymal Transition (EMT) signalling network and its responses to a kinase inhibitor library as a proof-of-principle study.

The AutoNET technologies and methodology would thus set the foundation for a new approach to address systems biology, drug discovery and personalised medicine. This long-term vision can only be achieved by the synergistic development of innovative tools in optoelectronics and cell biology, integrated into a coherent multidisciplinary analysis workflow.

In this ambitious project, Marc TRAMIER will be coordinator and the team will be mainly involved in the development of 2-color image correlation spectroscopy for protein-protein interaction and automation pipeline.

Biological application:

1- Spatiotemporal regulation of Aurora A activity using a new conformational sensor (ANR KinBioFRET 410 k€, ARED PhD 90 k€, ANR MEEN Asked grant) – Gaëtan HERBOMEL, Giulia BERTOLIN, Florian SIZAINE (in collaboration with Claude PRIGENT, IGDR). 264

It has been well established that activation of AurkA kinase depends on its autophosphorylation onto specific amino acid residues, such as Thr288. Thr288 phosphorylation is associated with a conformational change of the protein kinase (Bayliss et al., Mol Cell, 2003). We recently generated a conformational FRET biosensor in which the whole AurkA kinase is sandwiched with a fluorescent protein donor GFP and acceptor mCherry at the NH2- and COOH-terminal part, respectively. Few conformational biosensors have been developed so far, including the ones for MELK (Le Page et al., J Cell Sci. 2011), PKC gamma (Verbeek et al., J Cell Sci.2008,) and PTEN phosphatase (Lima-Fernandes et al., Nat Commun. 2014). By measuring the fluorescence lifetime of the donor alone (control GFP-AurkA) in comparison to the GFP lifetime for the biosensor (GFP-AurkA-mCherry), we were able to measure FRET activity. Indeed, when FRET occurs, the donor lifetime is significantly decreased. Quantitative FRET measurements were carried out by using the fastFLIM prototype developed by Marc TRAMIER’s team (Leray et al., PLoS One, 2013). Validation experiments of the AurkA conformational biosensor are presented in Fig3.

Fig3: Validation of AurkA conformational biosensor. A: In vitro deactivation of the biosensor using phosphatase treatment followed by its conformational changes and activation using ATP, as measured by FRET changes. B: In vitro kinase assay for wt-AurkA and the biosensor indicating a similar activity level. C: Representative intensity and lifetime images in live stable cell line expressing the biosensor or its control. The biosensor presents FRET and its localisation is identical to endogenous AurkA. D: Statistics of the live cell lifetime measurements for the biosensor and its control at different subcellular locations.

Since our conformational biosensor is an active AurkA kinase in vitro (fig3B), we also tested whether it was able to fully replace the endogenous AurkA. For this purpose, we generated a stable cell line expressing the biosensor construct made insensitive to siRNAs used to deplete the endogenous AurkA. Under conditions where the endogenous AurkA kinase was depleted, we observed that cell viability was unaffected and that the biosensor could indeed fulfil the functions of wt-AurkA (not shown). Altogether, with this original approach, we are for the first time in a position to follow the conformational changes and active states of AurkA in space and time during the cell division process. Then the continuation of the project will be to (i) study the conformational changes of this probe in tumor cell lines in each phase of the cell cycle and in different cellular sub-compartments; (ii) use the Aurora A biosensor to evaluate the effects of several available pharmacological inhibitors and currently used as anti-mitotic therapies in epithelial cancers.

2- Aurora-A and mitochondria (ARC Post-doc 150 k€) – Giulia BERTOLIN (in collaboration with Claude PRIGENT and Roland LE BORGNE, IGDR).

We are interested in a new role of Aurora A in mitochondria. This project was initiated by Giulia BERTOLIN (Post-doc ARC) in collaboration with Claude PRIGENT team by taking advantage of our expertise in quantitative fluorescence microscopy and proof-of-concept of new Aurora-A biosensors. Aurora A was shown to cooperate with the small GTPase RalA and its effector RalBP1 to promote mitochondrial fragmentation (organelle fission is essential to ensure the correct segregation of mitochondria in daughter cells). However, the molecular mechanism employed by Aurora A to induce the mitochondrial relocalization of RalA and RalBP1 and how this transport is mandatory to regulate mitochondrial fission, remains largely unknown. The aim of this project is to establish whether Aurora A shuttles at the mitochondrion per se and to decipher its role.

Very encouraging preliminary results were recently obtained revealing Aurora A mitochondrial localization and its link to mitophagy. In vivo drosophila experiment in Aurora A KO cells supports the working hypothesis that Aurora A is involved in the control of mitochondrial segregation to the two daughter cells during division.

2.4. Collaboration

265

2.4.1. Within the IGDR

Jacques PECREAUX (POMM project), Sébastien HUET (FRET by 2c-FCS, temporal focusing), Jean-Pierre TASSAN (tension sensor), Roland LE BORGNE (Aurora A in drosophila and tension sensor), Claude PRIGENT (Aurora A biosensor), Gwénaël RABUT (2c-FCS for E2-E3 interaction in ubiquitilation).

2.4.2. Other collaborations

AutoNET consortium (FRET HCS for cell signalling networks) - Sergio PADILLA-PARRA, University of Oxford (Gated HyD for FLIM) - Chiarra ZURZOLO, Pasteur Institute (FRET for apical protein interactions) - Leukos (wavelength selector transfer) - Photonlines (fastFLIM transfer) - Oxyus (laser test).

2.5. SWOT Analysis STRENGTHS Multidisciplinary team Scientific environment Several collaboration to improve development Connection with Imaging Facility MRic WEAKNESSES Only one permanent Spread resources in too many projects OPPORTUNITIES Economic transfer of the research Specific financing for valorization (SATT, Rennes Metropole, Région Bretagne) THREATS Technological developments needs equipement Difficulties to maintain grants

266

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2011 Padilla-Parra S, Audugé N, Coppey-Moisan M, Tramier M. Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell. Microsc Res Tech (2011) 74:788-93 (IF=1.6). Padilla-Parra S, Audugé N, Coppey-Moisan M, Tramier M. Non fitting based FRET–FLIM analysis approaches applied to quantify protein–protein interactions in live cells. Biophys Rev (2011) 3:63–70.

2012 Jourdan N, Jobart-Malfait A, Dos Reis G, Quignon F, Piolot T, Klein C, Tramier M, Coppey M, Marechal V. Live-cell imaging reveals multiple interactions between Epstein-Barr Nuclear Antigen 1 (EBNA-1) and cellular chromatin during interphase and mitosis. J Virol (2012) 86:5314-29 (IF=4.6). Padilla-Parra S, Tramier M. FRET microscopy in the living cell: Different approaches, strengths and weaknesses. BioEssays (2012) 34:369-76 (IF=4.8).

2013 Leray A, Padilla-Parra S, Roul J, Héliot L, Tramier M. Spatio-temporal quantification of FRET in living cells by fast Time-Domain FLIM: a comparative study of non-fitting methods. PLOS One (2013) 8:e69335 (IF=3.5).

2014 Hatte G, Tramier M, Prigent C, Tassan JP. Epithelial cell division in the Xenopus laevis embryo during gastrulation. Int J Dev Biol. (2014) 58:775-781 (IF=2.6). Padilla-Parra S, Audugé N, Coppey-Moisan M, Tramier M. Quantitative study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy. Methods Mol Biol. (2014) 1076:683-98 (IF=1.3). Paladino S, Lebreton S, Tivodar S, Formiggini F, Ossato G, Gratton E, Tramier M, Coppey-Moisan M, Zurzolo C. Golgi sorting regulates organization and activity of GPI proteins at apical membranes. Nat Chem Biol. (2014) 10:350-7 (IF=13.2).

2015 Padilla-Parra S, Audugé N, Tramier M, Coppey-Moisan. Time-domain fluorescence lifetime imaging microscopy: a quantitative method to follow transient protein-protein interactions in living cells. Cold Spring Harb Protoc. (2015) 2015(6):pdb.top086249 (IF=4.6).

Edited books Padilla-Parra S, Audugé N, Tramier M, Coppey-Moisan M. Time-Domain Fluorescence Lifetime Imaging Microscopy: A Quantitative Method to Follow Transient Protein–Protein Interactions in Living Cells. In: Yuste R, editor. Imaging: A Laboratory Manual. Columbia University (2011).

2. Patents (with licence)

ROUL J, PÉCREAUX J, TRAMIER M, Software "FLIMager" to Agence de Protection des Programmes IDDN.FR.001.140028.000.S.P.2012.000.31235 (2012).

ROUL J, PÉCREAUX J, TRAMIER M, "Procédé de pilotage multi-modules fonctionnels incluant un dispositif d’imagerie multi-longueur d’onde, et système de pilotage correspondant" INPI 14/52766 (2014).

3. Conferences (actual team members)

- International

2013 Marc TRAMIER: Focus on Microscopy 2013 – Maastricht (NL) – oral presentation

2015 Marc TRAMIER: Focus on Microscopy 2015 – Gottingen (GER) – oral presentation

- National 267

2014 Gaëtan HERBOMEL: Journées Dimacell 2014 – Besançon (FR) –oral presentation Claire DEMEAUTIS: Biosensors Meeting 2014 – Bordeaux (FR) – oral presentation Gaëtan HERBOMEL: MGT2014 – Paris (FR) – poster

2015 Gaëtan HERBOMEL: Biosensors Meeting 2015 – Grenoble (FR) – oral presentation

4. Funding

2011 IBiSA – Equipment fastFLIM - Coordinator M. TRAMIER 250 k€ 2011 Rennes Métropole – Equipment fastFLIM – Coordinator M. TRAMIER 50 k€ 2011 Institut Fédératif de Recherche 140 - Génétique fonctionnelle, Agronomie et Santé 6,7 K€ 2011 GDR 2588 CNRS 2 K€ 2011-2012 Région Bretagne SAD – fastFLIM - Coordinator M. TRAMIER 50 k€ 2012-2015 ANR Blanc – KinBioFRET – Coordinator M. TRAMIER 410 k€ 2013-2016 ARED PhD thesis fellowship Region Bretagne - Claire DÉMÉAUTIS 45 k€ 2014 Industrial collaboration Photonlines – Phototoxicity in microscopy – Coordinator M. TRAMIER 25 k€ 2014 BIOSIT - Structure fédérative de recherche en biologie santé 7 K€ 2014 IBiSA – Equipment Temporal focusing – Coordinator M. TRAMIER 150 k€ 2014-2015 Université européenne de Bretagne – UEB 3 K€ 2014-2015 Maturation SATT Ouest Valorisation – POMM – Coordinator M. TRAMIER 124 k€ 2014-2017 ARC 3-year Post-doc fellowship - Giulia BERTOLIN 150 k€ 2015-2017 Région Bretagne – ARED 90 K€ 2015-2018 ARED PhD thesis fellowship Region Bretagne & UR1 - Florian SIZAINE 90 k€

5. Training Practical courses: "Light microscopy applied to biology" organized with imaging facilities – Marc TRAMIER coordinator - 5 days, given 3 times: 16-20th May 2011 ; 12-16 November 2012 ; 24-28 March 2014. This training aims at providing to the trainees all the theoretical bases and first-hand practical covering all commonly used microscopy techniques in biology. This training was supported by CNRS, INSERM, INRA and Univ. Rennes 1. "Image analysis" organized with imaging facilities – Marc TRAMIER coordinator - 3 days, given 3 times: 12-14 December 2011 and January 9th 2012 ; 15-17 April 2013 and May 15th 2013 ; 10-12 December 2014 and January 12th 2015. This training aims at introducing to the trainee all the major image processing techniques. This training was supported by CNRS, INSERM, INRA and Univ. Rennes 1. "Protein interactions by FRET/FLIM and 2 color FCS" for expert organized with RTmfm – Marc TRAMIER coordinator – 3 days, given 1 time: 16-18 june 2014. This national advanced course aims at sharing expertise in protein-protein interaction methods in fluorescence microscopy. This training was supported by CNRS ANF (Action Nationale de Formation). "Advanced techniques in light microscopy to study the dynamics and molecular interactions: FRAP / FRET / FLIM / FCS / Photomanipulation" organized with imaging facilities – Marc TRAMIER coordinator – 4 days, given 1 time: 2-5 june 2015. This training aims at providing all the theoretical and practical bases for F-techniques. This training was supported by CNRS, INSERM, INRA and Univ. Rennes 1. Workshop – Schools: MiFoBio CNRS Thematic school (2010-2012-2014): Marc TRAMIER lecture (2010), Marc TRAMIER, Julien ROUL, Claire DEMEAUTIS, Gaëtan HERBOMEL, Giulia BERTOLIN practical sessions (2012-2014), Marc TRAMIER, Julien ROUL presentation of prototype (2012) EMBO practical course on protein-protein interactions – Porto (Portugal) – Marc TRAMIER lecture (2014) BTS / IUT 2014 - Melissa FAVRY- BTS 1e année biology (7 weeks) 2015 - Alexis DETAIN – BTS 2e année biology (2 months) Master 2011 - Thibault RICHARD - M2 biology (7 months), PhD at Stockholm University - Marine LAMBERT - M2 microscopy (5 months), Engineer in microscopy facilities 2012 - Adrien CLARYSSE - M1 biology (2 months) - Guillaume CORDIER - M2 physics (6,5 months), PhD at ICFO Barcelona 2013 - Karim MROUJ - M1 biology (2 months) - Claire DEMEAUTIS - M2 microscopy (6 months), PhD in the team 268

2015 - Nalhuirati MOHAMED - M2 biology (5 months) PhD Since 10/2013: Claire DEMEAUTIS – Dir. Thèse Marc TRAMIER Starting 10/2015: Florian SIZAINE – Dir. Thèse Marc TRAMIER Post-doctorants 2012-2013: Sergio PADILLA-PARRA, post-doctoral fellow 2 years & 6 months, recruited PI at University of Oxford UK. 2013-2015: Gaëtan HERBOMEL, post-doctoral fellow 2 years & 5 months, Post-doc at NIH, Bethesda (MD) USA. 2014-2017: Giulia BERTOLIN, post-doctoral fellow 3 years CDD 2011-2014: Julien ROUL, Engineer CNRS, contract 3 years & 3,5 months, recruited permanent Research Engineer at LAAS University of Toulouse. 2015-2016: Otmane BOUCHAREB, Engineer SATT OV, contract 1 year.

269

Team 20 "Genome duplication and maintenance" Leader: Pei-Yun Jenny WU

271

2.1. Team presentation

The Genome Duplication and Maintenance team was established in January 2012 with funding from the Association for International Cancer Research/AICR (now Worldwide Cancer Research), UK and from the Fondation pour la Recherche Médicale (French Foundation for Medical Research). The group also received a starting grant from the ATIP-AVENIR program of the CNRS/INSERM in 2013.

Genome duplication is a critical part of the cell cycle that is highly regulated to ensure proper cell growth and proliferation. The distribution of the sites of initiation of DNA replication, or origins, across the genome changes during development and differentiation, suggesting that the program of genome duplication is highly controlled. Alterations in the replication pattern have also been observed in a number of pathologies. However, the fundamental features driving origin selection and the importance of using specific replication programs for cellular functions remain surprisingly unknown. The research in our team aims to study different aspects of genome duplication and maintenance using the fission yeast Schizosaccharomyces pombe as a model system.

2.2. Assessment from January 1st 2010 to June 30th 2015

2.2.1. Flow Chart of the team (permanent people)

2.2.2. List of staff (people who will not be in the team in Jan 2017 are in blue)

2.2.2.1. Permanent staff

WU Pei-Yun Jenny CR1 CNRS SCHAUSI-TIFFOCHE Diane AI CNRS

2.2.2.2. Temporary staff

ESTRAVIS SASTRE Miguel Post-doc LANTERI Lilian PhD student (2014/2017) GOMEZ-ESCODA Blanca Post-doc PERROT Anthony PhD student (2012/2016)

2.2.3. Achievements

2.2.3.1. Scientific achievements

Major scientific achievements

Replication origin selection regulates the distribution of meiotic recombination Wu PY* and Nurse P, Molecular Cell (2014) 53:655-662. *corresponding author

The program of DNA replication, defined by the temporal and spatial pattern of origin activation, is altered during development and in cancers. However, whether changes in origin usage play a role in regulating specific biological processes remains unknown. We investigated the consequences of modifying origin selection on meiosis in fission yeast. Genome-wide changes in the replication program of premeiotic S phase do not affect meiotic progression, indicating that meiosis neither activates nor requires a particular origin pattern. In contrast, local changes in origin efficiencies between different replication programs lead to changes in recombination factor binding and recombination frequencies in these domains. We conclude that origin selection is a key determinant for organizing meiotic recombination, 272

providing evidence that genome-wide modifications in replication program can modulate cellular physiology. Our findings provide insight into how the organization of genome duplication may play a much broader role than anticipated in the biology of the eukaryotic cell.

Deregulation of the replication program and genome instability

In replication stress conditions, the S phase checkpoint protects the genome by stabilizing replication forks and inhibiting DNA synthesis. Both replication stress and checkpoint defects are hallmarks of tumorigenesis, and to study their combined effect, we established a model in which checkpoint- defective fission yeast cells lacking the ATR kinase Rad3 are subjected to replication stress through reduced nucleotide levels. This induces major changes in replication origin selection, with increases in replication initiation clustered in specific genomic domains. Strikingly, origin deregulation leads to the generation of DNA damage, potentially in the form of double-strand breaks (DSBs), at these sites. In these conditions, the formation of deleterious DNA lesions at these particular loci represents a major source of genome instability in these cells. Our studies provide insight into how the deregulation of DNA replication observed in tumor cells may contribute to the accumulation of genome alterations that is common to all cancers. We are currently preparing a manuscript describing this work for submission.

Quantitative control of CDK activity and origin selection

Despite the critical function of the conserved cell cycle cyclin-dependent kinase (CDK) Cdc2 in S phase entry and checkpoint regulation, surprisingly little is know about how CDKs may affect origin selection and the overall program of replication. Using a strain of S. pombe that allows us to precisely modulate CDK activity at any point during the cell cycle using chemical genetics, we aimed to uncover the quantitative relationship between CDK levels and origin usage. We analyzed the efficiency of origin activation across the genome in cells that are induced to enter S phase with varying levels of CDK activity. Interestingly, we observed a direct relationship between CDK activity and length of S phase, with higher levels of CDK activity driving faster genome duplication. Furthermore, our results show an overall increase in replication origin efficiencies across all three chromosomes in these conditions. Based on these results, we propose that CDK activity is a dose-dependent, limiting factor in the regulation of origin selection. To further address the functional importance of this control, we are currently investigating the impact of these changes in the replication program on genome stability. As deregulation of CDK activity is common to a number of cancers, our studies may help us understand its contribution to the acquisition of mutations during tumorigensis. We are currently preparing a manuscript describing this work for submission.

2.2.3.2. Scientific dissemination and influence

The team leader has been invited to present the work of the team at international conferences, including: 8th International Fission Yeast Meeting in Kobe, Japan (2015); “Views into Nuclear Function” Symposium in Patras, Greece (2014); 7th International Fission Yeast Meeting in London, UK (2013).

The team is supported by both national and international grants, including: Marie Curie Career Integration Grant, Seventh Framework Programme of the European Union; Starting research grant, ATIP- AVENIR program, CNRS-INSERM; Starting grant, "Amorçage de jeunes équipes", Fondation pour la Recherche Médicale (FRM), France; Research grant, Association for International Cancer Research (AICR – now Worldwide Cancer Research), UK.

The team participates in and is involved in the organization of an annual meeting for researchers using yeast in the greater Brittany area. This event allows for the sharing of research findings as well as resources and expertise among teams in the region.

The team has recruited international post-doctoral fellows who have received training in excellent laboratories and institutes.

2.2.3.3. Interaction with the economic, social and cultural environment

The team leader is a founding organizer of the "Frontiers in Biology" series of seminars, which aims to bring exceptional researchers to Rennes and the Brittany region. Members of the team also participate in the organization of this series. These seminars have been broadcast on the WikiRadio of the CNRS and have been the subject of reports in the regional news media. 273

The team collaborates with Cherry Biotech, a start-up based in Rennes, for the development of microfluidic devices for live-cell imaging. Together with another group at the IGDR, our team has been awarded a collaborative research grant from the regional government of Brittany for this project.

2.3. Projects, scientific strategies & perspectives (5 years)

Research interests

The maintenance of genome integrity is critical for cell growth and proliferation as well as for development and differentiation. Cells employ conserved pathways to ensure the accurate duplication and transmission of genetic information, including multi-layered regulation of DNA synthesis as well as checkpoint systems that verify the completion of replication prior to mitosis. Consistent with the crucial roles of these processes, errors in DNA replication have been linked to many human diseases, and defects in checkpoint control have been shown to contribute to cancer progression. In addition, changes in the program of replication, defined by the activation and distribution of the sites of replication initiation along the genome, occur during development and differentiation as well as in pathologies. In eukaryotes, these sites, or replication origins, are not equally used from one cell to another, and their specification has been the focus of a large body of work. However, the key parameters that determine the subset of origins that is fired in each cell as well as the importance of this replication program for cellular function are surprisingly unknown. We are interested in studying: 1) the core establishment and organization of the eukaryotic program of origin usage; 2) the interplay between the replication program and cellular physiology; and 3) the impact of the organization of genome duplication on the balance between genome maintenance and plasticity.

Balancing genome stability and plasticity through the organization of DNA replication

Genome duplication is central to the regulation of both genome maintenance and plasticity. During DNA replication, cells are particularly vulnerable due to the transient generation of single-stranded DNA that may result in deleterious alterations. DNA synthesis therefore relies on the tightly controlled, ordered assembly of conserved complexes, and it is coordinated with checkpoint mechanisms to ensure its completion and the repair of accompanying damage. Conversely, the potential for genomic instability intrinsic to DNA replication is an important contributor to genome plasticity and evolution. Intriguing evidence from both comparative genomic analyses and laboratory evolution experiments suggest that replication initiation itself is a mutagenic event and generates diversity for natural selection. Therefore, the organization of genome duplication may act as a rheostat through which genome maintenance and plasticity are balanced.

While studies of DNA replication and genome maintenance have largely focused on the multiple enzymes and modifications involved, it has become clear that higher-order chromosome organization and nuclear architecture play key roles in these processes. Replication is modulated not only by transcriptional and chromatin contexts but also by the nuclear microenvironment, which is critical for both DNA synthesis and repair. This architecture changes during development and in pathologies ranging from cancers to aging-related diseases and may participate in the accompanying physiological alterations. However, we are only beginning to understand how the content and spatial organization of distinct genomic regions contribute to faithful genome duplication while allowing sufficient plasticity for evolution. We aim to investigate the interface between genome architecture and duplication, with an emphasis on the regulation of the dynamic balance between stability and plasticity.

Dynamic architecture of genome duplication and checkpoint pathways

In replication stress conditions, the S phase checkpoint protects the genome by stabilizing replication forks and inhibiting DNA synthesis. Both replication stress and checkpoint defects are hallmarks of tumorigenesis, and to study their combined effect, we have established a model in which checkpoint- defective fission yeast cells lacking the ATR kinase Rad3 are subjected to replication stress through reduced nucleotide levels. This induces major changes in replication origin activation, with increases in initiation at 190 out of 900 total origins that directly lead to double-strand breaks at these sites contributing to a loss of genome integrity. Strikingly, these deregulated origins are strongly clustered in specific genomic domains, suggesting a differential regulation of replication and checkpoint pathways at the regional level. This unique system will therefore allow us to investigate the intricate relationship between local genomic content, local checkpoint operation, and higher-order genome organization. Furthermore, this will lead us to explore whether the checkpoint may act as a quantitative regulator of the equilibrium between the protection of the genome and its evolution. 274

Genome integrity and plasticity during physiological transitions

Quiescent states are common to many cells in the human body, and the ability to alternate between quiescence and proliferation is critical for development, differentiation, and tissue homeostasis/renewal. This transition is also integral to tumorigenesis and cancer recurrence: tumor cells unable to adapt to stress or to the changing environment can become quiescent, and they may abruptly return to proliferation and form metastases years later. Genome duplication is one of the first steps following quiescence exit, and events linked to this post-quiescent S phase may therefore contribute to the ability of cells to respond to environmental stimuli and challenges. Fission yeast cells enter quiescence when deprived of nitrogen, with changes in transcription and nuclear organization. Cells re- enter the division cycle upon addition of a nitrogen source, and the first S phase during this transition has two unique features: it is twice as long as in vegetatively growing cells, and our genome-wide analyses indicate that only around half of the normal complement of origins are used. These origins are remarkably organized in distinct genomic domains that may be crucial for optimal cell cycle re-entry, whether to promote early transcription of critical genes or to ensure rapid, high-fidelity duplication of essential genomic regions. Interestingly, these origin-rich domains are not the same as those described in the previous section, suggesting a different underlying multi-scale structure of the genome. This system will allow us to directly link distinct replication programs in specific physiological conditions to the architecture of the genome, and we will investigate how the organization of genome duplication during this transition participates in balancing genome integrity and plasticity in the immediate and long-term.

Interplay between DNA replication and meiotic recombination

Changes in the pattern of replication have been observed in Xenopus and Drosophila during development, in differentiating mouse and human cells, and in a number of cancers. However, despite numerous studies dedicated to unraveling the steps in replication initiation and the regulation of origin usage, the fundamental question of whether undergoing S phase with particular programs of replication has direct consequences on cellular function remains poorly explored. We have recently shown that origin selection in pre-meiotic S phase plays a major role in the organization of meiotic recombination (Wu and Nurse, Molecular Cell, 2014). Building on this work, we aim to decipher the steps in genome duplication that regulate the double-stranded DNA break (DSB) formation that is required for meiotic recombination. In addition, while our analyses of replication and recombination at the population level show that increasing origin activity in a genomic region leads to increased local recombination, it remains unknown how these processes interact in a given cell along individual chromosomes. This is the case for the domains of high origin efficiencies obtained from population studies, which do not represent actual origin firing in a single cell but rather a statistical view of genome organization. We are therefore developing methods to visualize both replication initiation and meiotic DSB formation on single chromosome fibers, with the goal of gaining critical and unprecedented insight into the interplay between origin selection and recombination in each cell.

Our projects will take advantage of a variety of approaches, including genomics and bioinformatic methodologies, single-molecule studies by DNA combing, computational modeling, analysis of higher-order chromosome conformation, and live-cell microscopy using microfluidics. Along with the expertise already present in our team, we have established several collaborations to pursue this work: 1) with Cherry Biotech, a start-up based in Rennes, for the development of microfluidic solutions for live-cell microscopy in different physiological conditions; 2) with Dr. Zoi LYGEROU at the , Greece, whose team has developed a computational model for DNA replication in fission yeast; and 3) with Dr. Jean- Christophe ANDRAU at the Institute of Molecular Genetics of Montpellier, France, who has excellent expertise in next-generation sequencing applications and analyses.

2.4. Collaboration

2.4.1. Within the IGDR

Dr. Damien COUDREUSE, Synthecell Team.

2.4.2. Other collaborations

National Dr. Jean-Christophe ANDRAU, Institute of Molecular Genetics of Montpellier, France - Dr. Daniel FISHER, 275

Institute of Molecular Genetics of Montpellier, France - Dr. Jeremy CRAMER, Cherry Biotech, France.

International Dr. Zoi LYGEROU, University of Patras, Greece.

2.5. SWOT Analysis STRENGTHS Unique model systems and approaches: genome-wide to single-cell Preliminary results put us in an ideal position to address research questions International research team, diverse backgrounds Funding sources in place for the next 3 years WEAKNESSES Projects take time to establish in a competitive field Currently using a single model organism Can be difficult for a new group to recruit postdocs OPPORTUNITIES Multidisciplinary collaborations in academia and in industry: fostering technical innovation and creativity Different lines of research with promising initial findings THREATS Funding sources run on short cycles (mostly 2-3 years) – may be insufficient time to establish projects and obtain results for a new team Administrative limitations of the CNRS may make it difficult to keep post-doctoral fellows until their projects are finished

276

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF)

2013 Gomez-Escoda B and Wu PY. The programme of DNA replication: beyond genome duplication. Biochemical Society Transactions (2013) 41:1720-5. (IF=3.194)

2014 Wu PY* and Nurse P. Replication origin selection regulates the distribution of meiotic recombination. Molecular Cell (2014) 53:655-662. *corresponding author (IF=14.078)

2. Patents (with licence)

N/A

3. Conferences (actual team members)

- International

2013 GOMEZ-ESCODA Blanca and WU Pei-Yun: Impact of replication origin selection on cellular physiology, 7th International Fission Yeast Meeting, London, UK, 2013, Session chair and invited speaker

2014 WU Pei-Yun: Impact of replication origin selection on cellular physiology, “Views into Nuclear Function” Symposium, Patras, Greece, 2014, Session chair and invited speaker WU Pei-Yun: Impact of replication origin selection on cellular physiology, EMBO Practical Course: Molecular genetics with fission yeast, Institut Pasteur, France, 2014

2015 WU Pei-Yun: Regulation of origin selection is critical for genome integrity, 8th International Fission Yeast Meeting, Kobe, Japan, 2015, Invited speaker

- National

2015 WU Pei-Yun: Impact of replication origin selection on cellular physiology, IGMM (Institut de Génétique Moléculaire de Montpellier), Montpellier, France, 2015 Invited seminar, Host: Jean-Christophe ANDRAU

4. Funding

2011-2014 Association for International Cancer Research (now Worldwide Cancer Research), UK 230 k€ 2012-2013 Projet ARC» research grant, Association pour la Recherche sur le Cancer (ARC), France 50 k€ 2012-2013 Stratégie d’attractivité durable (SAD), Brittany regional government 90 k€ 2012-2014 "Amorçage de jeunes équipes" Fondation pour la Recherche Médicale (FRM) 300 k€ 2012-2014 ATIP-AVENIR program, CNRS-INSERM 360 k€ 2013-2014 Aide à l’Installation Scientifique, city of Rennes 40 k€ 2014 BIOSIT 8 k€ 2014-2016 Brittany regional government, collaborative research grant 90 k€ 2014-2017 La Ligue contre le cancer & the Brittany regional government, PhD student fellowship 100 k€ 2014-2018 Marie Curie Career Integration Grant, 7th Framework Programme of the European Union 100 k€ 2015-2016 La Ligue contre le cancer (Comités d’Ille-et-Vilaine, du Finistere, et des Cotes d’Armor) 35 k€ 2015-2017 Stratégie d’attractivité durable (SAD), Brittany regional government, postdoctoral fellowship 72.9 k€

5. Training Master 2012 - Romain TROPEE, M1 University of Rennes 1 (4 months), PhD Since 10/2012: Anthony PERROT, PhD student University of Rennes 1 – Dir. Thèse Pei-Yun Jenny WU 277

Since 11/2014: Lilian LANTERI, PhD student University of Rennes 1 – Dir. Thèse Pei-Yun Jenny WU Post-doctorants 2011-2016: Blanca GOMEZ-ESCODA, post-doctoral fellow 4 years & 11,5 months 2012-2014: Christopher MILLINGTON, post-doctoral fellow 2 years & 4 months 2014-2017: Miguel ESTRAVIS SASTRE, post-doctoral fellow 3 years & 4 months

278

Team 21 "Protein ubiquitylation" Leader: Gwenaël RABUT

279

2.1. Team presentation

Gwénaël RABUT has been working as a project leader in the Cell Cycle team since his arrival at the IGDR in 2009. For the next contract, he is foreseen to develop a fully independent group. This group will investigate at the molecular level the functions and regulatory mechanisms of protein ubiquitylation, using budding yeast as a model organism and a combination of genetic, biochemical, proteomic and live cell imaging approaches.

2.2. Assessment from January 1st 2010 to June 30th 2015

This section does not apply to this team (cf team 17 Cell cycle).

2.3. Projects, scientific strategies & perspectives (5 years)

2.3.1. Liste of staff

2.3.1.1. Permanent staff

RABUT Gwénaël CR1 INSERM

2.3.1.2. Temporary staff

LE BOULCH Marie PhD student (2015-2018)

2.3.2. Projects, scientific stratégies & perspectives

Ubiquitylation is an essential posttranslational protein modification conserved in all eukaryotes. It encodes complex molecular signals that control the activity and degradation of a very wide array of cellular proteins. Defects in this process lead to unbalances in the cellular protein content and are associated with numerous human pathologies, including cancers and neurodegenerative diseases. The fundamental principles governing ubiquitylation are now understood; however we are far from knowing its full functional spectrum. For instance, many of the ubiquitylating and deubiquitylating enzymes that attach and remove ubiquitin to and from cellular proteins are not comprehensively studied and their specific substrates and functions are often described only partially, if at all. Our main objective is therefore to identify and characterize new cellular pathways regulated by ubiquitylation.

Current challenges in the field of protein ubiquitylation

The primary challenge in the field of protein ubiquitylation remains the identification of the substrates of given ubiquitylating enzymes. Currently, the most sensitive methods to identify ubiquitylated proteins are based on the use of affinity reagents to enrich ubiquitin modified proteins or peptides before mass spectrometry analysis. For instance, highly specific antibodies directed against the ubiquitin remnant peptides (diGly peptides) that are produced by trypsin digestion of ubiquitylated proteins have enabled the detection of thousands of ubiquitylated proteins in yeasts and cultured mammalian cells. This method combined with quantitative mass spectrometry assays such as SILAC is currently the most promising method to identify the substrates of ubiquitin ligases of interest, but it remains costly and technically difficult to implement. Furthermore, the identification of relevant substrates is hindered by the fact that ubiquitylated proteins are often rapidly degraded and only locally or transiently modified and hence the relevant diGly peptides are difficult to enrich. There is therefore a high rate of false-positives that demands secondary screens to identify the bona-fide substrates and limits the general use of such methods.

Another important challenge is to decipher the complexity of ubiquitin signals that operate in the cell. Indeed, ubiquitylation enzymes do not only attach ubiquitin to cellular proteins but also to ubiquitin itself, hence assembling polymeric chains of ubiquitin on their substrates. In this polymeric form, any of the seven lysine residues of ubiquitin or its N-terminus can be modified by a subsequent ubiquitin. This creates a great variety of ubiquitin chain architectures, comprising simple homotypic chains (with a single type of ubiquitin-ubiquitin linkage) but also complex heterotypic chains that can be mixed (chains with alternating linkages) or branched (chains comprising one or several ubiquitin moieties modified at two or 280

more sites). It is now evident that different types of poly-ubiquitylation as well as mono-ubiquitylation (modification of substrates with single ubiquitin moieties) function as distinct molecular signals in the cell. For instance, ubiquitin chains linked by the lysine 48 (K48) of ubiquitin are prototypical signals for proteasomal degradation while K63-linked ubiquitin chains are involved in other functions such as protein trafficking or non-proteolytic DNA-damage signalling. The biological relevance of other types of homotypic ubiquitin chains is starting to be elucidated while the possible specific roles of heterotypic chains essentially remain to be investigated.

How we plan to address those challenges in the next 5 years

During the next years, we plan to primarily focus our research on how ubiquitylation regulates essential nuclear functions. Indeed the nucleus is the cell compartment that hosts most of the genetic material and mis-regulation of nuclear activities causes development disorders, cancers and aging. Furthermore, the ubiquitylation machinery and the proteasome are concentrated in the nucleus and several nuclear ubiquitylation enzymes are orphan with no known substrates.

To identify novel nuclear proteolytic ubiquitylation substrates, we have engineered yeast strains to conditionally relocalize the proteasomes out of the nucleus. We have shown that depletion of nuclear proteasomes induces a dramatic enrichment of nuclear ubiquitylated proteins. We have purified these proteins and used mass spectrometry to identify more than 100 candidate nuclear ubiquitylation substrates. Using classical biochemical method, we have already validated the ubiquitylation of 13 out of 14 tested candidates, which demonstrates the efficiency of our approach. We will now start to work on the characterization of some of these novel nuclear ubiquitylation substrates with the aim to understand the mechanism (which enzymes are involved?), the circumstances (e.g. is it regulated during the cell cycle or induced by DNA damage?) and the functional consequences of their ubiquitylation. We will select the proteins to characterize based on their role in nuclear processes, their conservation in mammals and the possibility to establish internal or external collaborations. For instance, two of the proteins we plan to focus on during the coming years are Brn1 and Sld2, which are conserved regulatory subunits of the Condensin I and the DNA replication pre-initiation complexes (their human orthologues are hCAP-H and RecQL4). We anticipate that these projects will benefit from the in house expertise of Jenny Wu and Erwan Watrin, who are experts in the mechanisms regulating the initiation of DNA replication and in the biology of Cohesin and Condensin complexes, respectively.

To investigate how ubiquitylation enzymes encode specific ubiquitin signals, we have taken an original approach based on bimolecular fluorescence complementation (BiFC). We systematically screened ~600 possible pairs of ubiquitin conjugating and ligating enzymes in living yeasts, which enabled us to identify dozens of new putative enzyme couples. Importantly, this approach led us to uncover a new inner nuclear membrane (INM) associated protein degradation pathway (Khmelinskii et al, 2014). We showed that the INM-localized Asi-complex (consisting of the RING-domain proteins Asi1 and Asi3) is a ubiquitin ligase that functions together with the ubiquitin conjugating enzymes Ubc7, Ubc6 and Ubc4. We collaborated with the labs of Michael KNOP (ZMBH, Heidelberg, Germany) and Per LJUNGDAHL (University of Stockholm, Sweden) to demonstrate that these enzymes primarily target transmembrane proteins from the cellular endomembrane system that normally do not reside in the nucleus, suggesting that the Asi- complex acts to control the protein content of the INM. We are now analysing how the 3 ubiquitin conjugating enzymes Ubc4, Ubc6 and Ubc7 contribute to the function of the Asi-complex. Do these enzymes function redundantly or do they have distinct activities (e.g. to prime or elongate specific chains)? Do they serve to ubiquitylate identical or different substrates? Can they assemble heterologous ubiquitin chains with particular functions (Ubc6 is known to assemble primarily K11-linked ubiquitin chains in vitro, while Ubc7 is specialized in the assembly of K48 chains)? To address those questions we will analyse the ubiquitin chains assembled on different substrates of the Asi- complex, using commercial chain specific antibodies, ubiquitin mutants and the Ubiquitin Chain Restriction analysis method recently developed by the KOMANDER lab (UbiCRest method (Hospenthal et al. 2015)). To figure out how the different type ubiquitin chains assembled on Asi substrates regulate their function, we will in particular continue our collaboration with the laboratory of Michael KNOP to quantitatively assay the half-life and activity of differentially modified Asi substrates. We anticipate that this work on the ubiquitin signals assembled by the Asi-complex (which will be performed in collaboration with the KNOP lab), will enable us to acquire the expertise to then decipher the ubiquitin signals assembled by other pairs of nuclear ubiquitylation enzymes that we identified in our interaction screen. We will select the enzymes to investigate using the criteria described above for ubiquitylation substrates. For instance, two of the enzymes we plan to study soon are Psh1 and Tfb3. Psh1 is a ubiquitin ligase known to ubiquitylate and regulate the chromatin localization of the centromeric histone CENP-A (called Cse4 in yeast), while Tfb3 is a conserved subunit of the TFIIH complex, which is essential for transcription 281

initiation and nucleotide excision repair. Whenever possible, we will collaborate with other labs to deeply understand the biological functions of the ubiquitin signals assembled by these enzymes (e.g. we plan to collaborate with the team of Christian Jaulin at the IGDR, who is investigating the mechanisms regulating centromeric chromatin, to investigate the ubiquitin signals assembled by Psh1).

Conclusion and long term perspectives

Our team has established original approaches to investigate new nuclear functions of protein ubiquitylation and to dissect how ubiquitylation enzymes encode complex ubiquitin signals. We have demonstrated the efficiency of these approaches and have identified numerous novel ubiquitylation pathways. In the coming years, we plan to characterize at the molecular level some of these novel pathways, collaborating whenever possible with other labs at the IGDR or other institutes. We are using budding yeast as a model organism because it offers methodological advantages compared to cultured mammalian cells, but as the basic principles of ubiquitylation are fully conserved from yeast to humans, we anticipate that our findings will be highly relevant to the understanding of ubiquitin signaling in human cells. Since defective ubiquitin signaling is involved in numerous human diseases, further molecular characterization of ubiquitin signals could in the long term lead to original therapeutic opportunities. Although drug development is obviously not our field of expertise, we will be attentive to any possible translational perspective of our research.

2.4. Collaboration

2.4.1. Within the IGDR

We currently do not have formal collaboration with other IGDR teams. Yet, as highlighted in the above paragraph, our projects will certainly benefit from the expertise of several IGDR scientists and we foressee to collaborate with them depending on the progress of our research.

2.4.2. Other collaborations

We are currently actively collaborating with the following labs: - Michael KNOP (ZMBH, University of Heidelberg, Germany) - Per LJUNGDAHL (The Wenner-Gren Institute, University of Stockholm, Sweden) - Sebastian LEIDEL (Max-Planck-Institut für molekulare Biomedizin, Muenster, Germany).

2.5. SWOT Analysis STRENGTHS GR is internationally recognized for his expertise in protein ubiquitylation and live cell imaging The team is using original and complementary approaches Most methodological pipelines required for this project are well established in the lab The team has access to state of the art microscopy and thermophoresis equipment WEAKNESSES The team needs to secure long-term funding The team needs to recruit long-term lab members Structural biology expertise could be beneficial to address certain questions of this project OPPORTUNITIES The ubiquitin field is rapidly expanding with new research avenues GR has established strong international collaborations (e.g. Knop and Leidel labs in Germany) Several collaborations with other IGDR teams are possible Ubiquitylation is a transversal research field at the crossroads of many biological questions The ubiquitylation machinery is a niche for the development of novel therapeutic drugs THREATS The ubiquitin field is very competitive and progressing rapidly Methodological developments are still required to precisely investigate the complexity and function of ubiquitin signals

282

ANNEX LIST OF PRODUCTION: PUBLICATIONS (with IF), PATENTS (with licence), CONFERENCES (International & National), FUNDING (€) and TRAINING (name and level).

1. Publications 2010-2015 (with IF) (Within the team Cell Cycle)

2011 Burschowsky D, Rudolf F, Rabut G, Herrmann T, Peter M, Wider G. Structural analysis of the conserved ubiquitin- binding motifs (UBMs) of the translesion polymerase iota in complex with ubiquitin. J Biol Chem. 2011 Jan 14;286(2):1364-73. (IF 4.6) Rabut G, Le Dez G, Verma R, Makhnevych T, Knebel A, Kurz T, Boone C, Deshaies RJ, Peter M. The TFIIH subunit Tfb3 regulates cullin neddylation. Mol Cell. 2011 Aug 5;43(3):488-95. (IF 14.464)

2012 Rabut G. Introduction to the pervasive role of ubiquitin-dependent protein degradation in cell regulation. Semin Cell Dev Biol. 2012 Jul;23(5):481. (IF 5.971)

2013 Zemla A, Thomas Y, Kedziora S, Knebel A, Wood NT, Rabut G, Kurz T. CSNand CAND1-dependent remodelling of the budding yeast SCF complex. Nat Commun. 2013;4:1641. (IF 10.742)

2014 Khmelinskii A*, Blaszczak E*, Pantazopoulou M, Fischer B, Omnus DJ, Le Dez G, Brossard A, Gunnarsson A, Barry JD, Meurer M, Kirrmaier D, Boone C, Huber W, Rabut G#, Ljungdahl PO#, Knop M#. Protein quality control at the inner nuclear membrane. Nature. 2014 Dec 18;516(7531):410-3. *Equal contribution #Corresponding Authors. (IF 42.351)

2. Patents (with licence)

N/A

3. Conferences (Within the team Cell Cycle)

- International

2014 Gwénaël RABUT: The physiological network of ubiquitin conjugating and ligating enzymes probed by fluorescence complementation in yeast (ID 99). The 1st Proteostasis Meeting, the Centro de Investigación Principe Felipe, Valencia, Spain, November 5-7. (selected speaker).

2015 Gwénaël RABUT: Microscale Thermophoresis (MST) interactions and beyond. Theoretical and Practical Workshop, Institut Pasteur, Paris, March 3-6 (invited speaker).

- National

2012 Gwénaël RABUT: The TFIIH subunit Tfb3 regulates cullin neddylation - 10eme rencontre Levures Modeles Outils, Toulouse, France April 2-4 (selected speaker)

2013 Gwénaël RABUT: Institute of Biochemistry, ETH Zurich, Switzerland (invited by Matthias Peter) Gwénaël RABUT: Use of Bimolecular Fluorescence Complementation to probe the physiological network of ubiquitylating enzymes in yeast - International Centre for Genetic Engineering and Biotechnology. Trieste, Italie, November 8th.

2014 Gwénaël RABUT: Institut Jacques Monod, CNRS UMR 7592, Paris

2015 Gwénaël RABUT: LIOAD UMRS 791 - Nantes

283

4. Funding (Within the team Cell Cycle)

2010 SAD Région Bretagne ProtNEDD (Gwénaël RABUT) 50 k€ 2011 Rennes Metropole – Equipement & fonctionnement (Gwénaël RABUT) 75 k€ 2013 BIOSIT microscopy (Gwénaël RABUT) 3.7 k€ 2013-2015 ANR JCJC (Gwénaël RABUT) 200 k€ 2014 LNCC 4th year PhD Ewa BLASZCZAK (Gwénaël RABUT) 14.7 k€ 2014 AP UR1 (Gwénaël RABUT) 1.5 k€ 2015 BIOSIT thermophoresis (Gwénaël RABUT) 4 k€

5. Training (Within the team Cell Cycle) Licence 2013 - Marie LE BOULCH - L3 University Rennes 1 (6 weeks) 2014 - Eva LE RUN – - L3 University Rennes 1 (6 weeks) 2015 - Emilie EVEN - L3 University Rennes 1 (6 weeks) IUT/BTS 2013 - Julie LEGROS – BTS 1e et 2e année (2 months + 2 months) Master 2010 - Marie-Charlotte DUMARGNE - M1 University Rennes 1 (2 months) 2012 - Emeline PASQUIER - M2 University Rennes 1 (5 months) 2013 - Jordan AUGER - M1 University Rennes 1 (3 months) 2014 - Théo LEBEAUPIN - M2 University Rennes 1 (5 months) - Aaron BROOKS – M2 Erasmus (8 months) 2015 - Stephan NICOLE - M2 University Rennes 1 (6 months) PhD 26/06/2015: Ewa BLASZCZAK (bourse ministérielle + LNCC) – Dir. Thèse Gwénaël RABUT Starting 10/2015: - Marie LE BOULCH (bourse ministérielle) – Dir. Thèse Gwénaël RABUT Post-doctorants 2013-2014: Agata CENA, post-doctoral fellow 1 year – Direction Gwénaël RABUT CDD 2014-2015: Audrey BROSSARD –Engineer assistant CNRS, contract 1 year & 7 months - Direction Gwénaël RABUT

284