University Pierre and Marie Curie Doctoral school : Complexité Du Vivant - ED515 Nuclear Organization and Oncogenesis Unit (INSERM U993) Identification of new regulators for PML Nuclear Bodies By Thibaut Snollaerts Doctoral thesis of BIOCHEMISTRY AND MOLECULAR BIOLOGY OF THE CELL Supervised by Anne Dejean Presented and defended publicly on September 28th 2016 In front of a jury composed of: Dr. Laurent LE CAM INSERM research director Reviewer Dr. Dimitris XIRODIMAS CNRS research director Reviewer Dr. Mounira CHELBI-ALIX INSERM research director Examinator Dr. Robert WEIL CNRS research director Examinator Dr. Frederic DEVAUX UPMC university professor President Prof. Anne DEJEAN INSERM research director Thesis Director (Invited member) 1 La persévérance, c'est ce qui rend l'impossible possible, le possible probable et le probable réalisé. Perseverance is what makes the impossible possible, the possible probable and the probable realized Robert Half 2 Acknowledgements First, I would like to thank all the members of the jury: Laurent Le Cam, Dimitris Xirodimas, Mounira Chelbi-Alix, Robert Weil and Frederic Devaux who, despite a very busy schedule, took the time to take part in this thesis defense jury. I would like to thank Anne Dejean for giving me the opportunity to be part of her research team since my master internship, for giving me the chance to meet very interesting people, and for providing me with many opportunities. Thank you for the trust you put in me to carry out this complicated project as well as for providing the best working conditions, and collaborations to realize it. Thank you for all the things I was able to learn from you, your team and the world of science in which we are involved. I would like to thank the entire ONO team for their support during these past few, short, years: Thank you to the past members: Adrien, the basketball player from room 306, and Pierre for your help, advices and good sense of humor; Yoon Ra for your kindness and zeness; Ricardo for bringing a little bit of Mexican spirit to the lab; Louise-Mary for all your help with the administrative paperwork and Sophie aka Fofi, aka “la personne la plus désagréable du labo, mais qui en vrai, est plus ou moins relativement super sympa”. A big thank for your help and support to you and all the people I might have met during these years. I would like to give a special thank you to Hélène-Neyret Khan for giving me the opportunity to take over one of her projects. And also for her patience, kindness and understanding while teaching most of the techniques used for this project. Thank you for keeping contact, for supporting and helping me, even though, you moved on to another lab. Thank you to the present members of the team : Alexandra, merci pour ton aide précieuse sur ce projet notamment à compter des points verts sans devenir folle. Merci aussi pour ton soutien et ton franc parler tout au long de ces années. Thank you Elefteria (Elma) for your support, for your friendship and all your advices over the years. Thank you for listening, understanding, supporting and for being one hell of a crazy friend. Pablo, muchas gracias for 3 your kindness, enthusiasm, optimism and for our scientific discussions on the project; Juan Pablo, my statistics genius, mi amigo, for all your help inside and out of the lab; Ying for sharing crazy Chinese pastries; Jack for your help, good sense of humor and support; Ilan for bringing some craziness in the lab; Oliver for your insights in the project and your good spirit; Lucas for bringing a Canadian vibe to the lab; Pierre Francois and Greg, your communicative sense of humor, for all the laughs and good times; Jacob, king of the cloning, for your help and knowledge on the project; Pierre Tiollais, for all of your stories, Friday seminar organization and of course the design of this amazing fridge, I mean building! Thank you to all the interns for making this lab so lively. Thank you people from room 307; Agnes, qPCR master, for being there to listen and help me with my various problems. Thank you Pascal for sharing your passion of science and politics, for your availability under any circumstances, for your help, for listening and for everything else. Thank you Florence for helping us with all of the administrative paperwork. Thank you for your good sense of humor and energy. Merci à Chaty et Monique, qui parfois dans l’ombre, nous facilitent tant le travail. I would also like to thank all of my floor mates and in particular people from the Hepacivirus team: Barbara, the one and only unicorn from Britany; Pierick, aka the seagull, for the famous “Soirée Piscine!TM”; Dona, thank you for your support (long live ferrets and penguins!). Thank you to all the other members of the team: Christine; Stephanie; Mila; Aurora and Patrick. I would also like to extend that thanks to the fabulous people of our building: Claudia; Iratxe; Florence; Yu; but also from Pasteur: Mathilde; Lilliana; Emmanuelle and all the people I might have missed, thank you all. I would like to thank all my friends outside the Institute who have also supported me. Thank you Paul, for always having my back for all these years; Jessica for your support, help and Tanguy, for your good sense of humor and for making teaching so interesting; Dr David Moffet for introducing me to this crazy world of science. Thank you Raphael; Lola; Joel; Estelle; Leo; Elelta and of course all the friends that are not mentioned here. 4 Thank you Camille for your support, patience and understanding over the years. Thank you to your family for their support. I would like to thank my family in the broad sense for believing in me and supporting me, especially my parents for allowing me to pursue my dreams. Thank you for your patience and unwavering support. I would also like to give a special thanks to my brothers and sister who were always supporting me no matter what. A big extra thank you for Audrey who helped me a lot with corrections. Thank you all for being here, family rocks! I would like -to once again- thank all the people who helped me shape this thesis and make it what it is. Thank you all, family/friends for your support, it means a lot for me. Over the past years, you were always there for me, no matter the circumstances, no matter how hard or difficult the situation might have been. I am actually very proud to be one of your friend/family and I hope that I would never let you down. Thank you for the smiles; the good and bad jokes; for coffee breaks (thank you coffee machine), for reminding me to eat and sleep (thank you to the night guard for checking that I was still alive) and for everything. I would like to thank the -80 freezer for breaking on a Sunday night at 11:30pm; penguins because they are funny, and because I have one yelling at me every time I open my fridge; and otters, because I can and I like them. Finally, thank you reader, for taking the time to look at all the people that helped me in this project. I hope you will enjoy reading this work and find it as interesting as I did. 5 Collaborations Because this work is also the result of a collaborative effort, I would like to thank our collaborators for their help by providing their expertise and tools to this project. - Michael Howell from the “London Research Institute” and the members of the high throughput-screening platform, who helped realize the primary and secondary siRNA screening. - Michele Pagano from the “Howard Hughes Medical Institute” of New York, who shared knowledge on the SCF complex systems and F-Box proteins, as well as, implementing the first co-immunoprecipitating screening for the F-Box. - Florian Bassermann and his team, and in particular Vanessa Fernandez, from the School of Medicine at the Technical University of Munich who helped us with the in- vitro experiments and provided us with FBXO9 antibody. - All the people and scientists who I had the chance to encounter at the Pasteur Institute and in conferences, which led to discussion allowing to further this project. 6 Table of Contents Acknowledgements ................................................................................................................... 3 Collaborations ........................................................................................................................... 6 Table of Contents ..................................................................................................................... 7 Abbreviations .......................................................................................................................... 10 Introduction ............................................................................................................................ 11 I) Discovery of ProMyelocytic Leukemia protein (PML) ................................................ 11 1) Acute Promyelocytic Leukemia (APL) ................................................................. 11 2) The PML protein .................................................................................................... 15 3) PML Nuclear Bodies .............................................................................................. 19 II) Post-translational modifications ................................................................................ 23 1) Diversity of post-translational modifications ......................................................... 24 2) Phosphorylation ..................................................................................................... 26 3) Acetylation