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Investigating phase separation mechanisms for transcriptional control Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program IMPRS Molecular Biology of the Georg-August University School of Science (GAUSS) submitted by Marc Böhning from Fulda, Germany Göttingen 2019 Thesis Advisory Committee Prof. Dr. Patrick Cramer Department of Molecular Biology Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Henning Urlaub Research Group Bioanalytical Mass Spectrometry Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Steven A. Johnsen Department of Gastroenterology and Hepatology Mayo Clinic, Rochester (USA) Members of the Examination Board Prof. Dr. Patrick Cramer (1st reviewer) Department of Molecular Biology Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Henning Urlaub (2nd reviewer) Research Group Bioanalytical Mass Spectrometry Max Planck Institute for Biophysical Chemistry, Göttingen Further members of the Examination Board Prof. Dr. Markus Zweckstetter Translational Structural Biology in Dementia German Center for Neurodegenerative Diseases, Göttingen Prof. Dr. Argyris Papantonis Translational Epigenetics Laboratory University Medical Center Göttingen Prof. Dr. Herbert Jäckle Department of Molecular Developmental Biology Max Planck Institute for Biophysical Chemistry, Göttingen Dr. Peter Lenart Research Group Cytoskeletal Dynamics in Oocytes Max Planck Institute for Biophysical Chemistry, Göttingen Date of oral examination: 20th of November 2019 I Affidavit I, Marc Böhning, hereby declare that my dissertation entitled ‘Investigating phase separation mechanisms for transcriptional control’ has been written independently and with no other sources and aids than quoted. This dissertation or parts thereof have not been submitted elsewhere for any academic award or qualification. The electronic version of this dissertation is congruent to the printed versions. Göttingen, 30th of September 2019 _________________________________ (Marc Böhning) II “The living protoplasm […] is a liquid or rather a mixture of liquids in the form of a fine emulsion consisting of a continuous substance in which are suspended drops […] of different chemical nature.” Edmund B. Wilson, Science, July 1899 [1] III Acknowledgements First and foremost, I would like to thank Prof. Dr. Patrick Cramer for giving me the opportunity to conduct my PhD research in such an outstanding research environment. I’m extremely grateful for his continuous support and interest in all my projects. Patrick’s instant trust and continuous encouragement have motivated me throughout the entire time of my PhD research. I deeply appreciated the granted freedom to develop and peruse my own ideas and, and, at the same time, was really thankful for many insightful discussions that have been instrumental to overcome the difficulties and challenges. Parts of the work presented in this thesis were carried out in an extremely interactive and fruitful collaboration with the laboratory of Prof. Dr. Markus Zweckstetter, and would not have been possible in their entirety without the dedicated help of Dr. Marija Rankovic. I’m also very grateful to Markus Zweckstetter for his commitment and determination to push the project forward. A big thanks goes also to our oversea collaborators from the Tjian-Darzacq lab, especially to Dr. Claire Dugast-Darzacq and Prof. Dr. Xavier Darzacq, whose contributions have been crucial to the success of this work. The second part of the work presented in this thesis was conducted in a great collaboration with Dr. Ritwick Sawarkar and Prashant Rawat. I’m grateful for their keen dedication and insatiable curiosity that facilitated very rapid progress, and for keeping up with my numerous enquiries and questions. I’m looking forward to seeing it published soon. I’m grateful to all my lab internal collaborators, in particular Andrea Boltendahl for dedicated and excellent technical assistance, Dr. Seychelle Vos for numerous insightful discussions and for sharing her invaluable collection of protocols, plasmids and proteins, as well as Dr. Goran Kokic and Dr. Sandra Schilbach for sharing proteins. I thank all present and past members of Lab 116, Kerstin Maier, Petra Rus, Andrea Boltendahl, Dr. Sofia Battaglia, Dr. Carlo Bäjen, and Dr. Katharina Hofmann for daily discussions and for creating a truly enjoyable atmosphere. All work presented in this thesis was highly facilitated through an outstanding and reliable lab infrastructure provided and maintained by Kirsten Backs, Janine Blümel, Kerstin Maier, Petra Rus, Ute Neef, Thomas Schulz, Mario Klein, Angelika Kruse and Manuela Wenzel. IV I’m thankful to all my great colleagues within the Cramer lab and on the entire MPI campus, many of which I can count as my friends. I thank them all for lots of fun, entertainment and (non-) scientific discussions inside and outside the lab. Our weekly interdisciplinary phase separation journal club meetings at lunch with Dr. Johannes Söding, Dr. David Zwicker, Salma Sohrabi-Jahromi, Jan Kirschbaum and Matthew Grieshop have been a very fun and rewarding experience. I would like to especially thank Johannes Söding and Salma Sohrabi-Jahromi for numerous enlightening discussions. I would like to thank Jan Böhning, Kerstin Maier, Salma Sohrabi-Jahromi, Dr. Katharina Hoffmann, Dr. Goran Kokic, and Prashant Rawat for critically reading of parts of this thesis. I am grateful to the additional members of my Thesis Advisory Committee, Prof. Dr. Henning Urlaub and Prof. Dr. Steven Johnsen for providing valuable input and guidance during our meetings. I’m especially thankful to Henning Urlaub for examining this thesis as a second reviewer, for many interesting discussions and for granting me access to the mass spectrometers in his laboratory throughout the time of my PhD work. Thanks to my additional examination committee members Prof. Dr. Markus Zweckstetter, Prof. Dr. Argyris Papantonis, Prof. Dr. Herbert Jäckle, and Dr. Peter Lenart for their interest and time evaluating this thesis. Last but not least, I’m extremely grateful to my parents and my brother for their unconditional support, continuous encouragement, and sincere understanding throughout this time. V Publications Part of this work has been published or is in the process of being published: RNA polymerase II clustering through carboxy-terminal domain phase separation M. Boehning*, C. Dugast-Darzacq*, M. Rankovic*, A. S. Hansen, T. Yu, H. Marie-Nelly, D. T. McSwiggen, G. Kokic, G. M. Dailey, P. Cramer#, X. Darzacq#, M. Zweckstetter# (*) Equal contribution, (#) Corresponding author Nature Structural and Molecular Biology 25, 833–840 (2018) Author contributions: M.B. designed experiments, generated constructs, and prepared proteins unless otherwise noted. C.D.-D. designed experiments, established and characterized the RPB1 cell lines, and performed and analyzed the in vivo FRAP and SPT experiments. M.R. designed experiments, performed all phase separation assays, DIC and fluorescence microscopy, in vitro FRAP measurements and data analysis. A.S.H. designed, performed, and analyzed SPT experiments and helped with the in vivo FRAP analysis. H.M.-N. designed, performed, and analyzed 3D-PALM experiments. D.T.McS. performed cell-viability experiments and helped in performing 3D-PALM experiments. G.M.D. designed and cloned the different RPB1 expression vectors. G.K. prepared human TFIIH kinase complex. T.Y. performed CD and NMR experiments. C.D.-D., X.D., P.C., and M.Z. designed and supervised research. M.B., M.R., C.D.-D., P.C., X.D., and M.Z. prepared the manuscript with input from all authors. Stress-induced nuclear condensation of NELF drives transcriptional downregulation P. Rawat*#, M. Boehning*, B. Hummel, F. Aprile-Garcia, A. S. Pandit, N. Eisenhardt, A. Khavaran, E. Niskanen, S. M. Vos, J. J. Palmivo, A. Pichler, P. Cramer#, R. Sawarkar# (*) Equal contribution, (#) Corresponding author Manuscript in revision. Current author contributions: P.R. and R.S. initiated the project. P.R. performed imaging and molecular cell biology experiments. M.B designed and performed all in vitro experiments. S.M.V. purified recombinant NELF complex. B.H. performed all the computational analysis. F.A.G., A.S.P., A.K., N.E. and E.N. helped with the experiments. J.J.P., A.P., P.C. and R.S. supervised. P.R., M.B., P.C. and R.S. wrote the manuscript with inputs from all other authors. A detailed summary of items excerpted from published manuscripts can be found in the Appendix (‘List of items from publications’, Page 149). Excerpts from published manuscripts are additionally indicated at the beginning of each section. Co-author contributions are stated in the figure captions and/or Methods Section. The presentation of unpublished data that was not generated by the author of this thesis has been authorized by the co-authors who generated the data. VI Publications Contributions to other publications: Structure of activated transcription elongation complex Pol II-DSIF-PAF-SPT6 S. M. Vos, L. Farnung, M. Boehning, C. Wigge, A. Linden, H. Urlaub, P. Cramer# (#) Corresponding author Nature 560, 607-612 (2018) Author contributions: S.M.V. designed and conducted all experiments unless stated otherwise. L.F. established and conducted SPT6 preparation and crystallized the SPT6 tSH2 domain. M.B. determined linker phosphorylation sites by mass spectrometry. C.W. assisted in cryo-EM data collection. A.L. performed crosslinking–mass spectrometry,
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  • Amoebae and Amoeboid Protists Form a Large and Diverse Assemblage of Eukaryotes Characterized by Various Types of Pseudopodia

    Amoebae and Amoeboid Protists Form a Large and Diverse Assemblage of Eukaryotes Characterized by Various Types of Pseudopodia

    J. Eukaryot. Microbiol., 56(1), 2009 pp. 16–25 r 2009 The Author(s) Journal compilation r 2009 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2008.00379.x Untangling the Phylogeny of Amoeboid Protists1 JAN PAWLOWSKI and FABIEN BURKI Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland ABSTRACT. The amoebae and amoeboid protists form a large and diverse assemblage of eukaryotes characterized by various types of pseudopodia. For convenience, the traditional morphology-based classification grouped them together in a macrotaxon named Sarcodina. Molecular phylogenies contributed to the dismantlement of this assemblage, placing the majority of sarcodinids into two new supergroups: Amoebozoa and Rhizaria. In this review, we describe the taxonomic composition of both supergroups and present their small subunit rDNA-based phylogeny. We comment on the advantages and weaknesses of these phylogenies and emphasize the necessity of taxon-rich multigene datasets to resolve phylogenetic relationships within Amoebozoa and Rhizaria. We show the importance of environmental sequencing as a way of increasing taxon sampling in these supergroups. Finally, we highlight the interest of Amoebozoa and Rhizaria for understanding eukaryotic evolution and suggest that resolving their phylogenies will be among the main challenges for future phylogenomic analyses. Key Words. Amoebae, Amoebozoa, eukaryote, evolution, Foraminifera, Radiolaria, Rhizaria, SSU, rDNA. FROM SARCODINA TO AMOEBOZOA AND RHIZARIA ribosomal genes. The most spectacular fast-evolving lineages, HE amoebae and amoeboid protists form an important part of such as foraminiferans (Pawlowski et al. 1996), polycystines Teukaryotic diversity, amounting for about 15,000 described (Amaral Zettler, Sogin, and Caron 1997), pelobionts (Hinkle species (Adl et al.
  • Subulatomonas Tetraspora Nov. Gen. Nov. Sp. Is a Member of a Previously Unrecognized Major Clade of Eukaryotes

    Subulatomonas Tetraspora Nov. Gen. Nov. Sp. Is a Member of a Previously Unrecognized Major Clade of Eukaryotes

    Author's personal copy Protist, Vol. 162, 762–773, November 2011 http://www.elsevier.de/protis Published online date 1 July 2011 ORIGINAL PAPER Subulatomonas tetraspora nov. gen. nov. sp. is a Member of a Previously Unrecognized Major Clade of Eukaryotes Laura A. Katza,b,1, Jessica Granta, Laura Wegener Parfreya,b,2, Anastasia Ganta, Charles J. O’Kellyc, O. Roger Andersond, Robert E. Molestinae, and Thomas Neradf aDepartment of Biological Sciences, Smith College, 44 College Lane, Northampton, Massachusetts 01063, USA bProgram in Organismic and Evolutionary Biology, University of Massachusetts, 611 North Pleasant Street, Amherst, Massachusetts 01003, USA cFriday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, Washington 98250, USA dBiology, Lamont-Doherty Earth Observatory of Columbia University, Palisades, New York 10964, USA eAmerican Type Culture Collection, Protistology Collection, 10801 University Blvd., Manassas, Virginia 20110, USA fGeorge Mason University, PWII, Manassas, Virginia 10801, USA Submitted October 22, 2010; Accepted April 18, 2011 Monitoring Editor: Hervé Philippe. While a large number of aerobic free-living protists have been described within the last decade, the number of new anaerobic or microaerophilic microbial eukaryotic taxa has lagged behind. Here we describe a microaerophilic genus and species of amoeboflagellate isolated from a near-shore marine site off the coast at Plymouth, Massachusetts: Subulatomonas tetraspora nov. gen. nov. sp. This taxon is closely related to Breviata anathema based on both microscopical features and phylogenetic analyses of sequences of three genes: SSU-rDNA, actin, and alpha-tubulin. However, Subulatomonas tetraspora nov. gen. nov. sp. and B. anathema are morphologically distinctive, differ by 14.9% at their SSU-rDNA locus, and were isolated from marine and ‘slightly brackish’ environments, respectively.