THE ROLE OF CYCLIN B3 IN MAMMALIAN MEIOSIS by Mehmet Erman Karasu A Dissertation Presented to the Faculty of the Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy New York, NY November, 2018 Scott Keeney, PhD Date Dissertation Mentor Copyright © Mehmet Erman Karasu 2018 DEDICATION I would like to dedicate this thesis to my parents, Mukaddes and Mustafa Karasu. I have been so lucky to have their support and unconditional love in this life. ii ABSTRACT Cyclins and cyclin dependent kinases (CDKs) lie at the center of the regulation of the cell cycle. Cyclins as regulatory partners of CDKs control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Similar to somatic cell division, temporal regulation of cyclin-CDK activity is also important in meiosis, which is the specialized cell division that generates gametes for sexual production by halving the genome. Meiosis does so by carrying out one round of DNA replication followed by two successive divisions without another intervening phase of DNA replication. In budding yeast, cyclin-CDK activity has been shown to have a crucial role in meiotic events such as formation of meiotic double-strand breaks that initiate homologous recombination. Mammalian cells express numerous cyclins and CDKs, but how these proteins control meiosis remains poorly understood. Cyclin B3 was previously identified as germ cell specific, and its restricted expression pattern at the beginning of meiosis made it an interesting candidate to regulate meiotic events. Although its first characterization in mammalian cells was nearly 20 years ago, the functions of cyclin B3 have remained elusive. This thesis examines the role of mouse cyclin B3 in mammalian meiosis. Generation of highly specific monoclonal antibodies enabled characterization of cyclin B3 expression during spermatogenesis. Cyclin B3-deficient mice were generated by CRISRP-Cas9 mediated genome engineering. Unexpectedly, male mice lacking cyclin B3 did not show any detectable meiotic defects. However, mutant females were unable to produce progeny, indicating that cyclin B3 is iii essential for female fertility. Moreover, although expression patterns in spermatogenesis led to the expectation of a function for cyclin B3 during meiotic prophase, cyclin B3-ablated ovaries and follicular oocytes were structurally similar to controls, suggesting that meiotic prophase and development of oocytes up to entry into meiosis I are normal. Cyclin B3-deficient oocytes were also able to enter meiosis I when cultured ex vivo. However, they failed to complete the meiosis division I and arrested at the metaphase I to anaphase I transition with hallmarks of defective activation of the anaphase promoting complex/cyclosome (APC/C). Specifically, the arrested oocytes displayed high levels of securin and cyclin B1, high maturation promoting factor (MPF) activity and little or no separase activation. The arrested mutant oocytes could be rescued by inhibiting MPF activity or by injecting mRNA encoding wild-type cyclin B3 or a mutant form lacking its destruction box. Lastly, cyclin B3 homologs from frog, zebrafish and fruitfly could rescue the arrest in cyclin B3 ablated oocytes suggesting evolutionarily conserved roles for this cyclin, and opening up further possibility to study cyclin B3 in more experimentally tractable organisms such as frogs. Together, these studies establish a critical role for cyclin B3 in fine-tuning APC/C activity to promote the unique cell division program of oocyte meiosis. iv BIOGRAPHICAL SKETCH Mehmet Erman Karasu was born on November 8, 1988 in Izmir, Turkey. Erman grew up in Bornova, Izmir and graduated from Izmir Science High School in 2006. He obtained his Bachelor of Science degree in Molecular Biology and Genetics from Bilkent University (Ankara/Turkey). During his undergraduate education, Erman did several internships around the world, including SURP program in Gerstner Sloan Kettering Graduate School in 2010. In 2011, Erman moved to New York and started the doctoral program at Louis V. Gerstner Jr. Sloan Kettering Graduate School. In 2012, he joined the laboratory of Dr. Scott Keeney in Molecular Biology Program at Memorial Sloan Kettering Cancer Center, where he conducted his doctoral dissertation on investigating the role of cyclin B3 in mammalian meiosis. v ACKNOWLEDGEMENTS First and foremost, I would like to acknowledge and thank my mentor Scott Keeney for his guidance, support and patience. This thesis has been a long and not a smooth journey for me. For the first couple of years, I was not able to make any progress. However, he always stood by me and told me that I can complete this work. I was so lucky to work alongside such a talented and intelligent scientist. I am sincerely grateful that Scott took his time to teach me how to do science properly. I would like to thank my thesis committee members, Drs. Andrew Koff and Dirk Remus. In particular, I would like to thank Dr. Koff for the characterization of cyclin B3 in mammals and giving me unlimited access to Koff lab antibodies. I would like to thank Dr. John Petrini agreeing on for the chairman position in my thesis committee. I would like to also thank Dr. Debra Wolgemuth not only being my external committee member, but also for the several nice reviews and research papers regarding cyclins during the spermatogenesis. I would like to thank Keeney lab members, who made the Keeney Lab as a fun place to come and do science every day, including weekends. It was quite a joy to work with all of them and I will definitely miss each one of them in the future. I believe that they were not only colleagues to me but also close friends. I was so lucky to be a part of this amazing group of people. I cannot thank every one of them enough for all the support and assistance on this journey. I would like to thank particularly Boeke who worked on a gene that I identified in a yeast two-hybrid screen. Boeke took the project from the beginning vi and completed it, without him, we might not have heard of the gene called Ankrd31. I would like to thank my “French lab” as well. Nora Bouftas and Dr. Katja Wassmann are the ones I worked with to investigate cyclin B3 in oocytes. The Wassmann group hosted me for 7 months in Paris and taught me how to work with oocytes. Without their collaboration, chapter 3 would not be in this thesis. I cannot thank them enough for their assistance and patience with me. I would like to thank Corentin Claeys Bouuaert and Ryo Hayama for the discussion on the thesis chapters. In particular, I would like to thank Corentin for critical reading and for his help in editing thesis chapters. Finally, I would like to thank all my friends and family who have been with me on this journey. I would not have been able to finish this thesis without your support. I love you all. vii TABLE OF CONTENTS Introduction ............................................................................................................... 1 Cell cycle............................................................................................................................1 Cyclin-CDK complexes in mitosis .........................................................................................2 Cyclin D ................................................................................................................................................... 3 Cyclin E ................................................................................................................................................... 4 Cyclin A and B ......................................................................................................................................... 5 Regulation of cyclins in mitosis through proteasomal degradation ......................................7 Meiosis ..............................................................................................................................8 Double strand break formation ........................................................................................................... 10 Homolog synapsis through synaptonemal complexes ........................................................................ 11 Meiotic recombination and crossover designation ............................................................................. 12 Cyclin-CDK complexes in meiotic prophase I in budding yeast ........................................................... 13 Meiosis in mammals ........................................................................................................ 15 Oogenesis ............................................................................................................................................. 15 Resumption of meiosis I ....................................................................................................................... 17 M phase regulation through cyclin-CDK complexes and Metaphase I to Anaphase I transition ........ 18 Spindle assembly checkpoint mediated control of APC/C activity ....................................................... 20 Spermatogenesis ............................................................................................................. 20 Regulated cyclin expression during spermatogenesis ......................................................................... 21 Phenotypes of cyclin-CDK