GRuct rll Mechanisms of embryonic stem cell division and differentiation Josephine White Department of Molecular Biosciences (Biochemistry) The University of Adelaide Adelaide, South Australia Submitted for the degree of Doctor of Philosophy March, 2004 Thesis Summary The regulatory mechanisms governing dramatic proliferative changes during early mouse development are not well understood. This thesis aims to address this question using in vitro model systems of mouse embryogenesis. In particular, this thesis aimed to assess the function of the elevated, constitutive levels of cyclin dependent kinase 2 (CDK2) activity in embryonic stem (ES) and early primitive ectoderm-like (EPL) cells and the changes associated with differentiation into EPL embryoid bodies, in vitro equivalent of differentiation primarily to a mesodermal fate. It was determined that active CDK2 complexes associate with an increased proportion of substrates in pluripotent ES and EPL cells compared to EPL embryoid bodies. In addition, this thesis assessed the presence of other Gl CDK activity, determining that ES cells have high levels of constitutive CDK6 activity, which is refractory to inhibition by p16. Lineage specific decreases in CDK6 activity highlighted the complexities regulating cell proliferation during differentiation. Due to the reported constitutive E2F target gene expression in ES cells, this thesis also aimed to further analyse the regulation and activity of E2F transcription factors and pocket proteins in ES cells. It was demonstrated that constitutive phosphorylation of p107 and increased E2F-4 stability in ES cells contributes to increased levels of free E2F-4, that binds E2F target gene promoters in vivo. 'lhe importance of CDK regulation of pl07 in ES cells was demonstrated by analysis of ectopic expression of phosphorylation-resistant mutant p107. The increased sensitivity of EPL cells to ectopic p107 highlighted differences in pluripotent cell populations. In addition, it was determined that differential regulation of p107 during differentiation was associated with increased p107 binding to E2F target gene promoters and decreased E2F target gene expression. Differentiation associated changes in regulation and activity of cell cycle regulators demonstrated in this thesis are important for understanding the pre-gastrulating mouse embryo and to enable regulation of pluripotent cell differentiation for therapeutic use. Acknowledgments I would like to thank both past and present Head's of Department, Professor Peter Rathjen for allowing me to undertake a PhD and particularly Professor Graham Mayrhofer who rwas supportive in the latter stages. Thankyou to Steve Dalton for encouraging my interest in the wonderful world of the cell cycle. Thankyou for allowing me to make my own decisions, enabling me to become a better and more independent scientist. While it was challenging after you went overseas, I always appreciated the efforts you made to encourage me. Special thanks goes to Peter Cartwright. Thankyou for always being there to answer my endless questions and being patient when I challenged you, even though you were usually right. Thankyou for helping me become a much better scientist, technically and in critical thought. Your guidance has been fundamental and your continued help and encouragement, even after acareer change, has been invaluable. Thankyous must go to the 'old' lab for hours of entertainment and amusement. I am sure we will never be able to recreate those times. Special thanks for your understanding and patience during the many months of frustration and complaints about 'nightmare' ChIP assays and useless commercial antibodies. To Renate, by providing evidence that the perfect gels do exist, I will always live in your shadow. Thankyou for your support before, during and after the 'old' lab. I have missed our instant access to the mutual appreciation club. To Elaine, thankyou for sharing the development of the development project - what a team! Thankyou also for your listeniîg eaÍ, for singing with me for hours and for teaching me the virtues of having a constant supply of non-scientific reading material. To Dave, thankyou for being your lovely warrn self and for coping with my bad sense of humour. To Duane, thankyou for your laugh, which couldn't help but make anyone smile. Despite some shocking taste in music, you will always be the lab music man. To Cam, thankyou for extending my music collection to places I never thought I'd go, although there are still some shockers that will never enter my house! To Karen, thankyou for being great fun to have around, if only you had been with us longer. To Bujun, thankyou for keeping me company for a couple of months after the others had left - what a dynamic duo! Thankyou to my 'new' lab for welcoming me so warmly when I was experiencing destitution. To Murray Whitelaw, thankyou for making room in your very fuIl lab to accommodate me, supporting me and encouraging me to try different directions. To Anne, thankyou for so many things - your invaluable assistance with gel filtration, your kindness, your ever-ready listening ear and for providing a great role model (no, you are not too short!). To Dan, thankyou for always having time to answer any questions (no matter how ridiculous), to laugh or to make me laugh no matter how busy you were. To Susi, thankyou for always being there, particularly when sharing the experience of two most diff,rcult things - ChIP assays and thesis writing. Most importantly, thankyou for being my friend. To Fiona, thankyou for your quick wit, wonderful nature and making sure I remember there is always fun to be had. To Jodi, thanks for putting up with my constant drivel and encouraging me, by your example, to be more organised. To Anthony, thankyou for your jokes and your corny one-liners, despite any impressions to the contrary, I like them. To Cam, thankyou for being you - one of the loveliest blokes I know. To Sarah, thankyou for being your gorgeous self and providing hours of uninhibited information. To Seb, thankyou for proving that finishing does actually happen. To Alix, I couldn't have thought of a better bench replacement for Dan. Thankyou's also go to those that helped me scientifically who weren't in these labs. To Joy Rathjen, thankyou for always being there for advice on cell culture and to share my excitement when new techniques worked. To Boris Sarcevic, thankyou for providing -ê with purified cyclin-CDK complexes, particularly atthe last minute! To Patrick Humbert and Kristian Helin, thankyou for providing me withB2F4 antibodies. Special thanks goes to those who made working on the lab so much easier - John and Simon in TC, Brian, Serge and all the ladies in CSU. Thankyou so much_to Jan and Gail for helping me all my ordering needs, no matter how last minute they were. To Sharon Kolze, thãnkyou for being so supportive and helpful during all the money dramas. Thankyou also to Amanda, Sharon and many others, who have been there along the way. Thankyou to all the wonderful people around the department that made every day more enjoyable. Those that left before I did, Mike Lees, Kath Hudson, Mike Bettess and others, have been sorely missed. Those that remain, James, Nathan, Steve Kav, Colleen, Svetlana, Rebecca, Ken, Phil and many others, I look forward to more morning teas, talking constantly in TC and importantly Friday aftemoon drinks. Important thanks must go to all my friends who have made my life so pleasurable. I am so grateful to have such amazing friends. Thankyou for encouraging me to live life to the fullest and letting me live vicariously through your lives. To my lovely family - Thankyou for always being there and supporting me, even when I didn't have the energy to explain what was happening. To Mum and Dad, thankyou for everything. This entire experience reminded me how lucky I am to have had such wonderful parents. To Em and Dave, thankyou for your love and always being interested in my work even when I was reluctant to talk about it. To Mark and Tanya, th-ankyou for being there. I always enjoy our time together, if only it was more often. To Jo and pat, thankyou for your support and making me feel so welcome in your family. And of course, thanks must go to my study-buddy Abbey, whose wagging tail and attentiveness was so comforting during many hours of typing. Finally, thankyou to Tony - all the best adjectives (shift F7 and, beyond) would only begin to describe how important you are to me. I am so very lucky to share my life witir such a tremendous man. Thankyou for sharing a love people only dream of. TABLE OF CONTENTS CIIAPTER 1: Introduction 1.1 The mammalian cell cycle 1.2 Regulation of cyclin dependent kinase activity..... L2.1 Cyclin Binding..... 1.2.2 Phosphorylation of cyclin dependent kinases 1.2.3 Cyclin dependent kinases inhibitors... 1.3 Functions of cyclin dependent kinase activity... 1.3.1 Cyclin D-CDK416.... 1.3.2 Cyclin E-CDK2 1.3.3 Cyclin A-CDK2 10 1.4 Regulation of Gl/S phase progression 11 1.4.1 E2F Transcription Factors. 11 1.4.2 The pocket protein fami1y....... 15 1.5 Deregulation of the cell cycle in cancer 2l 1.ó The cell cycle and embryogenesis 22 1.6.1 Early mouse embryogenesis........... 23 1.6.2 Cell cycle regulation and early mouse embryogenesis........ 26 1.6.3 Invitro model systems of earlymouse embryogenesis....... 27 1.6.4 Cell cycle regulation of embryonic stem cells... 30 1 .6. 5 Cell cycle regulation during differentiation....... JZ 1.7 Aims JJ CHAPTER 2zMaterials and Methods 2. 1 Abbreviations..... 35 2.2 Bactenal Manipulations........... 2.2.1 Matenals 2.2.2 Buffers and Solutions 2.2.3 Bacterial Strains and Media.........