BIOLOGICAL FUNCTION OF E2F7 AND E2F8 IS ESSENTIAL FOR EMBRYO DEVELOPMENT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Jing Li, M.S. ***** The Ohio State University 2009 Approved by Dissertation Committee: Gustavo Leone, Ph.D., Advisor Susan Cole, Ph.D. ________________________________ Paul Herman, Ph.D. Advisor Tim Huang, Ph.D. Graduate Program in Molecular Genetics Copyright Jing Li 2009 ABSTRACT The novel E2F7 and E2F8 family members are thought to function as transcriptional repressors important for the control of cell proliferation in vitro. However, as the most recently indentified and least studied E2F members, their biological functions in vivo remain unknown. Here we have analyzed the consequences of inactivating E2f7 and E2f8 in mice. While loss of either E2f7 or E2f8 did not significantly affect mouse development, their combined ablation resulted in massive apoptosis, dilated blood vessels and severe placental defects, culminating in embryonic lethality by day 11.5. E2F7 and E2F8 formed homo-dimers and hetero-dimers that could recruit various co-repressor complexes to E2F binding sites of target promoters, including E2f1. Consistent with their important role in transcriptional repression, mouse embryonic fibroblasts (MEFs) deficient for E2f7 and E2f8 expressed abnormally high levels of E2f1 and other E2F-target mRNAs. These double knockout MEFs proliferated surprisingly well, but accumulated high levels of p53 protein and were hypersensitive to DNA damage-induced cell death. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis observed in double mutant embryos but failed to rescue their embryonic lethality. ii In order to identify the leading cause of fetal death, the critical tissues, related cellular processes and molecular mechanisms of E2F7 and E2F8 function, we utilized conditional knockout strategies to show that extra-embryonic function of E2F7 and E2F8 is both necessary and sufficient for embryo development, and thus define the placental abnormalities as the leading cause of embryonic lethality in E2f7-/-E2f8-/- embryos. Consistent with this genetic finding, cellular examination of double mutant placentas revealed ectopic DNA replication and inappropriate mitosis in certain cell lineages of placenta. We also provided two distinct molecular mechanisms of E2F7 and E2F8 function to explain these cellular phenotypes. On one hand, E2F7 and E2F8 could directly repress a novel transcription network that is critical for controlling DNA replication. On the other hand, E2F7 and E2F8 could indirectly regulate the expression of mitotic cyclins and therefore coordinate mitosis progression. We believe disruption of these pathways in E2f7-/-E2f8-/- placentas culminates in G1- S and G2-M specific defects and, presumably, leads to the profound placental abnormality and its associated fetal death. In summary, this study clearly provides the first in vivo evidence for the biological functions of E2F7 and E2F8. We demonstrate, as a unique repressive arm of the E2F program, E2F7 and E2F8 are not only critical for the control of apoptotic in the fetus, but also essential for the regulation of cell cycle progression in the placenta. We conclude that the synergistic function of E2F7 and E2F8 is essential for embryo development. iii DEDICATION To my mother and father with deep respect and love, and to all those whose constant support has helped me go through my graduate study. iv ACKNOWLEDGMENTS I would like to thank my advisor Dr. Gustavo Leone for his consistent support, the enthusiasm and knowledge he brought to me during my Ph.D. study. This study would have not been accomplished without his guidance. I am also grateful for having Dr. Susan Cole, Dr. Paul Herman, and Dr. Tim Huang in my committee. Their continuous mentorship and support in all my scientific endeavors are highly appreciated. The past and present members of the Leone lab are equally precious for me during my graduate study. I would like to give my thanks to Dr. Alain de Bruin, Dr. Pamela Wenzel, Shantanu Singh, Madhu Ouseph, Dr. Anthony Trimboli, Dr. Hui Wang, Dr. Leon Chong, Dr. Enrico Caserta, Dr. Soo-in Bae, and Lisa Rawahneh, not only for their advices and assistance during my graduate research, but also for the valuable discussions which have helped me become a better scientist. I can not thank enough my undergraduate students, Ran Cong, Grant Comstock and Branxton Forde. It was such a pleasant experience working with them, as a mentor, or more as a friend. v I sincerely thank my friends Xin Li, Fu Li, Hongtao Jia, Ainan Bao, Sudu Sharma, Judy Kuo and many others, who have always been there whenever I need advice or help. Last but not least, I will forever treasure the support and encouragement provided by my parents. Every accomplishment that I have ever achieved belongs to them. vi VITA 1996 – 2000……………………………………...B.S. Bioengineering Shanghai University 2000 – 2003……………………………………...M.S. Human Genetics Shanghai University 2003 – 2004……………………………………...University Fellow The Ohio State University 2004 – 2005……………………………………...Graduate Teaching Assistant The Ohio State University 2005 – 2009……………………………………..Graduate Research Assistant The Ohio State University PUBLICATIONS 1. Li, J., Chen, Y.G. and Kong, X.Y. 2001. New progress of serial analysis of gene expression. Sheng Wu Gong Cheng Xue Bao 17: 613-616. 2. Li, J., Hu, L.D., Wang, W.J., Chen, Y.G. and Kong, X.Y. 2003. Linkage analysis of the candidate genes of familial restless legs syndrome. Yi Chuan Xue Bao 30: 325-329. 3. Chang, Q., Pang, J.C., Li, J., Hu, L., Kong, X. and Ng, H.K. 2004. Molecular analysis of PinX1 in medulloblastomas. Int. J. Cancer 109: 309-314. 4. Maiti, B., Li, J., de Bruin, A., Gordon, F., Timmers, C., Opavsky, R., Patil, K., Tuttle, J., Cleghorn, W. and Leone, G. 2005. Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation. J. Biol. Chem. 280: 18211-18220. vii 5. Li, J., Ran, C., Li, E., Gordon, F., Comstock, G., Siddiqui, H., Cleghorn, W., Chen, H.Z., Kornacker, K., Liu, C.G., Pandit, S.K., Khanizadeh, M., Weinstein, M., Leone, G. and de Bruin, A. 2008. Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development. Dev. Cell. 14: 62- 75. 6. Lammens, T.*, Li, J.*, Leone, G. and De Veylder, L. 2009. Atypical E2Fs: new players in the E2F transcription factor family. Trends in Cell Biology 19: 111-118. *Co-first author. 7. Li, J., Ouseph, M., Comstock, G., Thompson, J., Forde, B., Chong, J. and Leone, G. E2F7 and E2F8 control proliferation and govern placental function during development. Dev. Cell. (In preparation). FIELDS OF STUDY Major Field: Molecular Genetics viii TABLE OF CONTENTS Page Abstract……………………………………………………………………….. ii Dedication……………………………………….................………………… iv Acknowledgements………………………………………………………….. v Vita…………………………………………………………………………….. vii List of Tables………………………………………………………………..... xv List of Figures………………………………………………………………… xvi List of Abbreviations…………………………………………………………. xix Chapters: 1 Introduction…………………………………………………………..….. 1 1.1 Cell cycle regulation……………………………………………………. 1 1.2 The E2F family of transcription factors………………………………. 2 1.3 Atypical E2Fs: new players in the E2F family…………………….…. 4 1.3.1 Molecular features of atypical E2F proteins……………..….... 5 1.3.2 Transcriptional regulation of atypical E2f expression……….. 7 1.3.3 Atypical E2F proteins and cell size control…………………… 10 ix 1.3.4 Atypical E2F proteins and cell cycle control………………..… 10 1.3.5 Atypical E2F proteins and DNA damage response………..… 13 2 Synergistic function of E2F7 and E2F8 is essential for embryonic development and cell survival…………………………………………. 19 2.1 Introduction……………………………………………………………… 19 2.2 Results…………………………………………………………………… 22 2.2.1 E2F7 and E2F8 are essential for embryonic viability………... 22 2.2.2 Genetic ablation of E2f7 and E2f8 in vivo induces massive apoptosis in the fetus…………………………………………………… 23 2.2.3 E2F7 and E2F8 form homo-dimers and hetero-dimers……... 24 2.2.4 E2f1 is a direct target of E2F7 and E2F8……………………... 25 2.2.5 E2f1 expression is deregulated in E2f7-/-E2f8-/- MEFs………. 26 2.2.6 E2f7-/-E2f8-/- cells are hypersensitive to DNA damage……… 28 2.2.7 Induction of apoptosis in E2f7-/-E2f8-/- embryos is dependent on E2F1 and p53………………………………………………………... 29 2.3 Discussion……………………………………………………………….. 30 2.4 Materials and Methods…………………………………………………. 34 2.4.1 Generation of E2f7 and E2f8 knockout mice…………………. 34 2.4.2 Quantitive reverse transcriptase PCR (RT-PCR)……............. 35 2.4.3 Affimetrix microarray analysis………………………………….. 35 2.4.4 Co-immunoprecipitation (Co-IP) assay…..……………….…... 36 2.4.5 Chromatin immunoprecipitation (ChIP) and sequential ChIP assays……………………………………………………………………. 36 x 2.4.6 Western blot and antibodies……………………………………. 38 2.4.7 Cell culture and viability assay…………………………………. 38 2.4.8 FACS analysis…………………………………………………… 38 2.4.9 BrdU and TUNEL assays…………………………...………… 39 3 Extra-embryonic function of E2F7 and E2F8 is essential for fetal survival…………………………………………………………………… 60 3.1 Introduction…………………………………….………………………… 60 3.2 Results…………………………………………………………………… 62 3.2.1 Loss of E2f7 and E2f8 leads to profound placental defects… 62 3.2.2 Wild type placenta is sufficient to support mutant fetuses to birth……………………………………………………………………….. 64 3.2.3 E2f7 and E2f8 are essential in the placenta………………….. 65 3.2.4 Loss of E2f7 and E2f8 in the placenta