Molecular function and regulation of the histone methyltransferase, Ezh2 By Susan C. Wu A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics Chapel Hill 2011 Approved by: Dr. Yi Zhang Dr. Yue Xiong Dr. William Marzluff Dr. Adrienne Cox Dr. Young Whang ©2011 Susan C. Wu ALL RIGHTS RESERVED ii ABSTRACT Susan C. Wu Molecular function and regulation of the histone methyltransferase, Ezh2 (Under the direction of Dr. Yi Zhang) EZH2 (Enhancer of zeste 2) is an important developmental regulator that plays an important role in numerous biological processes including multi-cellular development, stem cell biology, and cancer development. As an H3K27 histone methyltransferase, EZH2 is an epigenetic regulator that functions to repress transcription. It is often upregulated in human cancers and its oncogenic potential is thought to stem from its role in cell proliferation and repression of tumor suppressor genes. Hence, regulation of EZH2 function is important for normal cellular growth. In this dissertation, the molecular function and regulation of EZH2 are investigated. I demonstrate that genomic targets of EZH2 not only include protein- coding genes, but also encompasses lncRNAs (long non-coding RNAs), a class of RNAs that account for the vast majority of the mammalian transcriptome. Using a custom-designed microarray, I show that lncRNAs exhibit cell type specific expression patterns and that their expression levels correlate with the epigenetic marks, H3K4me3 and H3K27me3. Consistent with this finding, knockdown of EZH2 results in the derepression of many lncRNAs. With regard to EZH2 regulation, I provide evidence that EZH2 contains numerous phosphorylation sites although their functions are currently unknown. Characterization of two phosphorylation sites, threonines 345 and 487, has revealed important aspects of EZH2 iii regulation. Through in vitro and in vivo experiments, I show that phosphorylation of these two residues can be mediated by the cyclin dependent kinase, CDK1. Consistent with the cell cycle phase that exhibits peak CDK1 activity, mitotic cell extracts are enriched for phospho-EZH2. Importantly, when EZH2 is phosphorylated at T345 and T487, the stability of the protein is compromised as EZH2 is targeted for ubiquitination and degradation by the proteasome. Collectively, this work reveals a novel class of EZH2 targets and provides a mechanism by which EZH2 protein levels are regulated in the cell. iv To my father, Y. John Wu, who persistently instilled my interest in science at an early age. Without his support and the countless lectures encompassing broad scientific topics, I would not be where I am today. v ACKNOWLEDGEMENTS I must take a moment to acknowledge the extraordinary role that the following people had in shaping this work. First and foremost, I would like to thank Dr. Yi Zhang for his guidance during my tenure as a graduate student. I am indebted to him for his critical comments of my work as well as the opportunities that he has provided with me, both of which have pushed me to become a better scientist. I would also like to acknowledge my dissertation committee members: Drs. Yue Xiong, William Marzluff, Adrienne Cox, and Young Whang. Their continued support and scientific guidance were undoubtedly instrumental to the completion of this work. In addition, I would also like to thank Dr. Jean Cook who generously invested her time in providing me with the scientific expertise that I needed. I was also fortunate to work with an outstanding group of scientists on a day to day basis. Thank you to past and present members of the Zhang lab who provided me with both the encouragement and technical expertise that I needed over the years. In particular, I would like to thank Dr. Eric Kallin who I not only had the privilege of collaborating with, but who also contributed to my personal well-being. Drs. Nara Lee and Rob Klose were instrumental in my scientific development and I would be remiss to not thank them. I also have to thank vi Jake Dmochowski, an outstanding undergraduate student, who not only worked with me, but also kept me on my toes with his inquisitive questions. In retrospect, I have learned that graduate school is not for the faint of heart and that the challenges brought forth in front of me undoubtedly required the loving support of the wonderful people in my life. First and foremost, I have to thank my parents, Y. John and Grace Wu who have pushed me to realize my potential and unconditionally supported my decision to attend graduate school. I also have to thank my brothers Jeff and Greg as well as my sister-in-law, Helen, who have provided me with so much laughter over the years. I am extremely grateful to be a part of such an incredible family. In addition, I want to thank all of the friends that I have made during my time in Chapel Hill. In particular, I have to acknowledge Elizabeth Dorn who has been a fabulous co-pilot to have in this journey. Finally, none of this would be possible without the constant support and encouragement from my husband, Chris Winkler. I would like to thank him for his extraordinary patience and eternal positivity over the years. His companionship is truly special and I am sincerely grateful for it. vii TABLE OF CONTENTS Page LIST OF TABLES....................................................................................................................xi LIST OF FIGURES.................................................................................................................xii LIST OF ABBREVIATIONS.................................................................................................xiv CHAPTER I. INTRODUCTION..................................................................................................1 i. Epigenetic regulation of gene expression.................................................3 ii. Histone methylation.................................................................................5 iii. Polycomb group of proteins.....................................................................8 iv. Polycomb repressive complexes............................................................10 v. Functions of H3K27 methylation...........................................................15 vi. Recruitment of PRC2 to genomic targets...............................................18 vii. ES cell pluripotency, self-renewal, and differentiation..........................22 viii. EZH2 and human cancers......................................................................23 ix. Research summary.................................................................................25 x. References..............................................................................................35 II. ROLE OF H3K27 METHYLATION IN THE REGULATION OF LONG NON-CODING RNA EXPRESSION..................................................................50 viii i. Abstract..................................................................................................51 ii. Introduction............................................................................................52 iii. Results....................................................................................................54 iv. Discussion..............................................................................................60 v. Experimental procedures........................................................................63 vi. References..............................................................................................73 II. EZH2 IS A THREONINE PHOSPHORYLATED..............................................77 i. Abstract..................................................................................................78 ii. Introduction............................................................................................79 iii. Results....................................................................................................83 iv. Discussion..............................................................................................89 v. Experimental procedures........................................................................92 vi. References............................................................................................103 III. CDK1-MEDIATED PHOSPHORYLATION OF EZH2 LEADS TO DEGRADATION...............................................................................................108 i. Abstract................................................................................................109 ii. Introduction..........................................................................................110 iii. Results..................................................................................................113 iv. Discussion............................................................................................123 v. Experimental procedures......................................................................128 vi. References............................................................................................148 IV. SUMMARY AND CONCLUDING REMARKS..............................................152 i. Epigenetic regulation of long non-coding RNAs..................................153 ii. Long non-coding RNAs and ES cell biology.......................................156 ix iii. Investigating