University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 12-10-2015 Epigenetic Regulation of Neurogenesis By Citron Kinase Matthew irG genti University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Girgenti, Matthew, "Epigenetic Regulation of Neurogenesis By Citron Kinase" (2015). Doctoral Dissertations. 933. https://opencommons.uconn.edu/dissertations/933 Epigenetic Regulation Of Neurogenesis By Citron Kinase Matthew J. Girgenti, Ph.D. University of Connecticut, 2015 Patterns of neural progenitor division are controlled by a combination of asymmetries in cell division, extracellular signals, and changes in gene expression programs. In a screen for proteins that complex with citron kinase (CitK), a protein essential to cell division in developing brain, I identified the histone methyltransferase- euchromatic histone-lysine N-methyltransferse 2 (G9a). CitK is present in the nucleus and binds to positions in the genome clustered around transcription start sites of genes involved in neuronal development and differentiation. CitK and G9a co-occupy these genomic positions in S/G2-phase of the cell cycle in rat neural progenitors, and CitK functions with G9a to repress gene expression in several developmentally important genes including CDKN1a, H2afz, and Pou3f2/Brn2. The study indicates a novel function for citron kinase in gene repression, and contributes additional evidence to the hypothesis that mechanisms that coordinate gene expression states are directly linked to mechanisms that regulate cell division in the developing nervous system. Epigenetic Regulation Of Neurogenesis By Citron Kinase Matthew J. Girgenti B.S., Fairfield University, 2002 M.S., Southern Connecticut State University, 2004 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2015 ii Copyright by Matthew J. Girgenti 2015 iii APPROVAL PAGE Doctor of Philosophy Dissertation Epigenetic Regulation Of Neurogenesis By Citron Kinase Presented by Matthew J. Girgenti, B.S., M.S. Major Advisor:_____________________________________________________ Kent Holsinger, Ph.D. Associate Advisor:_________________________________________________ Charles Giardina, Ph.D. Associate Advisor:_________________________________________________ Stormy Chamberlain, Ph.D. Associate Advisor:_________________________________________________ James Li, Ph.D. University of Connecticut, 2015 iv This work is dedicated to my family. To my parents Jack and Eileen Girgenti for instilling in me a love of learning and to my wife Alicia Che for all of her love, encouragement, and support. v ACKNOWLEDGMENTS I would like to recognize those who aided me during the course of my dissertation work. I would like to thank my dissertation committee: Dr. Kent Holsinger, Dr. Charles Giardina, Dr. Stormy Chamberlain, and Dr. James Li for their insightful comments and hard questions. I would especially like to thank Dr. Jim Wohl, whose tireless dedication as Ombudsman and brilliant advice guided me through the toughest times in graduate school. I would also like to thank all of my undergraduates without whom this dissertation may never have been completed: Nicholas Heliotis, Amanda Collins, Christine Dougherty, Palak Shelat, Faseeha Altaf, Aditya Makol, and Shan Parikh. A special note of thanks to Dr. Aarti Tarkar for imparting with me her friendship and expertise in all things western blotting. Last but certainly not least, I would like to thank my family: my parents Jack and Eileen Girgenti for all of their support throughout my education; my brother Mark Girgenti, for all of our insightful discussions about mass spectrometry. And finally to my beautiful wife Alicia Che, whose brilliance never ceases to awe and inspire me both at home and in the lab. Matthew J. Girgenti University of Connecticut vi TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................... viii LIST OF FIGURES ............................................................................................... ix CHAPTER 1. INTRODUCTION ............................................................................ 1 Gene Expression States throughout Neurogenesis .............................................. 1 Citron Kinase and Neurogenesis .......................................................................... 3 Epigenetics and Development .............................................................................. 5 Histone Methylation ............................................................................................... 7 The Histone Methyltransferase G9a ..................................................................... 8 G9a Inhibitors ...................................................................................................... 10 Dissertation Organization .................................................................................... 11 CHAPTER 2. Citron Kinase protein is nuclear in neural progenitor cells and interacts with histone methyltransferase G9a .............................. 13 Abstract ............................................................................................................... 13 Introduction ......................................................................................................... 13 Materials and Methods ........................................................................................ 15 Results ................................................................................................................ 20 CitK is present in the nucleus of neural progenitors but masked by fixation ................................................................................... 20 CitK interacts with the histone methyltransferase G9a in neural progenitors ............................................................................... 21 Discussion ........................................................................................................... 30 CHAPTER 3. Chromatin-associated Citron Kinase Regulates Gene Expression in Rat Neural Progenitor Cells .................................................... 34 Abstract ............................................................................................................... 34 Introduction ......................................................................................................... 35 Materials and Methods ........................................................................................ 37 Results ................................................................................................................ 42 Identification of CitK genomic binding sites by ChIP-seq .................................... 42 Citron kinase regulates expression of thousands of genes ................................. 44 Citron kinase is required for G9a action but no localization to target genes ....... 46 Citron kinase and G9a co-localize to target genomic loci in S-phase ................. 48 Citron kinase function requires interaction with G9a ........................................... 49 Discussion ........................................................................................................... 70 vii CHAPTER 4. Assessing Citron Kinase’s ability to phosphorylate G9a In Rat Neural Progenitor Cells ......................................................................... 73 Abstract ............................................................................................................... 73 Introduction ......................................................................................................... 74 Materials and Methods ........................................................................................ 75 Results ................................................................................................................ 79 G9a phosphorylation levels are normal in mutant neural progenitor cells .......... 79 Citron kinase phosphorylates G9a in vitro .......................................................... 80 Phosphomimetic rescue in Citron kinase deficient neural progenitors ................ 81 Citron kinase does not phosphorylate G9a in NPs or HEK293T cells ................ 83 Discussion ........................................................................................................... 87 CHAPTER 5. Summary and Conclusion ........................................................ 90 Final Remarks ..................................................................................................... 94 APPENDIX A: LIST OF ABBREVIATIONS ...................................................... 95 APPENDIX B: PRIMERS ................................................................................... 98 B.1: Exon spanning primers used for real-time PCR of regulated transcripts ....................................................................................... 98 B.2: Promoter primers used for real-time PCR of CitK target genomic loci ............................................................................... 101 APPENDIX C: CitK Genomic Occupancy in Neural Progenitors ............... 106 APPENDIX D: Mass spectrometry Phospo-Peptides .................................. 129 D.1: In vitro Kinase Assay Phospho-peptides .................................................