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Understanding Heterochromatin Biology Through Histone Mutagenesis

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Understanding Heterochromatin Biology Through Histone Mutagenesis UNDERSTANDING HETEROCHROMATIN BIOLOGY THROUGH HISTONE MUTAGENESIS Taylor Joel Richard Penke 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 Curriculum in Genetics and Molecular Biology. Chapel Hill 2017 Approved by: Robert J. Duronio A. Gregory Matera Daniel J. McKay Brian D. Strahl Jeff J. Sekelsky © 2017 Taylor Joel Richard Penke ALL RIGHTS RESERVED ii ABSTRACT Taylor Joel Richard Penke: Understanding Heterochromatin Biology through Histone Mutagenesis (Under the direction of Robert J. Duronio) The relatively large genomes of eukaryotic cells must be organized and compacted within the nucleus while maintaining DNA accessibility for essential processes such as transcription, replication, and DNA repair. This organization is accomplished in large part through the interaction of DNA with histone proteins to form a structure known as chromatin. Chromatin organization is regulated through epigenetic mechanisms, such as histone post-translational modifications (PTMs) or the differential incorporation of variant or canonical histones into chromatin. These processes are regulated differently throughout the genome, leading to functionally distinct chromatin environments. Regions where DNA is “open” or more accessible are collectively referred to as euchromatin, whereas “closed” or inaccessible regions are classified as heterochromatin. Proper heterochromatin formation is essential for regulating numerous cellular processes including, cell division, nuclear organization, gene expression, and DNA replication. A defining feature of heterochromatin is methylation of lysine nine on histone H3 (H3K9me), a histone PTM that recruits Heterochromatin Protein 1 (HP1). Although H3K9 methyltransferases and HP1 are necessary for proper heterochromatin structure, the specific contribution of H3K9 to heterochromatin function and animal development is unknown. Using our recently developed platform to engineer histone iii genes in Drosophila, I generated H3K9R mutant flies, separating the functions of H3K9 and non-histone substrates of H3K9 methyltransferases. I observed that H3K9 plays an essential role in regulating the structure of pericentromeric heterochromatin and the repression of transposons, but not protein-coding gene expression. Furthermore, I generated a K9R mutation in the variant histone H3.3, revealing functional redundancies between variant H3.3K9 and canonical H3K9, though to differing extents in heterochromatin and euchromatin. Finally, I have used the H3K9R mutant as a tool to uncover general principles of genome regulation. Several previous studies have identified correlations between histone PTMs, transcription, and DNA replication; however, no causative relationship between these processes has been identified. The H3K9R mutant specifically disrupts pericentromeric heterochromatin providing a unique opportunity to determine the direct consequences of altered chromatin structure on replication. We demonstrated that changes in chromatin accessibility and most likely transcription are required but not sufficient for altered replication, influencing the framework through which we view genome regulation. iv To my family both old and new v ACKNOWLEDGEMENTS My journey through graduate school has benefitted from numerous individuals all of whom have my heartfelt thanks for making this experience personally and professionally fulfilling. First, I would like to thank Bob who has been an exemplary mentor and whose guidance has improved all aspects of my science. I am particularly grateful for the independence I was granted, his trust in my opinions and decisions, and his permission to pursue the scientific questions and skills I was interested in. The most telling aspect of Bob’s mentorship is his desire to see his students succeed both in and outside of lab, often at the expense of his own interests. This quality was evident throughout my career at UNC and especially as I decided on my next career step. I would also like to thank the members of my committee for their direction throughout many lab, histone group, and committee meetings. I thank Brian for sharing his passion for chromatin and a plethora of ideas never limited by perceived feasibility. Greg, thank you for instilling in me a meticulous attention to detail and being a great additional mentor. Jeff is one of the few Packers fans whose opinions I respect; I have appreciated the many suggestions you have given me. Finally, I am obliged to Dan for imparting many of his technical skills. Thank you for sharing many enjoyable scientific discussions and back of the fly box genetic schemes. I am also grateful to have shared my graduate school experience with many wonderful lab and pod members. I have been blessed to work alongside many talented vi scientists in the Matera, McKay, and Sekelsky labs. In particular, I would like to thank Mike Meers for establishing much of the scientific foundation that has facilitated what success I have had and for being a constant computational resource. Everyone in the Duronio lab has enriched my experience here, and although there are too many to name, I would like to thank Joy, Deirdre, and Este for going out of their way to welcome me to the lab and Mary for running the lab so efficiently. Finally, I would like to acknowledge Robin Armstrong as one of the most capable and enthusiastic scientists I have known. It has been a true pleasure to collaborate with you. Outside of lab I have been supported by great friends both in Carolina and back in Minnesota. At the risk of leaving someone out, I won’t name each of you, but I hope you all know I have cherished your friendship. As we continue to go separate ways, I hope we can remain in touch. Special thanks go out to Michael Bertucci for introducing me to the volleyball community at UNC and for remaining a close friend. I am also very grateful to have shared the adventure of moving to North Carolina with my former roommate and old friend Nathan Rodeberg. You made the transition to graduate school not only bearable but enjoyable, and I value the many experiences we have shared since. Finally, I am most thankful for the support of my family. To David and Nanci, I am incredibly grateful for the loving and welcoming environment you have shared with me. Charlotte has been and continues to be a home away from home. To my father and mother, I thank you for instilling in me the work ethic and ambition that I have heavily relied on throughout graduate school. Knowing I always have someone to fall back on has given me the confidence to leave home and push myself personally and vii professionally. To my sisters Dani and Grace, I hope I have been a good role-model even while living away. I now see in you both qualities that I myself look up to. To my wife and best friend Kelly, I thank you for encouraging me through every hardship and celebrating every triumph of graduate school. You have improved all aspects of my life, and I look forward to every adversity and accomplishment that the future brings us. viii TABLE OF CONTENTS LIST OF TABLES .......................................................................................................... xiii LIST OF FIGURES ........................................................................................................ xiv LIST OF ABBREVIATIONS .......................................................................................... xvii CHAPTER 1 – INTRODUCTION ..................................................................................... 1 Chromatin regulation of DNA-dependent processes .................................................... 1 A histone gene replacement platform enables metazoan histone genetics.................. 5 Historical perspective of heterochromatin .................................................................. 11 CHAPTER 2 – DIRECT INTERROGATION OF THE ROLE OF H3K9 IN METAZOAN HETEROCHROMATIN FUNCTION ..................................................... 20 Introduction ................................................................................................................ 20 Materials and Methods ............................................................................................... 23 Culture conditions ................................................................................................... 23 Immunofluorescence .............................................................................................. 24 Sample Preparation for Sequencing ....................................................................... 25 Sequence Data Analysis ......................................................................................... 25 Results ....................................................................................................................... 26 H3K9 is important, but not essential, for completion of Drosophila development ... 26 H3K9 regulates chromocenter organization and nucleosome occupancy .............. 33 H3K9 is required for HP1a localization to pericentromeric heterochromatin ........... 42 ix H3K9 prevents accumulation of transcripts from heterochromatic regions of the genome ............................................................................................ 50 H3K9 represses transposon activation and mobilization ........................................ 59 Discussion .................................................................................................................
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