The Identification of a New Rpd3 Deacetylase Complex Involved in the Yeast Oxidative Stress and Metabolism Pathways

Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2012 PHDS, Stress, And Starvation: The deI ntification Of A New RPD3 Deacetylase Complex Involved In The eY ast Oxidative Stress And Metabolism Pathways Lindsey A. Baker Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Baker, Lindsey A., "PHDS, Stress, And Starvation: The deI ntification Of A New RPD3 Deacetylase Complex Involved In The eY ast Oxidative Stress And Metabolism Pathways" (2012). Student Theses and Dissertations. Paper 144. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. PHDS, STRESS, AND STARVATION: THE IDENTIFICATION OF A NEW RPD3 DEACETYLASE COMPLEX INVOLVED IN THE YEAST OXIDATIVE STRESS AND METABOLISM PATHWAYS A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfi llment of the Requirements for the degree of Doctor of Philosophy by Lindsey A. Baker June 2012 © Copyright by Lindsey A. Baker 2012 PHDS, STRESS, AND STARVATION: THE IDENTIFICATION OF A NEW RPD3 DEACETYLASE COMPLEX INVOLVED IN THE YEAST OXIDATIVE STRESS AND METABOLISM PATHWAYS Lindsey A. Baker, Ph.D. The Rockefeller University 2012 The cellular pathways that govern survival in the face of diverse stresses rely on gene expression changes as one mechanism to respond to or protect against internal and external threats. Because eukaryotic DNA is packaged into chromatin, these gene expression changes depend on the targeting of regulatory proteins to specifi c regions of the genome to alter chromatin structure, promoting or repressing transcription. One protein domain involved in targeting chromatin regulators is the plant homeodomain, or PHD fi nger, a module that preferentially interacts with either methylated or unmethylated lysines on histones, and has important functions in human health. Despite recent advances in identifying the histone ligands for some PHD fi ngers as well as the functions of the proteins that contain them, for many other PHD fi ngers, including some of the 17 PHD fi ngers of the budding yeast Saccharomyces cerevisiae, these questions remain unanswered. In the research presented in this thesis, I sought to gain insight into the ligands and functions for three yeast PHD fi nger proteins, the Yng1 subunit of the NuA3 acetyltransferase complex, Jhd2, and Ecm5, the latter two both being homologous to the mammalian JARID family of histone demethylases. In Chapter 2, I demonstrate that the PHD fi ngers of these proteins interact with histone H3 enriched for different sites of methylation depending on the PHD and present results of an in vitro assay used to test whether any yeast PHD fi ngers possess ubiquitin E3 ligase activity, a function ascribed to the PHD-related RING domain. In Chapter 3, I discuss experiments performed to identify the protein interaction partners of Jhd2 and Ecm5, culminating in the discovery that Ecm5 interacts with the PHD fi nger protein Snt2 as well as the Rpd3 deacetylase, forming a complex I have named Rpd3(T). I also discuss experiments showing that the ecm5 knockout strain does not have obvious defects in many yeast pathways. In Chapter 4, I present evidence that Rpd3(T) complex members are involved in the cellular oxidative stress and metabolism pathways, and discuss chromatin immunoprecipitation experiments followed by high-throughput sequencing which were performed to map Ecm5 and Snt2 localization before and after hydrogen peroxide-mediated oxidative stress. I then discuss how the Ecm5 and Snt2 localization patterns relate to gene expression changes in wild-type cells after oxidative stress and in snt2 knockout cells. I compare Ecm5 and Snt2 localization patterns in rich media before and after oxidative stress to patterns in less rich media before and after nutrient stress induced by the TOR pathway inhibitor rapamycin. Finally, I discuss potential mechanisms through which Ecm5 and Snt2, either as a pair or as a part of the Rpd3(T) complex, may help to coordinate the cellular responses to oxidative and nutrient stresses, and the greater implications of this work. ACKNOWLEDGEMENTS First, I would like to thank my advisor, Dr. David Allis, for providing scientifi c guidance over the course of my graduate career and more importantly, providing moral support through the diffi cult parts of this project. Dave’s unquestioning faith in my abilities to become a successful scientist have inspired me to surpass what I intially thought I was capable of. I have particularly enjoyed his enthusiasm for all things chromatin and his love of puns (which I share). I also owe an immense debt of gratitude to my Faculty Committee members, Dr.’s Tom Muir and Michael Rout, who were always willing to listen to my ideas and offer helpful feedback. No matter how nervous I felt going into a Committee Meeting, their enthusiasm for my project always left me feeling elated afterwards. I am extremely thankful to Dr. Beatrix (Trixi) Ueberheide, who has been not only an amazing collaborator, but also a good friend. All of that cryo-grinding wouldn’t have been nearly as enjoyable without a fellow show tune lover to belt out songs with. Over the years, I have received scientifi c support from numerous sources without whom many of the experiments described in this thesis would not have been possible. I thank Dr. Sean Taverna for introducing me to the Allis lab, coaching me through my rotation, teaching me the art of Tetrahymena nuclear preparations, and writing me an awesome karaoke song. I thank Dr. Beth Duncan for being an excellent bay-mate, and for teaching me the ins and outs of qPCR. I thank Dr. Peter Lewis for showing me that radioactivity is not scary, guiding me through my fi rst northern blots, and always having useful suggestions for ways to improve my biochemical techniques. I also owe thanks to Dr. Ronen Sadeh for teaming up with me for in vitro ubiquitylation assays, for providing useful feedback throughout this project, for providing critical comments on this thesis, and for keeping the yeast alliance alive. I also would like to thank the other members of “yeast club,” Dr. Jung Ae Kim, who has become my go-to person in the lab for all questions relating to yeast, FACS, and DNA damage, and Dr. Christina Hughes, who provided useful feedback on this thesis, as well as many hours of stimulating coversation. I thank Dr. Laura Banaszynski for advice on high-throughput sequencing procedures and for her helpful suggestions regarding the fourth chapter of this work. I also thank Bryce Carey for being willing to become my metabolism buddy; it has been immensely satisfying to fi nally have someone to discuss the Krebs Cycle with. In addition, I have appreciated the interactions I have had with all iii the other Allis lab members, past and present, who have shaped the way I think about science. The Allis Lab Manager, Jamie Winshell, deserves special thanks. Without her untiring efforts to keep things in the lab running smoothly, the lab would have been a much different place in which to work. In addition, I have been happy to call Jamie a friend for many years now, and I am thankful for our many good conversations in our bay. I am hugely indebted to my two collaborators on my high-throughput sequencing projects: Dr. Deyou Zhang has always been willing to answer my queries with insightful and throrough comments, and Dr. Scott Dewell has been very helpful in teaching me to do my own data analysis, coming up with new ways to look at my data, and providing technical support. Without the contributions and support of these two collaborators, many of the insights presented in this work would never have been made. I must also thank Dr. Oliver Rando, who was willing to hire an undergraduate student with no lab experience, and his amazingly talented technician, Mike Dion, who taught me yeast genetics. The miccrococcal nuclease experiments I performed in Ollie’s lab fi rst sparked my interest in chromatin, and led me here. I am thankful for the fi nancial and administrative support I have received through the David Rockefeller Graduate Program. Throughout the years, the Dean’s Offi ce staff have been incredibly kind and helpful. I am also thankful to have received fi nancial support for this research through an NIH Cancer Training Grant. I want to conclude by thanking my friends and family for their untiring support during this journey. My father and my sister Jennifer (Little Sue) have been my life-lines through this process. Lastly, I must thank Chris for being so patient and caring with me throughout the last few years. I have been incredibly fortunate to have had a partner who is a joy to come home to, who wants to talk with me about my research, and who is so supportive. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................... iii TABLE OF CONTENTS........................................................................................................ v LIST OF FIGURES................................................................................................................ viii LIST OF TABLES.................................................................................................................. xi CHAPTER 1: GENERAL INTRODUCTION

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