UNIVERSITY OF CALIFORNIA, SAN DIEGO Bypass suppression defines critical genomic regulation by the NuA4 acetyltransferase complex A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Naomi Searle Committee in Charge: Professor Lorraine Pillus, Chair Professor Dong-Er Zhang, Co-Chair Professor Dong Wang Professor Eugene Yeo Professor Huilin Zhou 2017 Copyright Naomi Searle, 2017 All rights reserved. Signature Page The Dissertation of Naomi Searle is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California, San Diego 2017 iii DEDICATION Dedication This dissertation is dedicated to my family, for all of the support, love, guidance, and inspiration. iv TABLE OF CONTENTS Table of Contents Signature Page .................................................................................................. iii Dedication ......................................................................................................... iv Table of Contents .............................................................................................. v List of Abbreviations ........................................................................................ viii List of Figures ................................................................................................... ix List of Tables .................................................................................................... xii Acknowledgements ......................................................................................... xiii Vita .................................................................................................................. xv Abstract ........................................................................................................... xvi Chapter 1. Introduction ...................................................................................... 1 Tip60 is the catalytic subunit of the NuA4 acetyltransferase complex. ............ 2 Enhancer of Polycomb is broadly conserved .................................................. 4 Comparative studies between model organisms promote functional definition ........................................................................................................................ 6 From basic function to translational significance ........................................... 12 Bypass suppression is a powerful genetic tool .............................................. 15 Promoting a balanced acetylation state allows for the bypass of ESA1 ........ 16 Bypass suppression to define key functions of chromatin modifying proteins17 Acknowledgements ....................................................................................... 20 References .................................................................................................... 21 Chapter 2. Chromatin regulation by the NuA4 acetyltransferase complex is mediated by essential interactions between Enhancer of Polycomb (Epl1) and Esa1 ................................................................................................................ 30 Abstract ......................................................................................................... 31 Introduction .................................................................................................... 31 Materials and Methods .................................................................................. 32 Yeast Strains and Plasmids ......................................................................... 32 Growth Assays ............................................................................................ 32 Flow Cytometry ............................................................................................ 32 Lysate preparation ....................................................................................... 33 Immunoprecipitation .................................................................................... 33 v Immunoblots ................................................................................................ 33 RNA-seq sample preparation and analysis ................................................. 33 qPCR validation ........................................................................................... 33 Data availability ........................................................................................... 34 Results ........................................................................................................... 34 Bypass and function of essential piccolo-NuA4 subunits ............................ 34 ESA1 and EPL1 bypass strains have nearly identical gene expression profiles ......................................................................................................... 35 Epl1 promotes the chromatin association of Esa1 ...................................... 36 Defining the critical regions of Epl1 in vivo .................................................. 37 Physical association between Esa1 and Epl1 is required for activity .......... 38 Discussion ..................................................................................................... 39 Acknowledgements ....................................................................................... 42 Literature Cited .............................................................................................. 43 Supplemental Materials ................................................................................. 44 Supplemental References ........................................................................... 48 Acknowledgements ....................................................................................... 49 Chapter 3. Bypass suppression defines chromatin dynamics between NuA4 and the Hda1 deacetylase ............................................................................... 50 Introduction .................................................................................................... 51 Class I deacetylases .................................................................................... 51 Class II deacetylases ................................................................................... 54 Class III deacetylases .................................................................................. 55 Opposing activities of NuA4 and the HDACs .............................................. 56 Results ........................................................................................................... 57 NuA4 has distinct interactions with histone deacetylases ........................... 57 Phenotypes of epl1∆ sds3∆ are selectively suppressed by deletion of HDACs. ........................................................................................................ 59 Epl1 is opposed by Hda1 independent of Rpd3L ........................................ 66 Discussion ..................................................................................................... 70 Hos1 and Hos3 may act in opposition to NuA4 at the rDNA locus and spindle pole body, respectively .................................................................... 71 Epl1’s interactions with Hst2 and Sir2 are distinct from Esa1’s genetic Interactions .................................................................................................. 72 Functional antagonism between Rpd3 and Hos2 ........................................ 74 The Hda1-Rpd3-NuA4 functional axis ......................................................... 75 References .................................................................................................... 79 Chapter 4. Defining the Esa1 Transcriptional Regulome ................................ 85 Introduction .................................................................................................... 86 Esa1 is important for global transcription .................................................... 86 Esa1 is involved in regulating the expression of ribosomal protein genes .. 87 Esa1 in ribosome biogenesis ....................................................................... 88 vi Bypass suppression to define the Esa1 transcriptional ‘regulome.’ ............ 89 Results ........................................................................................................... 90 Differential expression analysis of ESA1 bypass mutant to define Esa1- regulated gene expression .......................................................................... 90 Ribosome biogenesis genes are regulated by Esa1 ................................... 94 A transcriptional basis for the hda1∆-mediated rescue of esa1∆ sds3∆ mutant phenotypes .................................................................................... 100 Discussion ................................................................................................... 109 Transcriptome analysis of ESA1 bypass ................................................... 109 Understanding the significance of Esa1 in ribosome biogenesis .............. 110 The role of Hda1 in balancing regulation of ribosome biogenesis ............. 112 References .................................................................................................. 123 Chapter 5. Conclusions and Future Directions .............................................. 127 Epl1 is a critical Esa1-cofactor ...................................................................