UC San Diego UC San Diego Electronic Theses and Dissertations Title Screening and Characterizing Amino Acid Metabolic Proteins for Functions in Chromatin Regulation Permalink https://escholarship.org/uc/item/8zb24311 Author Su, Xue Publication Date 2014 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Screening and Characterizing Amino Acid Metabolic Proteins for Functions in Chromatin Regulation A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Xue Su Committee in charge: Professor Lorraine Pillus, Chair Professor Michael Burkart Professor Beverly Emerson Professor Douglass Forbes Professor Amy Pasquinelli 2014 Copyright Xue Su, 2014 All rights reserved Signature Page The Dissertation of Xue Su is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2014 iii DEDICATION This thesis is dedicated to my grandmother, Shulan Shi, who would have been proud to see its completion. iv TABLE OF CONTENTS Signature Page ...................................................................................................................iii Dedication.......................................................................................................................... iv Table of Contents................................................................................................................ v List of Abbreviations ......................................................................................................... ix List of Figures.................................................................................................................... xi List of Tables ................................................................................................................... xiv Acknowledgements........................................................................................................... xv Vita.................................................................................................................................. xvii Abstract of the Dissertation ...........................................................................................xviii Chapter 1. Introduction ....................................................................................................... 1 Histone post-translational modifications ........................................................................ 3 Chromatin silencing and its regulatory mechanisms .................................................... 10 Crosstalk between metabolism and chromatin function ............................................... 13 Amino acid metabolism and chromatin regulation....................................................... 17 Overview of the thesis .................................................................................................. 17 References..................................................................................................................... 18 Chapter 2. The Gdh1 glutamate dehydrogenase regulates telomeric silencing through modulating α-ketoglutarate, recruitment of the SIR complex and histone H3 clipping... 21 Abstract......................................................................................................................... 21 Introduction................................................................................................................... 22 Results........................................................................................................................... 24 A screen for amino acid metabolic enzymes with chromatin function..................... 24 A subset of candidate genes has potential roles in chromatin silencing................... 26 Gdh1 is a positive regulator of telomeric silencing .................................................. 30 Both glutamate dehydrogenase paralogs contribute to telomeric silencing.............. 33 Gdh1’s silencing function depends on its metabolic activity ................................... 35 Elevated α-ketoglutarate levels result in telomeric silencing defects....................... 38 Gdh1 is a negative regulator of H3 N-terminal clipping in vivo............................... 41 v Gdh1 regulates telomeric silencing through both clipping- dependent and independent mechanisms .......................................................................................... 45 Discussion..................................................................................................................... 46 Deletion of four candidate metabolic proteins altered chromatin silencing ............. 46 α-ketoglutarate is an important metabolic regulator of telomeric silencing ............. 47 Gdh1 is a negative regulator of H3 N-terminal clipping .......................................... 48 Materials and Methods.................................................................................................. 50 Yeast strains and plasmids........................................................................................ 50 Growth assays and silencing reporter assays............................................................ 51 mRNA quantification................................................................................................ 51 Chromatin immunoprecipitation............................................................................... 52 Preparation of yeast nuclei and whole cell extract.................................................... 52 Protein immunoblotting ............................................................................................ 53 Supplementary Materials and Methods ........................................................................ 54 Yeast strains.............................................................................................................. 54 Silencing and rDNA recombination assays .............................................................. 54 Quantitative mating assay......................................................................................... 55 Immunoblotting and chromatin IP............................................................................ 55 Acknowledgements....................................................................................................... 70 References..................................................................................................................... 71 Chapter 3. Threonine metabolic enzymes Hom2 and Hom6 positively regulate rDNA silencing ............................................................................................................................ 78 Introduction................................................................................................................... 78 Results........................................................................................................................... 83 Independent assays support HOM6’s role in regulating rDNA silencing................. 83 Threonine but not methionine biosynthesis regulates rDNA silencing .................... 85 The catalytic mutant of Hom6 retains partial rDNA silencing function................... 87 Hom6 has a nuclear pool and nuclear-localized Hom6 contributes to its rDNA silencing function...................................................................................................... 88 Hom6 has a complex role in telomeric silencing...................................................... 88 Variability issues in this study .................................................................................. 91 Discussion..................................................................................................................... 94 The variability issue of the hom6Δ strain needs to be resolved................................ 95 Genetic interactions between different threonine metabolic genes .......................... 95 Hom6’s interaction with known chromatin regulators ............................................. 95 Analysis of additional Hom6 catalytic mutants........................................................ 96 Materials and Methods.................................................................................................. 96 Yeast strains and plasmids........................................................................................ 96 Dilution assays for growth and silencing.................................................................. 97 RNA extraction and cDNA preparation.................................................................... 97 References................................................................................................................. 102 Chapter 4. ARG82 regulates rDNA, telomeric and HM silencing ................................. 104 vi Introduction................................................................................................................. 104 Results......................................................................................................................... 107 Validation of Arg82’s function in HM silencing .................................................... 107 Further dissection of Arg82’s function in rDNA silencing .................................... 109 Arg82 is also required for the repression of an endogenous telomeric transcript .. 109 Structural-