Functional Characterization of the DExH-Box Helicase DHX9 Teresa Lee Department of Biochemistry McGill University Montréal, Québec, Canada October 2016 A thesis submitted to McGill University in partial fulfillment of the requirement of the degree of Doctor of Philosophy © Teresa Lee, October 2016 TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... 6 LIST OF TABLES ........................................................................................................................ 8 ABSTRACT ................................................................................................................................... 9 RESUMÉ ..................................................................................................................................... 11 PREFACE .................................................................................................................................... 13 CONTRIBUTION OF AUTHORS ........................................................................................... 14 ORIGINAL CONTRIBUTION TO KNOWLEDGE .............................................................. 15 ACKNOWLEDGEMENTS ....................................................................................................... 17 LIST OF ABBREVIATIONS .................................................................................................... 18 CHAPTER 1: GENERAL INTRODUCTION ......................................................................... 23 1.1 Introduction to DHX9 and DExD/H-box helicases........................................................ 24 1.2 Biochemical and structural characterization of DHX9 .................................................. 25 1.2.1 Discovery of DHX9 ................................................................................................. 25 1.2.2 DHX9 functional domains and structure ................................................................ 26 1.2.3 In vitro characterization of DHX9 .......................................................................... 32 1.3 Biological characterization of DHX9 ............................................................................. 35 1.3.1 Cellular localization of DHX9 ................................................................................ 35 1.3.2 DHX9 expression, half-life, and role in development ............................................. 36 1.4 Biological functions of DHX9 ....................................................................................... 37 1.4.1 DNA replication and maintenance of genomic stability ......................................... 37 1.4.2 Transcriptional regulation ...................................................................................... 39 1.4.3 Translational regulation ......................................................................................... 43 1.4.4 MicroRNA biogenesis and processing .................................................................... 45 1.4.5 RNA processing and transport ................................................................................ 46 1.5 Implications of DHX9 in human diseases ...................................................................... 51 1.5.1 Implication of DHX9 in cancer ............................................................................... 51 1.5.2 Role of DHX9 in viral infection .............................................................................. 53 1.5.3 Role of DHX9 in autoimmune disease .................................................................... 54 1.6 p53-dependent pathways regulating apoptosis and cell cycle progression .................... 54 1.6.1 The p53 tumor suppressor protein .......................................................................... 54 2 1.6.2 Regulation and activation of p53 ............................................................................ 55 1.6.3 p53-mediated transcriptional regulation ................................................................ 56 1.6.4 p53-dependent cell cycle arrest and senescence .................................................... 57 1.6.5 p53-dependent apoptosis ........................................................................................ 62 1.6.6 p53 and chemotherapy ............................................................................................ 65 1.6.7 Targeting the apoptotic machinery in chemotherapy ............................................. 67 1.7 DHX9 as a potential chemotherapeutic target ................................................................ 68 1.8 Overview and rationale for thesis ................................................................................... 69 CHAPTER 2: SUPPRESSION OF THE DHX9 HELICASE INDUCES PREMATURE SENESCENCE IN HUMAN DIPLOID FIBROBLASTS IN A P53-DEPENDENT MANNER..................................................................................................................................... 71 2.1 Preface to Chapter 2 ....................................................................................................... 72 2.2 Abstract .......................................................................................................................... 72 2.3 Introduction .................................................................................................................... 73 2.4 Results ............................................................................................................................ 75 2.4.1 Loss of DHX9 results in morphological changes, senescence-associated - galactosidase activity, and growth arrest in primary human cells ....................................... 75 2.4.2 DHX9 suppression leads to changes in some cell cycle regulatory and DNA damage response proteins ..................................................................................................... 79 2.4.3 p53 is essential for DHX9-induced senescence ...................................................... 81 2.4.4 DHX9 suppression is associated with a senescence signature ............................... 81 2.4.5 Structure/function analysis of DHX9 in the senescence response .......................... 83 2.4.6 Loss of DHX9 induces senescence through inhibition of DNA replication ............ 87 2.5 Discussion ...................................................................................................................... 95 2.6 Materials and Methods ................................................................................................. 101 2.6.1 Cell lines, cell culture and plasmids ..................................................................... 101 2.6.2 shRNA sequences .................................................................................................. 101 2.6.3 Growth curves ....................................................................................................... 102 2.6.4 Cell cycle analysis................................................................................................. 102 2.6.5 Senescence-associated -galactosidase assay ...................................................... 102 2.6.6 Immunoblot analysis ............................................................................................. 103 2.6.7 DHX9 cDNA rescue .............................................................................................. 103 2.6.8 Microarray analysis and validation...................................................................... 104 2.6.9 Genomic and nascent DNA isolation and quantitation ........................................ 105 2.6.10 Chromatin immunoprecipitation (ChIP) assays ................................................... 106 2.7 Acknowledgements ...................................................................................................... 107 3 CHAPTER 3: TUMOR CELL SURVIVAL DEPENDENCE ON THE DHX9 DEXH-BOX HELICASE ................................................................................................................................ 109 3.1 Preface to Chapter 3 ..................................................................................................... 110 3.2 Abstract ........................................................................................................................ 110 3.3 Introduction .................................................................................................................. 111 3.4 Results .......................................................................................................................... 112 3.4.1 DHX9 suppression reduces human cancer cell fitness ......................................... 112 3.4.2 Modeling DHX9 suppression in E-Myc lymphomas ........................................... 118 3.4.3 Modeling conditional DHX9 suppression in the mouse ....................................... 124 3.4.4 DHX9 knockdown in adult mice is well tolerated ................................................. 132 3.4.5 Assessing DHX9 suppression on global gene expression ..................................... 132 3.5 Discussion .................................................................................................................... 145 3.6 Materials and methods ................................................................................................. 149 3.6.1 Cell lines ..............................................................................................................