UNIVERSITY OF CALIFORNIA, SAN DIEGO A Dual Role for REV-ERB alpha in Th17 Cell Mediated Immune Response A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Fang-Chen Chang Committee in charge: Professor Ye Zheng, Chair Professor Jack Bui Professor Christopher K. Glass Professor Lifan Lu Professor Clodagh O’Shea Professor Elina Zuniga 2016 Copyright Fang-Chen Chang, 2016 All rights reserved The dissertation of Fang-Chen Chang is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2016 iii DEDICATION To my parents, for the opportunities they afforded me, and their unwavering support. And to Edmond, who spent many beautiful weekend afternoons waiting in the Salk parking lot. iv EPIGRAPH First, there is a mountain, then there is no mountain, then there is. —Traditional Buddhist saying, via Donovan (1967) v TABLE OF CONTENTS SIGNATURE PAGE .............................................................................................. iii DEDICATION ........................................................................................................ iv EPIGRAPH ............................................................................................................ v TABLE OF CONTENTS ........................................................................................ vi LIST OF FIGURES .............................................................................................. viii LIST OF TABLES .................................................................................................. x ACKNOWLEDGEMENTS ..................................................................................... xi VITA ..................................................................................................................... xii ABSTRACT OF THE DISSERTATION ................................................................ xiii Chapter One: Introduction ..................................................................................... 1 1.1 Th17 cells ..................................................................................................... 1 1.1.1 CD4+ T helper cells ............................................................................... 1 1.1.2 Th17 effector function and cytokine production ..................................... 3 1.1.3 Transcriptional regulation of Th17 differentiation .................................. 4 1.1.4 The role of Th17 cells in multiple sclerosis ............................................ 6 1.2 REV-ERBS .................................................................................................. 8 1.2.1 Nuclear hormone receptors ................................................................... 8 1.2.2 REV-ERBs ............................................................................................. 8 1.2.3 Endogenous and synthetic REV-ERB ligands ..................................... 10 1.2.4 Regulation of the circadian rhythm ...................................................... 11 1.2.5 Regulation of metabolism .................................................................... 12 1.2.6 Regulation of immune functions .......................................................... 14 1.3 Figures ....................................................................................................... 16 Chapter 2: Material and Methods ........................................................................ 18 2.1 Mice ........................................................................................................... 18 2.2 In vitro CD4 T cell differentiation ............................................................... 18 2.3 Flow cytometry .......................................................................................... 19 2.4 Reverse transcription and quantitative PCR ............................................. 20 2.5 Western blot .............................................................................................. 20 2.6 Retroviral transduction .............................................................................. 21 2.7 Dual luciferase reporter assay .................................................................. 21 vi 2.8 DNA binding assay ................................................................................... 21 2.9 Chromatin Immunoprecipitation ................................................................ 22 2.10 ChIP-sequencing .................................................................................... 22 2.11 RNA-sequencing and data analysis ........................................................ 23 2.12 EAE models ............................................................................................ 23 2.13 Histology analysis ................................................................................... 24 2.14 SR9009 treatment ................................................................................... 25 Chapter 3: Identification of REV-ERBa as a novel regulator in Th17 differentiation ............................................................................................................................. 27 3.1 Results ....................................................................................................... 27 3.2 Tables and Figures .................................................................................... 32 Chapter 4: The functional role of REV-ERBa in Th17 cell mediated autoimmune response .............................................................................................................. 44 4.1 Results ....................................................................................................... 44 4.2 Tables and Figures ................................................................................... 48 Chapter 5: In vitro and in vivo characterizations of REV-ERBa deficiency .......... 56 5.1 Results ....................................................................................................... 56 5.2 Tables and Figures .................................................................................... 59 Chapter 6: Discussion ......................................................................................... 65 6.1 Summary of findings ................................................................................. 65 6.3 Bridging the gain and loss of function phenotypes .................................... 68 6.4 Conclusion ................................................................................................. 72 6.5 Figures ....................................................................................................... 73 References .......................................................................................................... 76 vii LIST OF FIGURES Figure 1.1 Effector CD4+ T cell lineages .............................................................. 16 Figure 1.2 REV-ERBs and RORs play essential and opposing roles in the regulation of the molecular clock ................................................................... 17 Figure 3.1 REV-ERBa is up-regulated in Th17 cells ............................................ 32 Figure 3.2 REV-ERBa is up-regulated in human Th17 cells ................................ 33 Figure 3.3 Ectopic REV-ERBa expression inhibits Th17 differentiation ............... 34 Figure 3.4 REV-ERBs inhibit RORγt-dependent IL-17A expression .................... 35 Figure 3.5 Analysis of different REV-ERBα domains that are involved in repression of IL-17A expression .................................................................... 36 Figure 3.6 REV-ERBa over expression inhibits the expression of Th17 signature genes ............................................................................................................. 37 Figure 3.7 REV-ERBa directly inhibits IL17a expression ..................................... 38 Figure 3.8 Figure 2.8 REV-ERBa binds to a RORE within the il17a locus ........... 39 Figure 3.9 Figure 2.9 REV-ERBa binds to the endogenous Il17a locus ............... 40 Figure 3.10 REV-ERBa binds to the endogenous Il17a locus .............................. 41 Figure 3.11 REV-ERBa and RORgt compete for binding to the Il17a locus ......... 42 Figure 4.1 Doxycycline induces transgenic REV-ERBα expression in vivo, but does not affect EAE disease progression ..................................................... 48 Figure 4.2 In vivo induction of REV-ERBa expression in Th17 cells suppresses EAE disease progression .............................................................................. 49 Figure 4.3 Doxycycline treatment does not affect homing of transferred CD4+ T cells ............................................................................................................... 50 Figure 4.4 REV-ERB agonist SR9009 inhibits in vitro Th17 differentiation .......... 51 Figure 4.5 REV-ERB agonist SR9009 inhibits in vitro human Th17 differentiation52 Figure 4.6 REV-ERB agonist SR9009 enhances NCoR recruitment to the Il17a locus .............................................................................................................. 53 Figure 4.7 Treatment with REV-ERB agonist SR9009 suppresses EAE progression .................................................................................................... 54 Figure 4.8 Treatment with REV-ERB agonist SR9009 prevents relapse