PRMT5-CATALYZED ARGININE METHYLATION OF NF-κB p65 IN THE ENDOTHELIAL CELL INDUCTION OF PRO-INFLAMMATORY CHEMOKINES by DANIEL PELLERIN HARRIS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Paul E. DiCorleto, Ph.D. Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY January, 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Daniel P. Harris candidate for the Doctor of Philosophy degree *. Thomas N. Nosek, Ph.D., Committee Chair Paul E. DiCorleto, Ph.D. Cathleen R. Carlin, Ph.D. George R. Dubyak, Ph.D. Paul L. Fox, Ph.D. Mukesh K. Jain, M.D. July 27th, 2015 *We also certify that written approval has been obtained for any proprietary material contained therein. iii DEDICATION This dissertation is dedicated to my great-grandparents, Mark and Madeline Pellerin, and my parents Steve and Madeline Harris, for showing me how to live with grace and kindness. i TABLE OF CONTENTS List of Tables ....................................................................................................... 4 List of Figures ...................................................................................................... 5 Acknowledgements ............................................................................................ 6 List of Abbreviations ......................................................................................... 11 Abstract .............................................................................................................. 16 Chapter 1: Endothelial Functions .................................................................... 18 1.1 Introduction ............................................................................................. 18 1.2 The Vascular Endothelium ...................................................................... 19 1.2.1 Morphology and Barrier Function ..................................................... 19 1.2.2 Regulation of Vascular Tone ............................................................ 20 1.2.3 Regulation of Hemostasis ................................................................. 22 1.3 Endothelial Activation and Inflammation ................................................. 23 1.3.1 Leukocyte Adhesion Molecules ........................................................ 24 1.3.2 The Chemokines CXCL10 and CXCL11 ........................................... 26 1.4 Pro-Inflammatory Signaling: TNF and IFN-γ ........................................... 31 1.4.1 TNF Signaling ................................................................................... 31 1.4.2 IFN-γ Signaling and the Jak-STAT Pathway .................................... 32 1.4.3 The NF-κB Pathway ......................................................................... 33 1.5 The PTM Code and NF-κB ..................................................................... 33 Chapter 2: Protein Arginine Methylation ........................................................ 36 2.1 Project Goal: Identify PRMT5-Regulated Pro-Inflammatory Genes ........ 36 2.2 Arginine Methylation ................................................................................ 37 2.3 Protein Arginine Methyltransferase 5 (PRMT5) ...................................... 41 CHAPTER 3: Materials and Methods ............................................................... 43 3.1 Ethics Statement ..................................................................................... 43 3.2 Reagents ................................................................................................. 43 1 3.3 Cell Culture ............................................................................................. 47 3.4 Transient Transfection ............................................................................ 47 3.5 Preparation of Nuclear and Cytosolic Extracts ........................................ 47 3.6 Immunoprecipitation ................................................................................ 48 3.7 Immunoblotting ........................................................................................ 48 3.8 RNA Purification and Real-Time Quantitative PCR (qRT-PCR) ............. 49 3.9 CXCL10 Enzyme-Linked Immunosorbent Assay (ELISA) ...................... 51 3.10 Chromatin Immunoprecipitation (ChIP) and Re-ChIP ........................... 51 3.11 CXCL10 Promoter Activity Assay .......................................................... 54 3.12 Mass Spectrometry Methods ................................................................ 54 3.13 In Vitro Methyltransferase Assay .......................................................... 55 3.14 p65 Site-Directed Mutagenesis ............................................................. 55 3.15 NF-κB Point Mutant Reconstitution ....................................................... 56 3.16 Statistical Analysis ................................................................................ 56 Chapter 4: PRMT5-Catalyzed Methylation of NF-κB p65 Activates CXCL10 Expression ......................................................................................................... 57 4.1 Preface .................................................................................................... 57 4.2 Introduction ............................................................................................. 57 4.3 Results .................................................................................................... 60 4.3.1 PRMT5 Enhances TNF-induced CXCL10 Gene Expression ............ 60 4.3.2 PRMT5-Catalyzed SDMA-Containing Proteins Associate with the CXCL10 Promoter ........................................................................................ 64 4.3.3 NF-κB p65 is Symmetrically Dimethylated by PRMT5 ..................... 66 4.3.4 p65 is Methylated at 5 Arginine Residues ........................................ 69 4.3.5 p65 Association with the CXCL10 Promoter Requires p65 .............. 76 4.3.6 Methylation of the p65 RHD is Necessary for CXCL10 Induction ..... 78 4.4 Discussion ............................................................................................... 81 4.5 Acknowledgements ................................................................................. 85 2 Chapter 5: PRMT5-Mediated Methylation of NF-κB p65 at Arg174 Activates CXCL11 Gene Induction in EC Co-stimulated with TNF and IFN-γ ............... 86 5.1 Preface .................................................................................................... 86 5.2 Introduction ............................................................................................. 86 5.3 Results and Discussion ........................................................................... 89 5.3.1 PRMT5 Promotes CXCL11 Gene Expression .................................. 90 5.3.2 Expression of CXCL11 Requires p65 Arg174 .................................... 93 5.3.3 PRMT5 Catalyzes Dimethylation of p65 Arg174 ................................ 98 5.3.5 p65 Recruitment to the CXCL11 Promoter Requires Arg174 ........... 102 5.3.6 SDMA at the CXCL11 Promoter is PRMT5-Dependent .................. 104 5.3.7 p65 Arg174Lys Reduces SDMA at the CXCL11 Promoter ............... 106 5.4 Acknowledgements ............................................................................... 115 Chapter 6: Concluding Discussion ................................................................ 116 6.1 Discussion of Key Results ..................................................................... 116 6.2 The Methylarginine PTM Code of NF-κB p65 ....................................... 120 6.3 Implications for Atherosclerosis ............................................................ 122 6.4 Regulation of PRMT5 ............................................................................ 124 6.4 The Permanence of Arginine Methylation ............................................. 124 6.4.1 JMJD6: An Arginine Demethylase? ................................................ 127 6.4.2 Arginine Deimination ....................................................................... 127 References ....................................................................................................... 129 3 LIST OF TABLES TABLE 1 CXCR3, CXCL10, and CXCL11 involvement in disease. .................... 29 TABLE 2 Sequences of siRNAs used in the experiments. ................................. 45 TABLE 3 Details of the antibodies used in the experiments. .............................. 46 TABLE 4 Primer sequences used in cDNA amplification by qRT-PCR. ............. 50 TABLE 5 Primer sequences for ChIP experiments. ............................................ 53 TABLE 6 Methylated p65 peptides obtained from MS/MS experiments. ............ 75 4 LIST OF FIGURES FIGURE 1.1 Schematic of EC-leukocyte interactions in inflammation. ............... 25 FIGURE 2.1 PRMT enzymes catalyze formation of MMA, ADMA, and SDMA. .. 39 FIGURE 4.1 PRMT5 is necessary for the induction of CXCL10 and CX3CL1 in TNF-stimulated EC. ...................................................................................... 61 FIGURE 4.2 Knockdown of PRMT5 reduces CXCL10 transcription. .................. 63 FIGURE 4.3 PRMT5 activity leads to the association of SDMA-containing proteins with the CXCL10 promoter following TNF-stimulation. ................... 65 FIGURE 4.4 NF-κB p65 is methylated by PRMT5. ............................................