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...... 68 FIGURE 4.5 Five p65 arginine residues are dimethylated in EC...... 74 FIGURE 4.6 PRMT5 is necessary for p65 association with the CXCL10 promoter...... 77 FIGURE 4.7 SDMA-p65 is critical for CXCL10 induction...... 80 FIGURE 5.1 PRMT5 promotes expression of CXCL11 in EC co-stimulated with TNF and IFN-γ...... 92 FIGURE 5.2 TNF and IFN-γ mediated CXCL11 induction requires PRMT5- catalyzed p65 dimethylation at Arg174...... 96 FIGURE 5.3 PRMT5 knockdown reduces p65 association with the CXCL11 promoter...... 100 FIGURE 5.4 Mutation of Arg174 to lysine abrogates p65 association with the CXCL11 promoter...... 103 FIGURE 5.5 PRMT5-catalyzed dimethylation of p65 at Arg174 is necessary for p65 recruitment to the CXCL11 promoter...... 105 FIGURE 5.6 p65 Arg174 methylation is present on the CXCL11 promoter following co-stimulation...... 109 FIGURE 6.1 Model of SDMA-p65 in CXCL10 and CXCL11 transcription...... 117

5 ACKNOWLEDGEMENTS

When I was given the opportunity to work in Paul DiCorleto’s laboratory I knew it would be a tremendous opportunity. What I didn’t know is that it would also be a pleasure. Paul has provided me with full material support, and expert, incisive advice on my ideas and results. He has shaped my development as a scientist and his interest in me has made me a better person. Paul provided me with the freedom to explore my own ideas, make mistakes, learn from them, and eventually, to enjoy some success. I cannot thank Paul enough for the opportunities he’s afforded me, and for his exceptional patience.

I’ve had a phenomenally dedicated dissertation committee. I have long since lost count of the number of committee meetings we’ve had. Each time the group met I emerged with new ideas and renewed enthusiasm for my project.

The members of my committee are an accomplished group. Paul Fox is a true polymath. He is an endlessly creative scientist, a gifted speaker, and a skilled artist. The models he prepares of his laboratory’s results are elegant and make difficult concepts lucid. Cathy Carlin is the best teacher I’ve ever had at any level of my education. I have learned a tremendous amount about how to think about science by listening to her ask questions at seminars and journal clubs. She’s a rigorous and thorough scientist, and I admire her immensely. Mukesh Jain is both a top cardiologist and extraordinary vascular investigator. He’s committed to understanding cardiovascular function and dysfunction at all levels – from the

6 fundamental molecular and metabolic levels through to human pathology in his clinical practice. His passion for science is genuine. George Dubyak’s knowledge of the literature – especially in immunology and vascular cell biology studies - is immense. It’s fun to talk to him about the week’s articles in Nature or Science because he has always read them all and has insight to share. Tom Nosek played a vital role on my committee - he kept me on track. He let me know when I wasn’t doing a good job; he was there for me with an encouraging word when times were difficult; he congratulated me when things went well. That feedback – all of it – was valued and appreciated.

I thank several of my outstanding science professors from Albion College:

J. Dan Skean, Dean McCurdy, Ruth Schmitter, E. Dale Kennedy, and Ned

Garvin, for fostering and developing my interest in science.

I also thank a number of faculty members at both Case Western and

Cleveland Clinic for taking a personal interest in me. These faculty members include Jonathan Smith, Phil Howe, Bill Schilling, Corey Smith, Ulrich Hopfer,

Margie Chandler, Mattias Buck, Andrea Romani, Oliver Wessely, and Stan

Hazen.

Publishing my findings would have been difficult – and certainly would have been less convincing – without identification of the methylarginine residues

7 of NF-κB p65. The mass spectrometry experiments that identified these residues were performed by Belinda Willard in the Cleveland Clinic Proteomics Core

Facility. I thank Belinda for her expertise and skill in carrying out these critical experiments.

The administrative staffs of the Department of Physiology and Biophysics

(CWRU) and Department of Cellular and Molecular Medicine (CCF) have supported me and made my life easier. I thank Jean Davis, Morley Schwebel,

Judy Schiciano, and Raquel Delgado for their help. I also thank Jenienne Geist and Susan Dowhan for providing indispensible help with scheduling meetings.

I owe a special thanks to my colleagues in the DiCorleto laboratory, past and present, for creating such a fantastic working environment, and for so much helpful discussion and input on my science. These members include Matt

Waitkus, Mohammad Rajabi, Qiuyun Chen, Chad Braley, Joel Boerckel, Lisa

Dechert, Emily Tillmaand, Angela Money, Kristy Waite, Lori Mavrakis, Jianzhong

Shen, and numerous summer research students from John Carroll University. I also acknowledge Tyler Maxwell, an undergraduate from JCU, for working hard for a year under my direction. I hope Tyler learned as much from me as I learned about managing people from him.

8 Members of Paul Fox’s lab have been generous with their advice and friendship. I heartily thank Sandeepa Eswarappa, Peng Yao, Dalia Halawani,

Prabar Ghosh, Yi Fan, Fulvia Terenzi, Jie Jia, Abul Arif, Arnab China, Vasu

Komireddy, and Partho Ray for their contributions.

I don’t know what I would have done without Unnikrishnan

Chandrasekharan and Smarajit Bandyopadhyay. These men have been marvelously generous with their time, thoughts, and talents from my first day in the laboratory to the present moment. Unni and Smarajit have been my daily guiding lights. I’ve shared so much with them in the last 8 years. They’ve become wonderful friends and I will miss working with them greatly.

I have formed many close friendships during my graduate years. These friends include Krekwit Shinlapawittayatorn, Edward Agarwala, Jeff Hsu, Susan

Foy, Steve Foy, Rohit Shastry, Niko Balanis, Ross Anderson, Anish Ghodadra,

Sumantha Bhatt, Megan Ermler, Junjie Zhao, Pam Sangwung, Kapil Mandrekar, and my fellow Casseroles. I think that good friends are one of the best parts of life and I thank each of these individuals for their friendship.

My parents, Steve and Madeline Harris, sisters Alison Lubert and Emily

Harris, and brother-in-law Adam Lubert have all been tremendously supportive of me. I thank them with love and affection.

9 The studies presented in this dissertation were supported by National

Heart, Lung, and Blood Institute (NHLBI) grant P01-HL29582 (to P.E.D.). Support for the collection of HUVEC was made possible by a Case Western Reserve

University/Cleveland Clinic CTSA Grant (UL1TR000439) from the National

Center for Advancing Translational Sciences (NCATS). The Orbitrap Elite instrument was purchased with a National Center for Research Resources

(NCRR) shared instrument grant (1S10RR031537-01). NHLBI, NCATS, and

NCRR are components of the United States National Institutes of Health (NIH).

The funders had no role in study design, data collection and its analysis, publication decisions, or preparation of manuscripts, or of this dissertation.

10 LIST OF ABBREVIATIONS

A Alanine

ActD Actinomycin-D

Ala Alanine

ADMA Asymmetrical Dimethylarginine

Arg Arginine

ANOVA Analysis of Variance

β-Gal β-Galactosidase bp Base Pairs cDNA Complementary DNA

CCL2/MCP-1 Chemokine (C-C motif) Ligand 2/

Monocyte Chemoattractive Protein-1

-CH3 Methyl Group

ChIP Chromatin Immunoprecipitation

CMV Cytomegalovirus

CTL Cytotoxic T Lymphocyte

CXCL8/IL-8 C-X-C Motif Chemokine 8/Interleukin-8

CXCL10/IP-10 C-X-C Motif Chemokine 10/Interferon-Induced Protein

of 10 kDa

CXCL11/I-TAC C-X-C Motif Chemokine 11/Interferon-Inducible T-cell

Alpha Chemoattractant

CX3CL1/Fractalkine C-X3-C Motif Chemokine Ligand 1

11 CXCR3 Chemokine (C-X-C Motif) Receptor 3

Da Dalton

DC Dendritic Cell(s)

DMEM Dulbecco’s Modified Eagle’s Medium

DNA Deoxyribonucleic Acid

DTT Dithiothreitol

EC Endothelial Cell(s)

ECGS Endothelial Cell Growth Supplement

EDTA Ethylenediaminetetraacetic Acid

ELISA Enzyme Linked Immunosorbent Assay

E-SELECTIN Endothelial-Leukocyte Adhesion Molecule

ET-1 Endothelin-1

FBS Fetal Bovine Serum

FLUOROGRAPHY Fluorescence Enhanced Autoradiography

GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase

GAS γ-Interferon-Activated Site h Hour(s)

HEPES 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid

HRP Horseradish Peroxidase

HOXA9 Homeobox Protein A9

HUVEC Human Umbilical Vein Endothelial Cell(s)

IB Immunoblot

12 ICAM-1 Intracellular Adhesion Molecule 1

IFN-γ Interferon-gamma

IFN-γ-R Interferon-gamma Receptor

IgG Immunoglobulin G

IκBα Inhibitor of Kappa B-alpha

IP Immunoprecipitation

IPT Ig-like, Plexin, Transcription Factor Domain

IRF Interferon Regulatory Factor

ISRE Interferon Stimulated Response Element

K Lysine

LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry

LPS Lypopolysaccharide

Lys Lysine min Minute(s) mRNA Messenger RNA

MMA Monomethylarginine

NK Natural Killer Cell(s)

NKT Natural Killer T Cell(s)

NF-κB Nuclear Factor Kappa-Light-Chain-Enhancer of

Activated B cells

NO Nitric Oxide

NOS Nitric Oxide Synthetase

13 p21 Cyclin Dependent Kinase Inhibitor 1 p53 Tumor Protein 53 p65 Transcription Factor p65 (RelA)

PAGE Polyacrylamide Gel Electrophoresis

PBS Phosphate Buffered Saline

PCR Polymerase Chain Reaction

PRMT5 Protein Arginine Methyltransferase 5

PTM Post-Translational Modification

PVDF Polyvinylidene Fluoride qRT-PCR Quantitative Real-Time Polymerase Chain Reaction

R Arginine

RHD Rel Homology Domain

RNA Ribonucleic Acid

RNAi RNA Interference

RIPA Radioimmune Precipitation Assay

ROS Reactive Oxygen Species

SAH S-Adenosylhomocysteine

SAM S-Adenosylmethionine

SDMA Symmetrical Dimethylarginine

SDS Sodium Dodecyl Sulfate siRNA Small-Interfering RNA

SMC Smooth Muscle Cell(s)

14 STAT1 Signal Transducer and Activator of Transcription 1

STAT3 Signal Transducer and Activator of Transcription 3

TBST Tris-Buffered Saline and Tween-20

T-cell Thymocyte

TF Tissue Factor

Th1 Type-1 helper

TNF Tumor Necrosis Factor-α

TNF-RI TNF-Receptor I (p75)

TNF-RII TNF-Receptor II (p55)

UTR Untranslated Region

VCAM-1 Vascular Cell Adhesion Molecule-1

WT Wild Type

15 PRMT5-CATALYZED ARGININE METHYLATION OF NF-κB p65 IN THE

ENDOTHELIAL CELL INDUCTION OF PRO-INFLAMMATORY CHEMOKINES

by

DANIEL PELLERIN HARRIS

ABSTRACT

Inflammatory agonists differentially activate gene induction of chemokines in endothelial cells (EC). The molecular mechanisms that produce distinct chemokine induction profiles following EC activation are incompletely understood. The chemokines CXCL10/IP-10 and CXCL11/I-TAC both facilitate

Th1-type leukocytes recruitment to inflammatory lesions. CXCL10 and CXCL11 are strongly induced by IFN-γ. However, CXCL11 expression is not triggered by

TNF-α (TNF) exposure, whereas TNF potently induces the CXCL10 promoter.

Both genes display strong synergistic expression upon simultaneous exposure to

TNF and IFN-γ. In these studies we show that the arginine methyltransferase

PRMT5 is critical in the expression of CXCL10 by TNF and CXCL11 in response to co-stimulation with TNF and IFN-γ, but that PRMT5 operates through distinctive mechanisms to achieve activation of these promoters. Chromatin immunoprecipitation experiments revealed that proteins containing the symmetrical dimethylarginine (SDMA) modification catalyzed by PRMT5 are

16 found on each promoter following stimulation with pro-inflammatory agents.

Using immunoblotting and mass spectrometry approaches we found that NF-κB p65 (p65), a key transcription factor critical for both CXCL10 and CXCL11 induction, contains PRMT5-catalyzed dimethylarginine. PRMT5-mediated methylation is required for p65 association with each promoter.

A series of experiments revealed that methylation of different arginine residues on p65 is required for induction of CXCL10 versus CXCL11. CXCL10 induction requires methylation of Arg30 and Arg35 in response to TNF. In contrast, methylation of p65 at Arg174 is necessary for CXCL11 induction in EC co- stimulated with TNF and IFN-γ. Arg30, Arg35, and Arg174 are located in the rel homology domain of p65 but are present in regions associated with different biochemical functions. Arg30 and Arg35 are part of the DNA-binding domain.

Methylation of these residues probably enhances p65 DNA binding affinity. Arg174 is found in a domain involved in protein-protein interactions. Therefore, methylation of Arg174 is likely to promote an association between p65 and a coactivating component of the CXCL11 transcription complex.

In conclusion, I show that PRMT5 is a central player in chemokine gene expression by catalyzing the methylation of multiple arginine residues in p65.

Methylation of p65 at specific sites comprises a code that enables unique chemokine gene expression profiles in EC.

17 CHAPTER 1: ENDOTHELIAL FUNCTIONS

1.1 Introduction

Endothelial cells (EC) form a single cell layer thick lining of the entire luminal surface of vasculature called the endothelium. EC are in intimate contact with blood and underlying tissues, forming an extensive regulatory interface uniquely situated to monitor and respond to physiological and pathological drivers on both the local and systemic scale. The endothelial network exceeds 100,000 km in aggregate length, has an estimated surface area of 5,000 m2, and is present in all organs and tissues of the body (1).

The endothelium is not a passive body. Healthy endothelium under normal physiological conditions prevents vasospasm, resists leukocyte adhesion, inhibits smooth muscle cell (SMC) proliferation, and presents a non-thrombogenic surface. EC also act as a selectively permeable physical barrier to regulate passage of materials between the blood and tissue compartments (2).

Environmental stimuli can cause EC phenotypic changes resulting in enhanced endothelial permeability, leukocyte hyper-adhesiveness, presentation of thrombogenic surfaces, modulation of vascular tone, and production of vasoactive substances (1). Collectively, these changes are called endothelial activation. Such phenotypic plasticity can be localized or generalized, is usually adaptive and results in temporally restrained responses to physiological

18 conditions. Healthy endothelium will revert to the unactivated state upon resolution of the activating stimuli. These fluctuations are normal homeostatic mechanisms.

Maladaptive responses to environmental stimuli also occur. EC can become activated in the absence of stimuli, overrespond to insult, or fail to downregulate from the activated state. These maladaptive alterations are called endothelial dysfunction, and are involved in the initiation or progression of cardiovascular pathologies. I will briefly discuss major physiological roles of the endothelium. I will then describe the processes and consequences of EC activation and dysfunction.

1.2 The Vascular Endothelium

1.2.1 Morphology and Barrier Function

The endothelium is a simple squamous epithelium with cells elongated parallel to the direction of blood flow (3). EC are connected with tight, gap, and adherens junctions to allow for cell-cell communication, tissue organization, and mediate barrier functionality (4). The composition and number of junction varies according to vascular bed and anatomical location, enabling specificity of endothelial permeability (2). For example, cerebral vascular beds contain a higher number of tight junctions than vascular networks in other tissues. Tight

19 junctions function to stringently restrict macromolecule traffic across the endothelium, forming the blood-brain barrier.

Vascular permeability is controlled by extracellular stimuli through post- translational modifications (PTMs) of junctional proteins. These modifications include phosphorylation of components of the intercellular adhesions and cytoskeletal components, resulting in an increased number or enhanced size of intracellular gaps (2). These changes are local in nature, controlled, and reversible.

1.2.2 Regulation of Vascular Tone

EC regulate vascular tone by synthesizing and secreting vasodilatory or vasoconstrictive substances that operate on the underlying smooth muscle cells

(SMC). SMC were thought to be solely responsible for vascular tone regulation until the discovery a potent vasorelaxing agent in the early 1980s by Furchgott and Zawadzki, which they named endothelium derived relaxing factor (EDRF).

Subsequent studies identified EDRF as nitrous oxide, a gas produced by EC through the conversion of L-arginine to L-citrulline by NO synthetases (NOS) and nicotinamide adenine dinucleotide phosphate (NADPH) reducing equivalents.

Several NOS isoforms are present in EC. Type III NOS, or eNOS, is constitutively expressed in EC and produces small amounts of NO. Type II NOS, called inducible NOS (iNOS), is a high-capacity isoform upregulated in response to

20 inflammatory stressors such as IL-1β, or TNF in macrophages, SMC, or EC. A third type of NOS, nNOS, is exclusively expressed in neuronal cells. NO exerts a local vasorelaxing effect by diffusing across the vessel wall and into SMC, where it increases levels of cyclic guanosine monophosphate (cGMP) by activating guanylate cyclase. Higher levels of cGMP inhibit calcium entry into the SMC, decreasing vasoconstriction (5,6).

NO released into the vascular lumen confers anti-fibrotic properties and opposes leukocyte and platelet adhesion to the endothelium. NO has a short half-life and is inactivated by ROS and oxyhemoglobin. Reduced NO levels are characteristic of vascular disease and are a contributing factor in endothelial dysfunction. NO bioavailability is decreased in patients with coronary artery disease partly due to NO inactivation by elevated levels of ROS. Increases in NO levels oppose arterial plaque formation and may lead to regression of existing lesions (7,8).

EC also synthesize additional vasoactive metabolites, including eicosanoids. The most important eicosanoid involved in the regulation of vascular tone, prostacyclin, is a potent vasodilatory substance secreted by EC that acts on

SMC to decrease vascular tone (1). NO and prostacyclin antagonize a number of endothelial-derived vasoconstrictive substances. These substances, which include angiotensin II, endothelin-1 (ET-1), and platelet-derived growth factor

21 (PDGF) act as agonists of smooth muscle cell contraction (1,5). All three of these molecules are produced as pro-peptides and are proteolytically cleaved upon induction of signaling pathways by extracellular stimuli to allow the rapid induction of vasoconstriction (4).

1.2.3 Regulation of Hemostasis

Non-activated EC constitutively produce substances that oppose blood clot formation. These substances act on platelets and enzymes of the coagulation cascade. For example, EC produce thrombomodulin, a protein inhibitor that binds to the protease thrombin to prevent it’s pro-coagulant activity

(1). Prostacyclin (through the platelet prostacyclin receptor IP1) and NO released into the vascular lumen are also potent inhibitors of platelet aggregation and adhesion (1).

Healthy vessels tightly control the balance of pro- and anti-thrombogenic factors in favor of maintaining a non-thrombogenic surface under normal conditions. EC production of prostacyclin, thrombomodulin, and other anticoagulant factors is normally constitutive. In contrast, pro-coagulant factors are rapidly produced when faced with thrombogenic stimulation. Pro-coagulant factors include tissue factor (TF), platelet activating factor (PAF), and von

Willebrand factor (vWF) (1).

22 1.3 Endothelial Activation and Inflammation

Activation of EC in response to pro-inflammatory or pro-thrombotic agents or mechanical forces lead to the production of growth, regulatory, and vasoactive substances that are not produced by unstimulated EC (9). The activated state is characterized by increased intimal permeability, expression of leukocyte adhesion factors, and shifts in protein secretion. The EC activated state of healthy EC is usually a transient, adaptive response to physiological stressors.

The transition of the endothelium from its basal state to an activated phenotype is coordinated to a large extent by the NF-κB signaling pathway.

Activation of NF-κB results in the induction of hundreds of genes associated with the activated phenotype. Proteins produced by activated EC alter the ratios of antagonistic pro- and anti- inflammatory, thrombotic, growth, and dilatory/constrictive substances. In turn, these proteins influence EC interactions with immune cells, blood components, pericytes, SMC, and other EC. This dissertation concerns the roles of EC activation by TNF and by TNF and IFN-γ co-stimulation on the expression of selected pro-inflammatory chemokines. A discussion of EC activation in thrombosis, proliferation, angiogenesis, and vasoconstriction is beyond the scope of this work.

23 1.3.1 Leukocyte Adhesion Molecules

Leukocytes do not interact with healthy, unstimulated EC. Leukocyte adhesion molecules are either not present or are found at exceptionally low levels at the EC surface under basal conditions. They are rapidly induced at the genetic level, translated, and presented on the vascular surface upon stimulation.

Leukocyte rolling on the endothelial surface is a relatively weak interaction governed by the selectin family of proteins, P-, E-, and L-selectin. P-selectin is present in platelet secretory granules, as well as in the Weibel-Palade bodies of

EC, and is therefore rapidly displayed on the endothelial cell surface within minutes of agonist exposure (2). L-selectin is expressed on leukocyte populations and has affinity for ligands expressed on ECs and other leukocytes. E-selectin is only expressed in activated EC (Fig. 1.1). Stronger interactions mediated by the immunoglobulin superfamily tether the rolling leukocyte to the endothelium. The adhesion molecules include ICAM-1, -2, and -3, VCAM-1, and PECAM-1 (Fig.

1.1). ICAM-1 is constitutively expressed at low levels by EC. Upon activation

ICAM-1 and genes encoding similar members of the immunoglobulin family are rapidly upregulated.

24

FIGURE 1.1 Schematic of EC-leukocyte interactions in inflammation. Initial contact between the activated leukocyte and EC is random. Weak interactions between selectins on the EC surface and leukocyte counter-ligands mediate rolling along the endothelium. Higher affinity interactions lead to leukocyte arrest and firm adhesion. Activation of other molecules such as integrins enable subsequent emigration through the endothelial barrier and into the surrounding tissue. Chemokine gradients primarily function to recruit leukocytes to specific sites of cellular damage.

25 1.3.2 The Chemokines CXCL10 and CXCL11

The human genome encodes about 50 chemokines and 20 chemokine receptors. Chemokines are a family of structurally related small proteins (~8-10 kDa) that are similar to cytokines. They were originally named for their functions

(e.g. macrophage chemoattractive protein). Those names soon became confusing due to functional chemokine redundancies. A uniform naming system was introduced in 2000 based on positioning of the initial 2 N-terminal cysteine residues. Chemokines are now divided into 4 families (CC, CXC, CX3C, and XC; where X indicates any amino acid) (10).

Most chemokines are secreted proteins and function to regulate leukocyte cell trafficking. The name chemokine is an amalgam of the words chemoattractive and cytokine. Chemokines are distinguishable from other cytokines because they are the only superfamily members that transactivate G- protein-linked 7-transmembrane receptors (11). Interactions between chemokines and chemokine receptors are both promiscuous and redundant.

Promiscuity refers to the affinity of most chemokines for multiple receptors.

Chemokine receptors are redundant in that they may be activated by multiple ligands (12).

The three members of IFN-inducible family of chemokines CXCL9,

CXCL10, and CXCL11 each ligate the CXCR3 receptor. CXCR3 is enriched on

26 activated type-1 helper (Th1) CD4+ T cells, CD8+ cytotoxic effector cells, natural killer, NKT, plasmocytoid dendritic cells, and some B-cell subsets following activation by dendritic cells (13,14). CXCR3 is under the direct control of T-bet, the transcription factor that drives expression of genes encoding the Th1 and

CTL lineage (13). This dissertation focuses on CXCL10 and CXCL11, and will only discuss these chemokines in detail.

CXCL10 was first identified in 1985 as an early response gene activated by IFN-γ and initially named IP-10, for interferon–stimulated protein of 10 kDa

(15). It is a prominent contributing factor in multiple types of inflammatory diseases including atherosclerosis, coronary artery disease, multiple sclerosis, rheumatoid arthritis, psoriasis, asthma, and immune responses to solid organ transplant and infections (13) (Table 1). CXCL10 is not expressed by resting EC, but is induced to high levels in response to a wide variety of agonists, such as

TNF, IFN-α/β/γ, IL-1β, and LPS (13).

27 Table 1. Diseases involving CXCR3, CXCL10, and CXCL11. Disease/Mouse Model CXCR3 CXCL10 CXCL11 Autoimmune Psoriasis (16,17) ( 16) Sarcoid (18,19) (20,21) (19,22) Rheumatoid arthritis (20,23-25) (26,27) Asthma (28,29) (29-31) (29) Atherosclerosis (32,33) (34,35) (32) Multiple sclerosis (36-42) (43-46) Inflammatory bowel disease (47) (48,49) Idiopathic pulmonary fibrosis (50,51) (52) (53) Type I diabetes mellitus (54,55) (55-57) Systemic lupus erythematosus (58-60) (61,62) Cigarette smoke injury/COPD (63-66) (63,66) ( 63) Myocarditis (67)

Transplantation Heart transplant (68-72) (73,74) Lung transplant (75,76) (75-77) (77) Graft-versus-host disease (78,79) (80) Small bowel (81) (81)

Infections Leprosy (82) Tuberculosis (83,84) Influenza (85,86) (85,86) Toxoplasma gondii (87,88) Malaria (89,90) (91-93) Dengue (94) (94,95) Hepatitis B and C (96,97) (97-99) (100) Herpes simplex (101,102) (101-104) Human immunodeficiency virus (105) (105-107) (106) Leishmania (108) (109) Chlamydia trachomatis (110,111) (110) Lyme (112) (113,114) West nile virus (115) (115)

Cancer Renal (116) (116) (116) Colon (117) Melanoma (118) (118) Lymphoma (119-121) (122) Breast (123) (123)

28 TABLE 1 CXCR3, CXCL10, and CXCL11 involvement in disease. CXCR3 and its ligands CXCL10 and CXCL11 are involved in numerous pathological conditions. Listed in this table are selected diseases falling into the autoimmune, infectious, and cancer categories. This table is adapted from Groom and Luster (13).

29 CXCL11 is transcribed upon stimulation by EC, astrocytes, monocytes, neutrophils, and keratinocytes (124). The CXCL11 promoter contains an IRF1,

STAT3, NF-κB1, ISRE, and GAS elements. CXCL11 gene induction is activated in response to IFN-γ and IFN-β but not IFN-α or TNF (125). The nonclassical heterodimer STAT1-STAT3 (as opposed to the canonical STAT1-STAT2 heterodimer) is required for gene induction in response to IFN-γ (13). CXCL11 is more potent than CXCL10 as it is able to trigger CXCR3 internalization, calcium mobilization, and chemotaxis at lower doses (13,126,127). CXCL10 and CXCL11 bind different extracellular regions of the CXCR3 receptor, and require different intracellular regions for internationalization. Activation of CXCR3 by CXCL10 triggers receptor internalization via the carboxyl-terminal domain and beta- arrestin-1 binding domains. In contrast, CXCR3 internalization following CXCL11 ligation principally requires the CXCR3 third intracellular loop (128). Additionally,

CXCL11 is able to activate the CXCR7 receptor (129,130), while CXCL10 cannot.

Diseases involving CXCL11 are listed in Table 1. Care must be taken in evaluating results from CXCL9-/- and CXCL10-/- mice on the C57BL/6 or mixed backgrounds because wild type C57BL/6 mice contain a point mutation in the

CXCL11 gene that results in an early stop codon (13). These mice are null for

CXCL11, but are competent to respond to injected CXCL11.

30 1.4 Pro-Inflammatory Signaling: TNF and IFN-γ

The mammalian immune system is generally divided into two arms: Innate and adaptive. The innate arm of the immune system functions to detect and clear pathogens through activation of the members of the Toll-like receptor (TLR) family of pattern-recognition receptors. Activation of these receptors results in induction of type-1 interferon (IFN-α and IFN-β), which have antiviral, anti- proliferative, and immunomodulatory functions. The second arm of the immune system is the cell-based adaptive immune system. This system is composed of B and T lymphocytes and utilizes antigen receptors to recognize components of infectious agents, and to integrate signals from the cytokine environment to mount an inflammatory response (131).

1.4.1 TNF Signaling

Most mammalian cell types have the ability to produce and secrete TNF.

TNF activates two distinct cell surface receptors, TNF-RI (p55) and TNF-RII

(p75). TNF-RI is ubiquitously expressed. In contrast, TNF-RII expression is restricted to EC and some monocytic populations. Both TNF receptors have a highly homologous extracellular N-terminal ligand-binding domain consisting of multiple cysteine-rich repeats, a transmembrane region, and a cytoplasmic domain (132). Signaling emanating from the receptors is thought to be different due to the complete lack of similarity between the intracellular domains. TNF-RI, for example, has a death domain, whereas TNF-RII does not. Despite the absence of homology, however, signaling commonalities exist, including the

31 requirement of the adaptor protein TRAF2 for signaling from both receptors. Both receptors are capable of activating the NF-κB pathway.

1.4.2 IFN-γ Signaling and the Jak-STAT Pathway

IFN-γ (type II interferon) is a small (19 kDa) secreted cytokine crucial in both innate and adaptive immune responses. It is an activator of major histocompatibility complex (MHC) class I and II presentation, and of macrophage activation (133,134). It is primarily produced by NK and NKT cells in innate immune responses, and by Th1-type CD4+ and CD8+ CTL following development of antigen-specific immunity. IFN-γ secretion is considered a defining characteristic of the Th1 lineage. However, dendritic cells and macrophages are also able to produce IFN-γ, a process thought to be important in autoactivation

(133). IFN-γ is biologically active as a homodimer. It ligates a heterodimer of IFN-

γ-receptor 1 and IFN-γ-receptor 2. Receptor activation activates the kinase function of Janus kinases (JAKs), which autophosphorylate, and then phosphorylate tyrosine residues on the IFN-γ-receptors. Signal transducer and activator of transcription (STAT) proteins are recruited to the receptor phosphotyrosine residues via their SH2 domains and are phosphorylated by the

JAKs (133,134). Phosphorylated STAT proteins form homodimers and heterodimers, translocate to the nucleus, and associate with promoter elements such as GAS and ISRE motifs.

32 1.4.3 The NF-κB Pathway

EC reactions to pro-inflammatory stimulation are coordinated to a considerable degree by the NF-κB pathway. The NF-κB family includes five transcription factors, RelA (p65), RelB, c-Rel, NF-κB1 (p50), and NF-κB2 (p52), and their inhibitors, the IκB subunits, all of which are ubiquitously expressed in mammals. Characteristic of NF-κB family members is a common domain essential for dimerization and DNA binding, the rel homology domain. NF-κB transcription factors are present in cytosolic inhibitory complexes when the pathway is not activated. Upon pathway induction signaling emanating from cytokine receptors such as the TNF-receptor lead to κB inhibitor phosphorylation by the IκB kinase complex (IKK), ubiquitinated, and targeted for proteasomal degradation. Newly liberated NF-κB factors dimerize and translocate to the nucleus, where they bind the loose, decameric consensus sequence

GGGRNNTYCC (R = G or A; Y = C or T; N = any nucleotide) that comprises the

κB sequence of target promoters (135).

1.5 The PTM Code and NF-κB

Protein post-translation modification (PTM) is the covalent addition of chemical functional groups or macromolecules to protein-incorporated amino acids. Protein modifications vary in their permanence and may be present at low stoichiometry (136). There are over 450 known types of PTM in the UniProt database (137,138). The variability of PTM chemistry and physical properties is

33 immense, and includes differences in modification charge, polarity, hydrophobicity, volume, and shape (139). PTM may alter protein conformation and influence protein-substrate, protein-protein, or protein-nucleic acid interactions. Changes in a protein’s intramolecular interactions may potentially resulting in “propagation shifts” as existing contacts are broken and new ones established, an effect that leads neighboring atoms to readjust their molecular associations (139).

As a single PTM can alter a protein’s function, it stands to reason that various combinations of modifications can impart increasingly distinct functionality to protein subsets (139,140). This concept is known as the “protein modification code”. The language of the PTM code is complex and involves cooperative, multisite, order-dependent, antagonistic, and mutually exclusive modifications (139). Combinatorial PTMs result from the activities of distinct signaling pathways and enable the modified protein to function as a signal integrator.

A PTM code confers tremendous advantages to an organism as it greatly expands the diversity and functionality of the proteome far beyond the number of gene products. It allows a cell to operate with a fewer number of genes and allows for rapid responses to physiological or pathological changes by the addition or removal of modifications.

34 The transcriptional activity of NF-κB is regulated by a variety of PTMs, including phosphorylation, acetylation, ubiquitination, and lysine methylation

(141). PTMs regulate important aspects of NF-κB biology, such as protein localization, DNA-binding affinity, and the duration and strength of transcription

(142,143). A major unresolved question in the study of NF-κB remains how the various PTMs of NF-κB enable specific gene activation, differential kinetics, transcription magnitude, and inducer specific responses (135).

35 CHAPTER 2: PROTEIN ARGININE METHYLATION

2.1 Project Goal: Identify PRMT5-Regulated Pro-Inflammatory Genes

At the outset of this project our laboratory had recently performed a mass spectrometric screen for protein-interacting partners of the homeobox containing transcription factor HOXA9. We had a longstanding interest in HOXA9 because due to its roles in co-activating gene expression of the leukocyte adhesion molecule E-Selectin (144). The critical experiment involved immunoprecipitating overexpressed HOXA9 from EC. Immunoprecipitated proteins were separated by

SDS-PAGE, silver-stained, and identified by mass spectrometric analysis. We were able to identify the arginine methyltransferase PRMT5 from that experiment.

Additional experiments showed that PRMT5 positively influenced gene induction by TNF of the leukocyte adhesion molecules E-Selectin and VCAM-1 by associating and methylating HOXA9 on target promoters (145).

The goal of this project was to identify TNF-induced, PRMT5-regulated genes involved in EC pro-inflammatory processes. The initial approaches were to transfect primary human EC with PRMT5 siRNA, expose the cells to TNF, and assess gene expression using real-time PCR. We chose to investigate induction of chemokines due to the roles of family members in promoting and sustaining inflammation. I will introduce arginine methylation, and then specifically discuss roles of PRMT5.

36 2.2 Arginine Methylation

It is estimated that 1-2% of numerous prokaryotic and eukaryotic genomes encode methyltransferases that modify protein-incorporated amino acids at arginine, lysine, histidine, glutamate, glutamine, asparagine, D-aspartate/L- isoaspartate, cysteine, and both N-terminal and C-terminal residues (136,146). In human cells, lysine and arginine methylation are probably the most prevalent type of protein methylation (146).

Methylation of protein-incorporated arginine residues is catalyzed by members of the protein arginine methyltransferase (PRMT) family. Mammalian genomes encode 9 PRMT family members (146). PRMT family members are highly conserved throughout the eukaryotic lineage and are found in protozoa, fungi, plants, and animals (147,148). PRMT catalyze the transfer of a methyl group from the donor S-adenosylmethionine (SAM, Ado-Met) to the nitrogen atoms of the arginine guanidinium group, producing methylated arginine and S- adenosylhomocysteine (SAH, Ado-Hcy).

Mammalian PRMT enzymes are divided into two types based on the methylation pattern they are capable of producing on the arginine side chain.

Both types I and II PRMT5 enzymes can add a single methyl group to either of the two terminal nitrogen atoms, producing mono-ω-N-methylarginine (MMA).

Type I PRMT catalyze the addition of a second methyl group to the same

37 nitrogen atom to produce di-ω-N,N-dimethylarginine (asymmetric dimethylarginine, ADMA). Type II enzymes are capable of adding a second methyl group to the opposite terminal nitrogen atom, producing a product with methyl adducts on both terminal nitrogen residues (di-ω-N,N’-dimethylarginine; symmetrical dimethylarginine, SDMA). The structures of MMA, ADMA, and

SDMA are shown in Fig. 2.1. Arginine methylation usually occurs in RGG

(arginine-glycine-glycine) or GAR (glycine-arginine-rich) motifs (149). Arginine methyltransferases are capable of modifying multiple substrates and often methylate a single substrate at more than one residue (150).

38

FIGURE 2.1 PRMT enzymes catalyze formation of MMA, ADMA, and SDMA. All mammalian PRMT are capable of adding a single methyl group to either terminal nitrogen atom of the arginine side chain (middle structure). Type I PRMT catalyze the addition of a second methyl group to the monomethylated nitrogen atom, producing asymmetrical dimethylarginine (ADMA; top right structure). Most mammalian PRMT catalyze ADMA formation. Type II PRMT form symmetrical dimethylarginine (SDMA; bottom right structure) by adding a second methyl group to the opposite terminal nitrogen atom. PRMT5 is the dominant type II arginine methyltransferase in humans. Each methyltransferase reaction transfers the methyl group from S-adenosylmethionine (SAM) to protein-incorporated arginine, and produces S-adenosylhomocysteine (SAH). Structures were drawn using ChemBioDraw 14.0 (CambridgeSoft).

39 Methylation eliminates potential hydrogen bond donors from the arginine side chain, adds physical bulk, and increases hydrophobicity, but does not alter the residue’s positive charge. Hydrogen atoms of the added methyl group are able to form productive hydrogen bond pairs and thereby facilitate new hydrogen bond networks. Methylarginine-containing residues alter binding interfaces, and have been shown to both impede and promote protein-substrate interactions, depending on the context (147,151-153).

A general signaling paradigm is that there are PTM “writers” that deposit the modification (e.g. tyrosine kinases), “readers” that associate with the modified protein to affect a biological outcome (e.g. SH2-domain containing proteins), and

“erasers” that remove the mark as part of downregulatory mechanisms (e.g. phosphatases). In arginine methylation, PRMT family members are the writers.

The only known methlyarginine reader is the Tudor domain. Tudor domains form a “cage” of aromatic residues that (150,154). The mammalian genome encodes

~30 Tudor domain containing proteins that are either specific for methyllysine or methylarginine. Methylarginine-specific Tudors recognize MMA, ADMA, and

SDMA. The permanence of arginine methylation remains an unresolved issue.

Arginine methylation has historically been considered a permanent modification that persists for the marked protein’s lifetime.

40 The purposes of the projects discussed in this dissertation were to ascertain mechanisms of PRMT5 in the activation of EC inflammatory gene expression. A brief presentation of some of the major roles of PRMT5 in transcriptional activation and RNA processing follows.

2.3 Protein Arginine Methyltransferase 5 (PRMT5)

PRMT5 (EC 2.1.1.125) is the dominant type II (MMA and SDMA- producing) methyltransferase in eukaryotic cells (147). PRMT5 was first identified in humans via a yeast two hybrid screen for Janus kinase binding proteins, and was named Jak2 binding protein 1 (JBP1) (155). PRMT5 is ubiquitously expressed in mammalian cells. Germline knockout of PRMT5 in mice is embryonically lethal (156).

Much of the PRMT5 literature describes PRMT5 as a factor that negatively regulates transcription via methylation of histones. Various PTMs of the flexible, basic histone tails - including arginine methylation - affect transcriptional outcomes by influencing higher order chromatin structure, chromatin accessibility, protein recruitment, and the rate of promoter assembly (147,157-

160). PRMT is capable of methylating various residues on histones H2A, H3, and

H4 (147,158). In some cases PRMT5-catalyzed histone methylation leads to the recruitment of DNA methyltransferases and epigenetic silencing of specific genes

(161).

41 In contrast, methylation of non-histone DNA-binding proteins such as

HOXA9 (145) and p53 (162) results in transcriptional enhancement. At present, only one example of PRMT5 histone methylation positively regulating gene transcription is present in the literature to our knowledge. Arg8 of histone H3 found at the myogenin promoter is dimethylated in muscle cells. This methylation facilitates formation of a transcription complex involving the Brg1 ATPase and the myogenic transcription factor, MyoD (163).

PRMT5 has several roles in the RNA processing. PRMT5 is critical in the formation of the spliceosome, a complex necessary for pre-mRNA exon splicing and m7G cap formation (164-169). In this process, small nuclear ribonucleoproteins (snRNPs) are exported from the nucleus to the cytosol where they bind the seven Sm proteins B/B’, D1, D2, D3, E, F and G to form a ring- shaped core domain. This complex is then reimported into the nucleus where it forms a mature spliceosome complex (164,165). The spliceosome functions to recognize intron-exon boundaries and catalyze spicing. Studies in Arabidopsis show that PRMT5 facilitates alternative splicing by increasing the efficiency of snRNP interactions with weak 5´ splice sites of pre-mRNA (170). The 5´ splice site is recognized through direct RNA-RNA interactions between the U1 snRNA and the splice site. SmB, SmD1, and SmD3 form direct contacts close to the splice site to stabilize the RNA interaction.

42 CHAPTER 3: MATERIALS AND METHODS

3.1 Ethics Statement

HUVEC are isolated from umbilical cords collected by MetroHealth

Hospital and Hillcrest Hospital. The cords are not linked to any patient identification, and isolated EC are pooled. The Cleveland Clinic Foundation

Institutional Review Board has confirmed that our use of discarded, de-identified human tissue is exempt from review under National Institutes of Health guidelines.

3.2 Reagents

Primary human EC were isolated from discarded patient samples as described previously (171). Fetal bovine serum was from Atlas Biologicals (Fort

Collins, CO). Targefect F-2 and peptide enhancer transfection reagents were purchased from Targeting Systems (El Cajon, CA). siRNAs complementary to the coding sequence and 3´-UTR of PRMT5 and the NF-κB p65 3´-UTR were designed using the Whitehead Institute siRNA design tool

(http://sirna.wi.mit.edu/). siRNA sequences are listed in Table 2. All experiments were repeated using two different siRNA sequences for each target to minimize the potential of off-target effects. siRNAs were synthesized by Ambion and contain the Silencer Select modifications. Nontargeting control Silencer Select siRNA (4390843) was purchased from Ambion. ChIP was performed using a kit from Millipore (Billerica, MA; catalog no. 17-295). Antibodies used for ChIP, IP,

43 and Western blots are listed in Table 3. Recombinant TNF from R&D Systems was used at 2 ng/ml. Recombinant IFN-γ from R&D Systems was used at 450

U/ml. Human p65 cDNA was acquired from Addgene (construct 21966). PCR was performed to insert an N-terminal KpnI site followed by a FLAG sequence

(amino acid sequence: DYKDDDDK) in-frame with the start codon. The resulting

FLAG-tagged cDNA was subcloned into the KpnI and HindIII sites of pCDNA3.

The FLAG-tagged fusion protein is expressed under control of the CMV promoter.

44 Table 2. siRNA sequences. mRNA Target Sense Sequence NF-κB p65 (3´ UTR; siRNA #1) 5´-GGAUUCAUUACAGCUUAAUUU-3´ NF-κB p65 (3´ UTR; siRNA #2) 5´-GCUCUUUCUACUCUGAACUUU-3´ PRMT5 (3´ UTR) 5´-GCUCAAGCCACCAAUCUAUUU-3´ PRMT5 (CDS) 5´-GAGGGAGUUCAUUCAGGAAUU-3´

TABLE 2 Sequences of siRNAs used in the experiments. The listed siRNA sequences were transfected into EC at a final concentration of 50 nM. Sequences were designed using the Whitehead Institute siRNA design tool (http://sirna.wi.mit.edu/) and synthesized by Ambion. Protein knockdown for all siRNAs was greater than 90% by 48 hours post-transfection.

45 Table 3. Antibodies used for immunoblotting, immunoprecipitation, and ChIP. Antigen Company ID Species Clonality App. β-Tubulin Sigma-Aldrich T8328 Mouse Monoclonal IB CXCL11 R&D Systems MAB672 Mouse Monoclonal IB FLAG tag Sigma-Aldrich F1804 Mouse Monoclonal IP, ChIP GAPDH Cell Signaling #2118 Rabbit Monoclonal IB HOXA9 Millipore 07-178 Rabbit Polyclonal ChIP NF-κB p65 Millipore 06-418 Rabbit Polyclonal IB NF-κB p65 Cell Signaling #6956 Mouse Monoclonal IP, ChIP PRMT5 Santa Cruz sc-22132 Goat Polyclonal IB PRMT5 Millipore 07-405 Rabbit Polyclonal IP, ChIP SDMA Millipore 07-412 Rabbit Polyclonal IB, ChIP

TABLE 3 Details of the antibodies used in the experiments. All antibodies used in the immunoblot (IB), immunoprecipitation (IP), and chromatin immunoprecipitation (ChIP) experiments presented in this dissertation are listed here. Abbreviations: App., application; Cell Signaling, Cell Signaling Technologies; ID, commercial product identifier.

46 3.3 Cell Culture

Primary human EC were isolated from human umbilical cords (171). Cells from multiple individuals were pooled and used at early passages (P2-4). Cells were cultured in MCDB 107 medium (Sigma Life Sciences) supplemented with

15% FBS (Atlas Biologicals), 150 µg/ml endothelial cell growth supplement

(ECGS), and 90 µg/ml heparin (Sigma-Aldrich). Recombinant TNF-α (R&D

Systems) was used at 2 ng/ml. Recombinant IFN-γ (R&D Systems) was used at

450 U/ml. Cells were serum starved in MCDB media for 3 hours prior to the addition of agonists.

3.4 Transient Transfection

Cells were transfected within 24 h of passage. Both siRNA and cDNA were transfected in serum free DMEM using targefect F-2 (5 μl/ml) and peptide enhancer (5 μl/ml) from Targeting Systems (El Cajon, CA). siRNA was transfected at a final concentration of 50 nM. cDNA was transfected at 250 ng/ml.

All transfections were incubated at 37 °C for 4 h, followed by replacement of the

DMEM-transfection solution with complete medium containing 15% FBS. Cells were cultured for 24–48 h to allow for cDNA expression or protein knockdown.

3.5 Preparation of Nuclear and Cytosolic Extracts

Cells were grown to confluence on 150-mm plates. Cells were separated into nuclear and cytosolic fractions using the CellLytic NuCLEAR Extraction Kit

47 (Sigma) according to the manufacturer’s instructions. Briefly, cells were harvested with a cell scraper, swelled in hypotonic lysis buffer, and lysed with

IGEPAL-630. Following centrifugation, the supernatant was saved as the cytosolic fraction. The pellet was washed three times with hypotonic lysis buffer and lysed in 420 mM NaCl high salt extraction buffer to collect the crude nuclear fraction.

3.6 Immunoprecipitation

Cells were lysed in 1X radioimmune precipitation assay buffer (RIPA; 1%

Nonidet P-40 (NP-40), 0.1% SDS, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM

NaCl) containing protease inhibitors. Endogenous proteins were immunoprecipitated using anti-PRMT5 or anti-p65 antibody from Millipore and protein A/G-agarose beads (Pierce). Overexpressed N-terminal tagged FLAG- p65 was pulled down using anti-FLAG agarose magnetic beads (Sigma).

3.7 Immunoblotting

Lysates were resolved on 10% bis-tris acrylamide gels (145). Proteins were transferred to PVDF membranes and blocked with 5% nonfat milk or protein-free blocking buffer (Pierce; catalog no. 37570). Blots were washed with

1X TBST or milli-Q water and incubated with the appropriate HRP-conjugated secondary antibody.

48 3.8 RNA Purification and Real-Time Quantitative PCR (qRT-PCR)

After transfection, cells were allowed to recover for 48 h. Following TNF treatment cells were lysed with 350 μl of buffer RLT containing 1% 2- mercaptoethanol and processed with the RNeasy kit (Qiagen) to collect RNA. cDNA was synthesized from RNA using SuperScript First-Strand Synthesis reagents (Invitrogen). cDNA was diluted 6-fold, and real-time PCR was performed with Fast SYBR Green PCR Reagents (PE Applied Biosystems) using an ABI StepOnePlus quantitative real-time PCR machine. Primers are separated by an intron and amplified 100–150 base pairs of spliced products. Primer sequences are listed in Table 4. Results were normalized to GAPDH expression following the -ΔΔCt method. For the mRNA stability experiment, actinomycin-D

(Sigma) was used at 1 μg/ml. Amplification, melt curves, and sequenced products indicate a single product identical to the reference sequence.

49 Table 4. qRT-PCR primers for cDNA amplification. Message Primer Sequence F 5´-GCTTAATTTGACTCAACACGGGA-3´ 18S rRNA R 5´-AGCTATCAATCTGTCAATCCTGTC-3´ F 5´-TCTCTTCCTCCACCACTAATGCA-3´ CCL2/MCP-1 R 5´-GGCTGAGACAGCACGTGGAT-3´ F 5´-CATCATGCGGCAAACGCGCA-3´ CX3CL1/Fractalkine R 5´-AGCAGCCTGGCGGTCCAGAT-3´ F 5´-GGCCGTGGCTCTCTTGGCAG-3´ CXCL8/IL-8 R 5´-TGTGTTGGCGCAGTGTGGTCC-3´ F 5´-GCAGAGGAACCTCCAGTCTCAGCA-3´ CXCL10/IP-10 R 5´-GCTGATGCAGGTACAGCGTACAGT-3´ F 5´-CCACGTGTTGAGATGTGAGTGA-3´ CXCL10 pre-mRNA R 5´-TGGCATACGCAGTTCTGAAGTCAG-3´ F 5´-TGAGTGTGAAGGGCATGGCT-3´ CXCL11/I-TAC R 5´-GCTTTTACCCCAGGGCCTAT-3´ F 5´-TCAACAGCGACACCCACTCC-3´ GAPDH R 5´-TGAGGT CCACCACCCTGTTG-3´

TABLE 4 Primer sequences used in cDNA amplification by qRT-PCR. All primers amplified ~100-150 base pairs of the intended mRNA. Products were capillary sequenced and match the reference sequence. All primers were designed using the NCBI Primer-BLAST tool.

50 3.9 CXCL10 Enzyme-Linked Immunosorbent Assay (ELISA)

Cells were plated in six-well dishes and transfected. After 24 h, 1 ml of fresh medium was applied. At 40 h, cells were stimulated with TNF for 8 h. At the end of treatment, conditioned medium was collected and assayed for the presence of CXCL10 using a sandwich ELISA kit (R&D Systems; DIP-100) and read with an iMark plate reader (Bio-Rad).

3.10 Chromatin Immunoprecipitation (ChIP) and Re-ChIP

Cells were cultured in 10-cm dishes, treated with TNF, and crosslinked with 1% formaldehyde (Sigma) in MCDB medium. Immunoprecipitated DNA was purified using the PrepEase DNA Cleanup kit (Affymetrix, Santa Clara, CA).

Primers for the CXCL10 proximal promoter amplify a 182-bp fragment between -

186/+5 containing the κB2, κB1 (distal and proximal κB sites, respectively), AP-1,

CAAT, and TATA elements. Primers amplifying the proximal promoters containing the κB sites of CX3CL1 and MCP-1 were also designed (Table 5).

The ChIP assay was standardized using E-selectin promoter primers (144) and

NF-κB p65 antibody. DNA was quantified in 15 μl quantitative real-time PCR reactions using Power SYBR green (Applied Biosystems) with 40 cycles of amplification.

In the sequential ChIP (Re-ChIP) experiments the complexes from the initial IP were eluted at 37°C for 30 minutes with 10 mM DTT. Following

51 centrifugation the supernatant was removed to a fresh tube and diluted 20 times in Re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl

(pH 8.1). The second IP antibody (or antiserum was then added, followed by crosslink reversal and DNA purification as outlined above.

52

Table 5. qRT-PCR primers for ChIP promoter amplification. Promoter Sequence F 5´-CCCATTTGCTCATTTGGTCTCAGC-3´ CCL2 R 5´-GCTGCTGTCTCTGCCTCTTATTGA-3´ F 5´-GGCATGTTCCCAGCTTGTGGCAGG-3´ CX3CL1 R 5´-GTTGCCAAGGAACCAAGCCGGC-3´ F 5´-AGGAGCAGAGGGAAATTCCGTAAC-3´ CXCL10 R 5´-AACGTGGGGCTAGTGTGCCA-3´ F 5´-TGGAAGCAGGAAAGGTGCAT-3´ CXCL11 R 5´-TGGAAGGAGTAGAAATGCTGAACA-3´

TABLE 5 Primer sequences for ChIP experiments. The primer sequences amplifying ~100-150 base pairs of the proximal promoters region of the given chemokines are listed. Each amplified segment contains at least one κB binding site. Primers were designed using the NCBI Primer-BLAST tool.

53 3.11 CXCL10 Promoter Activity Assay

Cells were plated in triplicate in 24-well plates and transfected with a plasmid encoding -875 to -97 bp of the human CXCL10 promoter (GL-IP10) upstream of firefly luciferase in promoterless pGL3-basic. The GL-IP10 construct was a gift of Richard Ransohoff (172). Cells were cultured for 24 h and then treated with TNF for 6 h, washed twice with MCDB medium, and lysed in 60

μl/well passive lysis buffer (Promega). 20 μl of lysate was pipetted in triplicate into 96-well luciferase plates, activated with luciferase substrate (Promega), and measured with a Dynex luminometer.

3.12 Mass Spectrometry Methods

Coomassie or silver-stained bands of overexpressed p65 were excised

(~65 kDa) and digested in-gel with trypsin, chymotrypsin, or GluC, and analyzed by capillary column LC-tandem mass spectrometry to identify methylated peptides. Enzymatic digestion was carried out at room temperature overnight.

We used a Finnigan LTQ (linear trap quadrapole) ion trap mass spectrometer

(ThermoFinnigan, San Jose, CA) and a self-packed 9 cm x 75 μm inner diameter

Phenomenex Jupiter C18 reversed phase capillary chromatography column.

Collision-induced dissociation (CID) spectra were analyzed using the NCBI human non-redundant protein database. Targeted experiments were also performed involving selected reaction monitoring (SRM) of specific p65 peptides, including unmodified and arginine methylated forms. Chromatograms derived

54 from these data were assessed in relation to known fragmentation patterns. Peak areas were used to quantitate the extent of methylation.

3.13 In Vitro Methyltransferase Assay

Active, recombinant human PRMT5 expressed in HEK293 (Sigma-Aldrich,

SRP0145), full-length recombinant human p65 produced in HEK293 (OriGene,

TA301086), and the methyl donor S–adenosylmethionine (New England Biolabs,

B9003S) were incubated for 90 min at 37 °C in a 30 μl volume according to published methods (145). In each reaction 0.3 μg of PRMT5, 1.0 μg of p65, and

S-adenosylmethionine was added to a final concentration of 80 μM. Buffer conditions were 25 mM HEPES (pH 7.6), 5 mM MgCl2, 100 mM NaCl, and 2 mM dithiothreitol (DTT). Reactions were terminated with the addition of 10 μl of 4X

Laemmli buffer. Arginine methylation was detected by immunoblotting for SDMA.

3.14 p65 Site-Directed Mutagenesis

A FLAG tag was added to the N-terminus of the RelA-pCMV4 construct

(173) (Addgene construct 21966) via PCR and digestion with Kpn1 and Bsm1.

Arginine residues identified as methylation targets were mutated to lysine via the

GeneArt Mutagenesis System (Invitrogen).

55 3.15 NF-κB Point Mutant Reconstitution

Arginine-to-lysine and arginine-to-alanine mutant constructs were co- transfected along with a CMV-lacZ reporter plasmid and 3´-UTR targeting sip65 introduced to knockdown endogenous p65. Cells were maintained for 45 h and then stimulated with TNF for 3 h. RNA was collected, reverse-transcribed, and analyzed for chemokine expression.

3.16 Statistical Analysis

Results are presented as means ± S.E. Comparisons between groups were evaluated by one- or two-way analysis of variance followed by Bonferroni post hoc tests to evaluate pairwise comparisons. Multiple comparisons were performed by Student’s t-test. A p value ≤ 0.05 was selected as the cutoff for significance. p values are expressed in the figures as follows: *, p ≤ 0.05; **, p ≤

0.01; ***, p ≤ 0.005. For all experiments, n = 3–4, with the exception of the mass spectrometric analysis (n = 2).

56 CHAPTER 4: PRMT5-CATALYZED METHYLATION OF NF-κB p65

ACTIVATES CXCL10 EXPRESSION

4.1 Preface

Results presented in this chapter were published in my first-author article entitled “Tumor Necrosis Factor (TNF)-α Induction of CXCL10 in Endothelial

Cells Requires Protein Arginine Methyltransferase 5 (PRMT5)-mediated Nuclear

Factor (NF)-κB p65 Methylation,” published in The Journal of Biological

Chemistry (174). According to JBC policy, this material can be included in a

Ph.D. dissertation without additional permissions.

4.2 Introduction

Activation of endothelial cells (EC) in response to inflammatory agonists lead to the production of growth, regulatory, and vasoactive substances that are not produced by unstimulated EC (9). Studies from our laboratory and others identify arginine methylation as a contributing factor in induction of the inflammatory genes E-selectin, VCAM-1, IL-2, CXCL8/IL-8 and IκBα (145,175-

177). Arginine residues in mammalian proteins can carry 0, 1, or 2 methyl groups on their terminal (ω) nitrogen atoms. Methylation eliminates a potential hydrogen bond, adds bulk, and increases hydrophobicity, but does not alter the residue’s positive charge. Methylarginine residues may impede or promote protein- substrate interactions (147,151-153). Protein arginine methyltransferase 5

57 (PRMT5) is the major enzyme that catalyzes SDMA formation in mammalian cells, and also produce monomethylated (MMA) products. PRMT5 regulates transcription through multiple mechanisms, including alterations in chromatin structure and methylation of proteins involved in transcription, elongation, and splicing (147).

To complement our previous studies of arginine methylation in the expression of leukocyte adhesion molecules (145), we chose to examine roles of

PRMT5 in expression of chemoattractive cytokines (chemokines). Most pro- inflammatory chemokines are secreted proteins that promote recruitment, adherence, and extravasation of circulating leukocytes (13,14). We determined that the chemokine CXCL10 requires PRMT5 to achieve full gene induction in response to TNF. CXCL10 has been extensively studied since its discovery in

1985 by Luster et al. (15) due to its contributions to numerous pathologies involving Th1-type inflammation, including atherosclerosis, coronary artery disease, multiple sclerosis, rheumatoid arthritis, psoriasis, asthma, and immune responses to solid organ transplant and infections (13) (Table 1). Despite its prominence in such a wide variety of pathologies, only one published study to our knowledge has examined CXCL10 expression in EC (178). CXCL10 is rapidly induced in response to TNF, IFN-α/β/γ, IL-1β, or LPS (13). Secreted CXCL10 recruits and retains type-1 helper (Th1) CD4+ T cells, CD8+ cytotoxic effector cells, natural killer, NKT, plasmocytoid dendritic cells, and some B-cell subsets at

58 inflammatory lesions (13,14). CXCL10 also has non-immune cell effects as a potent SMC mitogen and chemotactic agent, and as a vascular angiostatic factor

(179,180).

CXCL10 induction is driven in part by NF-κB transcription factors, known as master regulators of inflammation and immunity (142). In canonical NF-κB signaling the latent cytosolic transcription factor NF-κB is activated by kinases that phosphorylate p65 and trigger the degradation of inhibitory IκB subunits.

These events free NF-κB to translocate into the nucleus where it associates with

κB sites in target promoters (142).

Multiple types of post-translation modifications of p65 are established, including roles for phosphorylation, acetylation, and ubiquitination. Roles of many of these modifications have been identified, including as regulators of protein localization, DNA-binding affinity, interactions with other proteins, and the duration and strength of transcription (142,143). However, a major unresolved question in the study of NF-κB remains how the various post-translational modifications of NF-κB enable specific gene activation, differential kinetics, transcription magnitude, and inducer-specific responses (135). Many of these processes are governed by post-translational modifications of NF-κB. We report here that the arginine methyltransferase PRMT5 post-translationally modifies the

59 p65 subunit of the NF-κB Rel-homology domain, a step imperative to the

CXCL10 induction by TNF.

4.3 Results

4.3.1 PRMT5 Enhances TNF-induced CXCL10 Gene Expression

Our group has previously reported that PRMT5 positively regulates expression of leukocyte adhesion molecules VCAM-1 and E-selectin in TNF- stimulated EC (145). Here we explored the regulatory role of PRMT5 in the TNF- mediated induction of chemokines involved in vascular disease pathogenesis. To identify PRMT5’s role in inflammatory chemokine gene expression we compared

TNF-induced mRNA levels of chemokines in PRMT5-intact versus PRMT5- depleted EC. Transfection of PRMT5 siRNA yielded greater than 80% knockdown of PRMT5 protein (Fig. 4.1A). Depletion of PRMT5 in EC led to a substantial decrease (> 60%) in induction of both CXCL10 (Fig. 4.1B) and

CX3CL1/fractalkine (Fig. 4.1C) message levels. Thus, PRMT5 is necessary to achieve full induction of these chemokines in stimulated EC. In contrast, expression of both CXCL8/IL-8 (Fig. 4.1D) and CCL2/MCP-1 (Fig. 4.1E) were unaffected by the absence of PRMT5. We conclude that PRMT5 is required for the expression of specific pro-inflammatory chemokines, including CXCL10 (Fig.

4.1B) and CX3CL1 (Fig. 4.1C) in EC. In this study we focused on elucidating the mechanism of PRMT5’s contribution to CXCL10 expression.

60

FIGURE 4.1 PRMT5 is necessary for the induction of CXCL10 and CX3CL1 in TNF-stimulated EC. (A) Western blot analysis of PRMT5 levels from control and siPRMT5 transfected cells are shown in A, and quantified at right (p < 0.01). (B-E) Real-time quantitative PCR was performed to measure mRNA of the following chemokines from control and siPRMT5 transfected EC: (B) CXCL10 (p < 0.0001); (C) CX3CL1/Fractalkine (p < 0.001); (D) CXCL8/IL-8 (p > 0.05); and (E) CCL2/MCP- 1 (p > 0.05).

61 To differentiate between potential roles for PRMT5 in transcriptional versus post-transcriptional processes we measured CXCL10 pre-mRNA in TNF- stimulated EC with quantitative real-time PCR. The levels of CXCL10 pre-mRNA in PRMT5-depleted cells were significantly lower compared to control cells (Fig.

4.2A) indicating that regulation occurs at the level of transcription. In some circumstances chemokine protein production is modulated through regulating mRNA half-life (181). To assess a possible role for PRMT5 in CXCL10 mRNA stability we blocked de novo transcription with actinomycin-D and observed no significant difference in message stability with and without PRMT5 (Fig. 4.2B).

To provide further evidence for PRMT5’s role in CXCL10 expression we transfected EC with a promoter-reporter construct consisting of a 1 kb fragment of the CXCL10 promoter proximal to the transcription start site cloned upstream of the luciferase reporter. Results from this assay showed significantly diminished promoter activity from PRMT5-depleted cells compared with control cells (Fig.

4.2C). We also analyzed CXCL10 protein levels via ELISA assay in EC conditioned media following 8 hours of TNF-stimulation. EC lacking PRMT5 showed significant reduction as compared with control siRNA transfected cells

(Fig. 4.2D). This finding is consistent with the mRNA result presented in Fig.

4.1B. Taken together, we conclude that PRMT5 regulates TNF-stimulated induction of CXCL10 at the transcriptional level.

62

FIGURE 4.2 Knockdown of PRMT5 reduces CXCL10 transcription. (A) CXCL10 pre-mRNA levels were measured following TNF-stimulation (p < 0.005). (B) CXCL10 mRNA stability was quantified following stimulation (3 h) and application of actinomycin-D (1 μg/ml). (C) CXCL10 promoter activity was measured by luciferase luminescence intensity (p < 0.005). (D) ELISA was used to measure levels of CXCL10 in conditioned media (p < 0.0005).

63 4.3.2 PRMT5-Catalyzed SDMA-Containing Proteins Associate with the

CXCL10 Promoter

Considering PRMT5’s positive contribution to CXCL10 transcription, we next asked whether the SDMA modification could be detected at the CXCL10 promoter. To address this question we employed chromatin immunoprecipitation

(ChIP) assays using an antibody specific for SDMA, and PCR primers that amplify a region of the proximal promoter critical for CXCL10 induction (Fig.

4.3A). As shown in Fig. 4.3B, our ChIP results demonstrated the presence of

SDMA-containing proteins on the CXCL10 proximal promoter in TNF-stimulated

EC. Importantly, when PRMT5 is knocked down in TNF-stimulated cells promoter amplification was at background level (Fig. 4.3B). These results therefore suggest that PRMT5 symmetrically dimethylates at least one protein associated with the CXCL10 promoter in response to TNF-stimulation in EC.

64

FIGURE 4.3 PRMT5 activity leads to the association of SDMA-containing proteins with the CXCL10 promoter following TNF-stimulation. (A) Illustration depicting the CXCL10 promoter 250 bp upstream of the transcription start site. Major transcription factor binding sites are noted, including the two functional κB sites, which are numbered according to their position relative to the transcription start site. Arrowheads under the promoter denote the ChIP primer binding sites. (B) The CXCL10 promoter was immunoprecipitated in a ChIP assay using anti-SDMA modification antibody. PRMT5-depletion blunted TNF induced association of SDMA-containing proteins with the CXCL10 promoter. CXCL10 promoter amplification was also performed in ChIP assays with anti-PRMT5 (C) (p > 0.05), and anti-HOXA9 (D) (p > 0.05) IP antibodies.

65 In a previous investigation we showed that PRMT5 and HOXA9 associate on the promoters of E-selectin and VCAM-1 upon TNF exposure in EC. We investigated whether CXCL10 induction occurs though the same mechanism

(145). We performed ChIP experiments to assess whether HOXA9 could be identified together with PRMT5 at the CXCL10 promoter. We did not detect

CXCL10 promoter enrichment of either HOXA9 (Fig. 4.3C) or PRMT5 (Fig. 4.3D) in unstimulated or stimulated EC. Together, the ChIP results indicated that

PRMT5 catalyzes methylation of at least one DNA-binding protein that associates with the CXCL10 promoter following TNF-induced activation of EC. Methylation appears to occur prior to association with the CXCL10 promoter.

4.3.3 NF-κB p65 is Symmetrically Dimethylated by PRMT5

To identify SDMA-modified proteins that associate with the CXCL10 promoter following TNF activation we immunoprecipitated transcription factors known to associate with the CXCL10 proximal promoter (Fig. 4.3A), such as NF-

κB p65, p300, and STAT1 (172,182). Of these factors, only NF-κB p65 was symmetrically dimethylated in both the stimulated and unstimulated states as detected by the anti-symmetrical dimethylarginine antibody (Fig. 4.4A). To test whether the arginine methylation detected on p65 is catalyzed by PRMT5 we depleted PRMT5 with siRNA, immunoprecipitated p65, and assessed SDMA levels by western blot. PRMT5 knockdown resulted in a substantial decrease (>

80%) in arginine methylation levels of both untreated and TNF-stimulated lysates

66 (Fig. 4.4B). We performed an in vitro methyltransferase assay using human recombinant PRMT5 and p65 to test whether PRMT5 is capable of directly methylating p65. We observed an increase in p65 methylation as detected by the anti-SDMA antibody following the methyltransferase reaction (Fig. 4.4C). To assess whether arginine methylated forms of p65 translocated to the nucleus, cytosolic and nuclear fractions from control and TNF-treated cells were prepared.

Endogenous p65 was immunoprecipitated from each fraction and analyzed by western blot. Results show that the SDMA-containing forms of p65 are located in the cytosol in the absence of TNF. After stimulation these methylated p65 species migrate to the nucleus (Fig. 4.4D).

67

FIGURE 4.4 NF-κB p65 is methylated by PRMT5. (A) Immunoprecipitated endogenous p65 was probed by western blot for the SDMA modification following TNF-stimulation. Densitometry of the SDMA signal is shown at right (p > 0.05). (B) Endogenous p65 was immunoprecipitated from control and siPRMT5 transfected cells and probed for SDMA by western (left) and quantified by densitometry (right; p < 0.01). (C) In vitro methyltransferase assay analyzed by western blot with anti-SDMA antibody (left), and quantified in the panel at right (p < 0.01). (D) Endogenous p65 was immunoprecipitated from cytosolic and nuclear extracts and probed with the anti-SDMA antibody.

68 4.3.4 p65 is Methylated at 5 Arginine Residues

Mass spectrometric analysis was used to identify the specific dimethylarginine residues in p65. FLAG-tagged p65 was overexpressed in EC, immunoprecipitated and subsequently visualized on a Coomassie-stained gel

(Fig. 4.5A). Simultaneously, we probed a small sample of these immunoprecipitates for SDMA by western blot to detect the modification levels in these samples (Fig. 4.5B). p65-containing bands in the Coomassie-stained gel were digested with trypsin, chymotrypsin and GluC, and analyzed by mass spectrometry to detect mono- and dimethylarginine. Comparison between the mass spectra of the (23)IIEQPKQRGMoRFRYKCE(39) unmodified peptide (Fig.

4.5C) versus its MMA-containing (Fig. 4.5D) and SDMA-containing forms (Fig.

4.5E) suggested methylarginine residues at Arg30 or Arg35. Mass shift consistent with dimethylation was detected at Arg174 in the (174)RLPPVLSHPIF(184) peptide (Fig. 4.5F). Dimethylation of Arg304 is present in the (304)RTYETFK(310) peptide (Fig. 4.5G). Both monomethylation (Fig. 4.5H) and dimethylation (Fig.

4.5I) were found in the (330)RIAVPSR(336) peptide at Arg330. In all, 5 different arginine loci containing either MMA or dimethylarginine were identified (Table 6).

These residues are Arg30 or Arg35, and Arg174 present in the Rel homology domain (RHD), Arg304 in the nuclear localization signal, and Arg330, present in an interdomain region (Fig. 4.5J). No significant change in methylation levels of any of these residues was observed in response to TNF.

69

70

71 72

73 FIGURE 4.5 Five p65 arginine residues are dimethylated in EC. (A) Lysates from wild type p65 transfected-EC were immunoprecipitated with anti-FLAG antibody, resolved by SDS-PAGE, and stained with Coomassie blue. (B), top panel: A fraction of the immunoprecipitate in A was probed separately for anti-p65 by immunoblot. The membrane was then stripped and reprobed with anti-SDMA. Bottom panel: Pre-immunoprecipitated input material was probed with anti-FLAG antibody to show FLAG-p65 expression. The lower molecular weight band in A and B appears to be related to FLAG-p65. (C-E), Full-length FLAG-p65 bands were analyzed by mass spectrometry for the presence of methylarginine residues. MS/MS spectra of the unmethylated (C), monomethylated (D), and dimethylated (E) peptide (23)IIEQPKQRGMoRFRYKCE(39) containing Arg30 and Arg35. (F) MS/MS spectra of the (174)RLPPVLSHPIF(184) peptide containing Arg174. (G) MS/MS spectra of the (304)RTYETFK(310) including Arg304. (H) MS/MS spectra of the (330)RIAVPSR(336) containing Arg330. (I) MS/MS spectra of the (330)RIAVPSR(336) peptide. (J) The location of methylated arginine residues are shown on a diagram of the human p65 domain structure.

74

Table 6. Methylated p65 peptides obtained from MS/MS experiments. Peptide Enzyme Residue Type IIEQPKKQRGMoRFRYKCE GluC Arg30, Arg35 MMA, DMA PLRLPPVLSHPIFDNR Trypsin Arg174 DMA RLPPVLSHPIF Chymotrypsin Arg174 DMA RLPPVLSHPIFDNRAPN Chymotrypsin Arg174 DMA RTYETFK Trypsin Arg304 DMA RIAVPSR Trypsin Arg330 MMA, DMA

TABLE 6 Methylated p65 peptides obtained from MS/MS experiments. Presented here are the proteases used and corresponding methylarginine- containing peptides of p65 detected in the MS/MS experiments. Methylated arginine residues are shown in bold in the first column.

75 4.3.5 p65 Association with the CXCL10 Promoter Requires p65

We performed a ChIP assay in the absence of PRMT5 and assessed the association of p65 with the CXCL10 promoter. Under control transfection conditions, we observed ∼4-fold increase in p65 promoter association upon addition of TNF. In contrast, in the absence of PRMT5, promoter amplification was not altered significantly between the TNF-treated versus unstimulated conditions (Fig. 4.6A). A similar pattern was observed with p65 association with the CX3CL1 promoter (Fig. 4.6B). However, p65 association with the MCP-1 promoter upon TNF-stimulation was similar in both the control and PRMT5 knockdown conditions (Fig. 4.6C).

76

FIGURE 4.6 PRMT5 is necessary for p65 association with the CXCL10 promoter. (A-C) In a ChIP assay, protein-DNA complexes were immunoprecipitated with anti-p65 antibody from control and PRMT5 siRNA transfected, TNF-stimulated EC. (A) Enrichment of the CXCL10 promoter was enhanced upon TNF- stimulation in control-transfected cells (p < 0.05), but not in PRMT5-depleted conditions. (B) p65 association with the CX3CL1 promoter was also increased upon application of TNF in control transfected cells (p < 0.05). (C) Association of p65 with the MCP-1 promoter was not altered by PRMT5 depletion.

77 4.3.6 Methylation of the p65 RHD is Necessary for CXCL10 Induction

We next tested the hypothesis that at least one arginine residue must be methylated to induce CXCL10 expression by TNF using site-directed arginine to lysine point mutants. This conservative mutation preserves the positive charge at the position, but prevents methylation by arginine methyltransferases. Following transfection in EC, the mutant constructs express p65 at levels comparable to the wild type (Fig. 4.7A). Additionally, we used siRNA targeted against the 3´ UTR of p65 to knockdown endogenous protein levels without affecting expression of the transfected p65 cDNA (Fig. 4.7B). To identify specific dimethylarginine residues required for CXCL10 induction, EC were transfected with either wild type or mutant p65 cDNA along with the above mentioned p65 siRNA and treated with

TNF. RNA was isolated and analyzed for specific chemokine mRNA expression.

R30K and R35K mutants were ineffective in recapitulating wild type levels of

CXCL10 expression (Fig. 4.7C). Expression of R304K significantly increased

CXCL10 expression, and all other Arg-to-Lys mutants led to levels of CXCL10 not statistically different from the wild type reconstituted levels (Fig. 4.7C).

Similar results were obtained for expression of CX3CL1 (Fig. 4.7D). In contrast, expression of MCP-1 by R30K and R35K mutants was not different from wild type, indicating these mutants are functional (Fig. 4.7E). R174K, R304K, and

R330K enhanced MCP-1 expression relative to wild type (Fig. 4.7E).

78

79 FIGURE 4.7 SDMA-p65 is critical for CXCL10 induction. (A) p65 arginine-to-lysine (R-to-K) mutants at Arg30, Arg35, Arg174, Arg304, and Arg330 were expressed and immunoblotted for p65. Quantification of the immunoblot is shown at right (p > 0.05). (B) Lysates from p65 3´ UTR targeting siRNA and control transfection are presented (left) and quantified (right; p < 0.05). (C-E) Knockdown of endogenous p65 and reconstitution with R30K, R35K, R174K, R304K, and R330K was performed in EC. TNF-mediated induction of CXCL10 (C), CX3CL1 (D), and MCP-1 (E) in EC reconstituted with p65 mutants is shown.

80 4.4 Discussion

We previously demonstrated the requirement of PRMT5 for the induction of the EC-leukocyte adhesion molecules E-selectin and VCAM-1 (145). Here we report a critical role of PRMT5 in specific chemokine gene induction in EC. The mechanism we report here differs from our previous report of E-selectin and

VCAM-1 induction in that HOXA9 is not involved, and that PRMT5 is not detected by ChIP on the promoter itself. We report that PRMT5 is capable of methylating

NF-κB p65, both in vitro and in cultured EC. Mass spectroscopy approaches identify that NF-κB p65 is methylated at multiple residues in EC. We show that p65 recruited to the CXCL10 and CX3CL1 promoters in response to TNF was methylated by PRMT5 at arginine residues. Furthermore, we show that arginine methylation of Arg30 and Arg35 of the p65 Rel-homology domain is critical for

TNF-stimulated induction of the pro-inflammatory chemokine CXCL10. Together, we propose that PRMT5 methylation of p65 at Arg30 and Arg35 enhances p65 association and transcription of a subset of TNF-responsive pro-inflammatory genes in EC.

Most reports of PRMT5’s role in transcription describe PRMT5 as a transcriptional repressor that operates through SDMA modification of histones

H2A, H3, and H4 (147,158). In contrast, methylation of non-histone DNA-binding proteins such as HOXA9 (145) and p53 (162) results in transcriptional enhancement. In CXCL10 expression, we show that SDMA-containing proteins

81 associate with the promoter only when PRMT5 is present and following TNF- simulation. Through immunoprecipitation, mass spectrometry, and siRNA-based approaches we demonstrate that PRMT5 methylates NF-κB p65 at multiple arginine residues. Methylation of arginine residues 35, 174, 304, and 330 has not been reported in the literature. The fifth, Arg30, was recently reported by Wei et al. (183) and is also catalyzed by PRMT5. Arginine methylated p65 is present at low abundance. However, small quantities of post-translationally modified species are sufficient to initiate physiological responses, particularly in the context of transcription. Through replacement of endogenous p65 with Arg-to-Lys mutants, we show that dimethylation of Arg30 and Arg35 is critical for TNF-induced

CXCL10 expression in EC. Of the two residues, methylation of Arg35 appears to be of greater significance in CXCL10 induction. Both R30K and R35K mutants remain competent to induce MCP-1 expression, however, which shows that these mutants are still transcriptionally functional. We also find that reconstitution with the R304K mutant resulted in significantly increased CXCL10, CX3CL1, and

MCP-1 expression in EC as compared with the wild type. We suggest that Arg304 methylation may be an inhibitory modification that interferes with cofactor interactions. Alternatively, the presence of lysine instead of arginine at that location alone may be enough to change p65 conformation and enhance expression of these genes.

82 Arg30 and Arg35 are present in p65ʹs Rel-homology domain (RHD), a motif necessary for dimerization, DNA binding, and cytosolic localization of p65 (142).

Arg35 directly contacts the 3´ subsite of κB sites (184). However, the 3´ subsite for p65 is not well-conserved, requiring conformational flexibility of κB dimers to allow for base-specific interactions with the DNA (184). As reported by Wei et al.,

Arg30 dimethylation probably enhances DNA affinity (183). Methylation of Arg35 may function though a similar mechanism, in that one or both of the CXCL10 κB elements may require methylarginine in contrast to the κB element/s present in the promoters of other chemokines.

We show that p65 is methylated under non-stimulated conditions in EC.

We did not detect changes in methylation levels by anti-SDMA western blot following TNF-exposure. However, the anti-methylarginine antibody may be interacting with several p65 methylarginine sites and changes at individual sites may be masked or not be detected by the antibody. Conditions may exist whereby individual methylarginine sites are modulated.

The permanence of arginine methylation is an open question. Historically, arginine methylation has been seen as a permanent modification that may provide a mechanism for long-lived transcriptional effects. Roles for arginine demethylases have yet to be established. In this case p65 methylation does not appear to be enhanced by TNF. Termination of methylarginine-containing p65

83 signaling could be achieved through canonical NF-κB downregulatory mechanisms, including cytosolic re-sequestration by IκB upon nuclear export, or degradation through proteolytic mechanisms (185).

A major active area of NF-κB research focuses on determining the underlying molecular mechanisms of specific gene induction. A primary driver of context-dependent gene expression is the post-translational modifications of p65, including phosphorylation, acetylation, lysine methylation, and ubiquitination

(141,142,186). Individual modifications can also influence the presence or absence of subsequent modifications through post-translational modification crosstalk. Comprehensive study of histone post-translational modification in mouse brain reveals that post-translational modifications are present in defined combinations with different ratios (187). Studies have noted that methylation of

H3 at Arg2 enhances the probability of Lys4 acetylation, and together, these modifications control interaction with other proteins (188,189). Post-translational modification crosstalk has been demonstrated in non-histone substrates as well.

Arginine methylation of the nuclease FEN1 inhibits nearby phosphorylation and enhances binding to the DNA repair enzyme PCNA (190). In another example, arginine methylation of EGFR by PRMT5 at Arg1175 enhances trans- autophosphorylation of Tyr1173 leading to increased ERK activation (191).

Arginine methylation of p65 is likely to be a factor in other combinatorial mechanisms regulating p65 functionality and promoter targeting.

84 Development of strategies to limit CXCL10 production by inhibiting PRMT5 or the PRMT5-p65 interaction may ultimately be beneficial in the management of inflammatory and immune pathologies. Inhibitor utilization will have to be weighed against a potential increase in susceptibility to other diseases. For example, while CXCL10 is atherogenic and promotes development of coronary artery disease (34,35,192,193), it protects against the formation and rupture of abdominal aortic aneurysms (AAA) in a murine model (194).

This study is the first to demonstrate that PRMT5 enhances expression of

CXCL10, a chemokine regulator of Th1 effector populations at sites of infection and inflammation. This occurs through PRMT5-mediated methylation of NF-κB at

Arg30 and Arg35. Methylarginine-containing p65 subsequently interacts with the

CXCL10 promoter where it may enhance promoter association. Future studies are warranted to address the underlying molecular mechanism of p65 Rel- homology domain methylation in gene specific transcriptional activation.

4.5 Acknowledgements

We thank Lisa Dechert, Chad Braley, and Emily Tillmaand for isolating EC used in this study.

85 CHAPTER 5: PRMT5-MEDIATED METHYLATION OF NF-κB p65 AT ARG174

ACTIVATES CXCL11 GENE INDUCTION IN EC CO-STIMULATED WITH TNF

AND IFN-γ

5.1 Preface

The results presented in our manuscript entitled “PRMT5-mediated

Methylation of NF-κB p65 at Arg174 is Required for Endothelial CXCL11 Gene

Induction in Response to TNF-α and IFN-γ Costimulation” published in PLoS

One are presented in this chapter. Rights to the PLoS One manuscript’s contents are retained by the article’s authors and can be reproduced under the Creative

Commons Attribution (CC BY) license.

5.2 Introduction

Activation of EC by inflammatory substances results in stimuli-specific induction of proteins that participate in EC-leukocyte interactions and leukocyte recruitment to inflammatory foci (9). We have previously reported that arginine methylation of transcription factors catalyzed by PRMT5 potentiates expression of the leukocyte adhesion molecules VCAM1 and E-selectin and the chemokines

CXCL10 and CX3CL1 in response to TNF (145,174). PRMT5 is a member of the protein arginine methyltransferase (PRMT) family and catalyzes the covalent addition of methyl groups to the two terminal nitrogen atoms of protein- incorporated arginine. Type I and II arginine methyltransferases can add a single

86 methyl group to arginine, producing monomethylarginine (MMA). Type I PRMT add a second methyl group to the same nitrogen atom to produce di-ω-N,N- dimethylarginine (asymmetric dimethylarginine, ADMA). In contrast, type II PRMT are capable of adding a second methyl group to the opposite terminal nitrogen atom of arginine, producing a product with methyl adducts on both terminal nitrogen residues (di-ω-N,N’-dimethylarginine; symmetrical dimethylarginine,

SDMA) (195-197). MMA, SDMA, or ADMA at a particular site may result in opposing transcriptional outcomes depending on cellular context (198,199). The presence of ADMA or SDMA is therefore not definitively predictive of transcriptional activity.

PRMT5 is a member of the protein arginine methyltransferase (PRMT) family and catalyzes the covalent addition of methyl groups to either of the two terminal nitrogen atoms of protein-incorporated arginine, producing monomethylarginine (MMA) or symmetrical dimethylarginine (SDMA) (147) (Fig.

2.1). PRMT5 is the primary enzyme responsible for formation of SDMA in mammals. Addition of a methyl group to the arginine side chain increases steric bulk and hydrophobicity, and eliminates a potential hydrogen bond, but does not alter arginine’s positive charge (139,147,153). These chemical changes imparted by PRMT5 function to enhance or impede protein-substrate interactions by modulating interaction surfaces (167-169,197,199-204).

87 Combinations of posttranslational modifications (PTMs) such as arginine methylation, phosphorylation, and acetylation enable a “PTM code” that transduces context-specific information to the nucleus, facilitating nuanced control over transcriptional responses (137,138,205-207). Symmetrical dimethylation of the transcription factors p53 (162,208,209) and NF-κB p65

(174,183) by PRMT5 have been shown by multiple groups to enhance expression of specific genes. We reported previously that expression of CXCL10 in response to TNF in EC requires PRMT5-catalyzed arginine methylation of the transcription factor p65 at Arg30 and Arg35 (174). These residues are located in the proximal region of the p65 rel homology domain and are part of the p65 DNA- binding core. Arg35 in particular directly interacts with the 3´ subsite of the κB promoter element (184). Methylation of these residues likely enhances p65-DNA binding by facilitating hydrophobic interactions between the methyl groups and

DNA base pairs (174,183,184).

Our previous study also identified other residues methylated by PRMT5 on p65, such as Arg174, a residue accessible to the cytosol that is located in a region important for mediating protein-protein interactions (142). We posited that p65

Arg174 methylation is also important for mediating stimuli-specific chemokine gene expression. To test this hypothesis we first suppressed PRMT5 levels in EC using RNAi, and stimulated the cells with TNF, IFN-γ, and TNF plus IFN-γ to identify chemokines requiring PRMT5 for induction. We discovered that PRMT5

88 is essential for induction of the chemokine CXCL11 when co-stimulated with TNF plus IFN-γ. CXCL11 is transcribed upon stimulation by EC, astrocytes, monocytes, neutrophils, and keratinocytes (124). Ligation of CXCL11 to the classical CXCR3 receptor enriched on activated Th1-type (type-1 helper) CD4+ and CD8+ cytotoxic T-lymphocytes (CTL), CD4+ and CD8+ memory cells, natural killer (NK), natural killer T cells (NKT), dendritic cells, and some B cells results in recruitment of leukocyte populations to inflamed sites (12-14). CXC11 further increases the polarity of lymphocytes at inflammatory lesions by antagonizing the

CCR3 receptor enriched on Th2-type lymphocytes (210). CXCL11 participates in numerous pathologies including atherosclerosis, organ transplant, inflammatory arthritis, inflammatory bowel disease, psoriasis, asthma, hematopoietic malignancies, and responses to infection (11-13,211).

Additional characterization of the role of PRMT5 in the induction of

CXCL11 revealed a requirement for methylation of p65 at Arg174. Together with our previous report (174), our results show that arginine methylation of p65 residues by PRMT5 comprises a critical aspect of the PTM code governing the specificity of inflammatory chemokine gene expression.

5.3 Results and Discussion

In our previous publication we identified that symmetrical dimethylarginine

(SDMA) formation catalyzed by PRMT5 on Arg30 and Arg35 of p65 is necessary for induction of CXCL10 by TNF in EC (174). Methylation of these residues is

89 part of a signaling mechanism enabling transcription of the chemokines CXCL10 and CX3CL1, and not other pro-inflammatory chemokines, such as CXCL8/IL-8 or CCL2/MCP-1. We also discovered methylation at other p65 arginine residues, such as Arg174, and postulated that arginine methylation of this residue may be necessary to induce other pro-inflammatory chemokines in a stimulus-dependent manner. In particular, we explored the effects of EC co-stimulation by TNF plus

IFN-γ as this combination of agonists synergistically induces CXCL10

(172,212,213) and CXCL11 (214-216). Coincident stimulation of EC with TNF and IFN-γ is physiologically relevant as both factors are simultaneously present in localized regions of inflammation under pathological conditions, such as atherosclerosis (217).

5.3.1 PRMT5 Promotes CXCL11 Gene Expression

We tested our hypothesis by measuring chemokine mRNA expression levels in control versus PRMT5-depleted EC. Cells were serum-starved for 48 h post siRNA transfection and then stimulated with TNF, IFN-γ, and TNF plus IFN-

γ. Total RNA was isolated and real-time PCR was performed to measure chemokine mRNA levels. Knockdown of PRMT5 efficiently suppressed PRMT5 protein levels by greater than 90% (Fig. 5.1A, p < 0.001). We found that transcription of CXCL11 mRNA was significantly blunted in PRMT5-depleted EC in response to TNF plus IFN-γ co-stimulation (~30% of the mRNA elicited from the control cells, p < 0.05, Fig. 5.1B). PRMT5-depletion did not significantly alter

90 CXCL11 mRNA abundance in response to IFN-γ stimulation alone (p > 0.05). We observed a significant decrease in CXCL10 mRNA induction when both agonists were presented individually, and in combination (Fig. 5.1C, all p < 0.05). TNF plus IFN-γ-mediated induction of CXCL10 (Fig. 5.1C), or CCL2 (Fig. 5.1D) was not significantly affected by PRMT5 depletion. We verified a positive role for

PRMT5 in CXCL11 induction at the protein level by immunoblot (Fig. 5.1E).

CXCL11 levels in PRMT5-depleted, TNF plus IFN-γ-treated EC was reduced by about 90% of the TNF plus IFN-γ-treated control cells (p < 0.001; Fig. 5.1E).

These results indicate that PRMT5 is necessary for CXCL11 induction in response to TNF and IFN-γ co-stimulation.

91

FIGURE 5.1 PRMT5 promotes expression of CXCL11 in EC co-stimulated with TNF and IFN-γ. (A) PRMT5-specific siRNA depleted protein levels in endothelial cells (EC) by ~90% as measured by immunoblotting (left panel) and densitometry (right panel). Expression of the chemokines CXCL11 (B), CXCL10 (C) and CCL2 (D) were measured by real-time PCR of RNA isolated from EC transfected with the indicated siRNAs and subsequently stimulated with TNF, IFN-γ, or TNF plus IFN- γ for 3 hours. An immunoblot showing CXCL11 protein expression in PRMT5 or control siRNA-transfected cells after 3 h of stimulation is shown at left in (E). *, p < 0.05; **, p < 0.01; ***, p < 0.005; error bars represent S.E. (n = 3-4).

92 5.3.2 Expression of CXCL11 Requires p65 Arg174

We then asked whether p65 Arg174 must be present in order to transactivate CXCL11. We co-transfected siRNA targeting the 3´ UTR of p65 along with cDNA encoding wild type p65, p65 Arg174Ala, or p65 Arg174Lys.

PRMT5 is incapable of methylating alanine and lysine residues (196,218-220).

Transfection of p65 siRNA depleted ~80% of endogenous p65, and reconstitution of either Arg174 point mutant recapitulated endogenous levels of p65 (Fig. 5.2A, quantified in right panel). EC reconstituted with p65 mutants were then stimulated with TNF, IFN-γ, or TNF plus IFN-γ, and CXCL11 mRNA levels were measured with quantitative real-time PCR. Wild type p65 reconstituted-EC co-stimulated with TNF and IFN-γ showed synergistic CXCL11 induction. The synergistic induction of CXCL11 by TNF or IFN-γ in wild type-reconstituted EC was ~92 fold increased over unstimulated cells. Importantly, CXCL11 induction was dramatically reduced in cells reconstituted with either Arg174Ala (~14 fold increase over unstimulated, p < 0.05) or p65 Arg174Lys (~18 fold increase over unstimulated, p < 0.05; Fig. 5.2B). CXCL11 mRNA levels were not significantly different in wild type or Arg174 mutant reconstituted EC upon stimulation with TNF or IFN-γ in isolation. These results suggest that p65 Arg174 is critical for the synergistic induction of CXCL11 in response to TNF and IFN-γ co-stimulation. In contrast, expression of CXCL10 in cells expressing p65 WT or either Arg174 mutant stimulated with either agonist alone or in combination was comparable to

EC expressing wild type p65 (Fig. 5.2C). This finding was unsurprising given our

93 previous findings that methylation of Arg30 and Arg35, but not Arg174, is necessary to transactivate CXCL10 (174).

94

95

FIGURE 5.2 TNF and IFN-γ mediated CXCL11 induction requires PRMT5- catalyzed p65 dimethylation at Arg174. siRNA targeting the 3´ UTR of p65 was co-transfected in EC with cDNA encoding wild type, Arg174Ala, or Arg174Lys p65. Knockdown of endogenous p65 and reconstitution with p65 constructs is shown by immunoblot and densitometric analysis (A). EC reconstituted with wild type and p65 mutants were treated with the indicated pro-inflammatory agonists for 3 hours followed by RNA collection and real-time PCR analysis to determine the mRNA levels of CXCL11 (B) and CXCL10 (C). In (D), FLAG-p65 was immunoprecipitated from EC transfected with two different PRMT5 siRNAs (sequence 1, lane 4; sequence 2, lane 5) following stimulation with TNF and IFN-γ for 30 minutes. Immunoprecipitates were separated by PAGE and the gel was silver-stained. FLAG-p65 bands were excised for mass spectrometric analysis. (E) Mass spectrometric quantitation of the peak area of dimethylated Arg174 present in the tryptic 172PLRLPPVLSHPIFDNR185 peptide relative to the unmodified peptide. The 28 in the peptide sequence refers to the +28 Da mass shift consistent with dimethylation (2 x 14 Da) of Arg174. (F) MS/MS spectra of the 633 Da triply charged ion identified in the tryptic digestion of NF-κB p65. The mass of this peptide is consistent with the addition of two methyl groups to the (172)PLRLPPVLSHPIFDNR(185) peptide. This spectra contains several

96 unmodified C-terminal y ions, all of which are consistent with dimethylation at Arg174. *, p < 0.05; ***, p < 0.005; error bars represent S.E. (n = 3).

97 5.3.3 PRMT5 Catalyzes Dimethylation of p65 Arg174

We next sought to determine whether Arg174 methylation levels are modulated in response to co-stimulation with TNF and IFN-γ. We introduced wild type FLAG-p65 cDNA along with control or PRMT5 siRNA. After 40 hours we stimulated the cells, prepared lysates, and immunoprecipitated p65 using the

FLAG tag. IP products were separated using PAGE and silver-stained (Fig.

5.2D). The 65 kDa protein bands were excised, digested with trypsin, and fragmented by collision induced dissociation. A mass spectra of the

172PLRLPPVLSHPIFDNR185 peptide containing dimethylarginine at Arg174 is provided in Fig. 5.2F. Peptides were also subjected to mass spectrometric selected reaction monitoring experiments to quantitate relative levels of arginine methylation. We found that p65 Arg174 dimethylation was undetected under unstimulated conditions, but was present in ~0.6 percent of the

172PLRLPPVLSHPIFDNR185 peptide (Fig. 5.2E). We did not detect monomethylation with or without treatment with TNF and IFN-γ. Additionally, we determined that Arg174 dimethylation was catalyzed by PRMT5, as depletion of

PRMT5 using two different sequences in TNF plus IFN-γ treated cells diminished

Arg174 methylation to ~50% and ~33% of the control transfected, co-stimulated levels (Fig. 5.2E), respectively. Together, these findings suggest a requirement for PRMT-mediated p65 Arg174 methylation in the expression of CXCL11, but not

CXCL10, when EC are co-stimulated with TNF and IFN-γ. We do not contend that Arg174 methylation is the only p65 PTM necessary for CXCL11 activation, as

98 other modifications such as phosphorylation, also contribute to CXCL11 induction.

5.3.4 PRMT5 is Essential for p65 Recruitment to the CXCL11 Promoter

We next performed ChIP to determine whether PRMT5 is obligatory for p65 association with the CXCL11 promoter following TNF plus IFN-γ co- stimulation. We immunoprecipitated chromatin from PRMT5-intact or -depleted

EC using an anti-p65 antibody. We then amplified CXCL11 promoter fragments using primers encompassing the κB site (Fig. 5.3A). p65 was found to be associated with the CXCL11 promoter upon TNF plus IFN-γ exposure (~7 fold increase) but PRMT5-depletion decreased p65 binding to the CXCL11 promoter significantly (~2 fold increase, p < 0.01) as compared with the unstimulated condition (Fig. 5.3B). We were unable to detect p65 on the CXCL10 promoter in

PRMT5-depleted EC (Fig. 5.3C). In contrast, p65 association with the CCL2

(MCP-1) promoter following co-stimulation was unaffected by PRMT5 knockdown

(Fig. 5.3D), in line with our observations that PRMT5 is not involved in CCL2 expression in response to TNF, IFN-γ, or TNF plus IFN-γ stimulation (Fig. 5.1D).

These results reveal that PRMT5 activity is critical for p65 recruitment to the

CXCL10 and CXCL11 promoters when EC are co-stimulated with TNF and IFN-

γ.

99

FIGURE 5.3 PRMT5 knockdown reduces p65 association with the CXCL11 promoter. The location of major regulatory elements and primer binding sites used for the chromatin immunoprecipitation (ChIP) assay of the CXCL11 promoter are illustrated in (A). IFN-γ response elements include the gamma interferon activation site (GAS) and interferon-sensitive response element (ISRE). The major TNF responsive element is the NF-κB binding site. ChIP assays were performed to assess p65 binding with the proximal promoter elements encompassing the κB binding sites of CXCL11 (B), CXCL10 (C), and CCL2 (D)

100 in PRMT5-intact or -depleted EC stimulated with TNF plus IFN-γ for 30 minutes. *, p < 0.05; **, p < 0.01; error bars represent S.E. (n = 3).

101 5.3.5 p65 Recruitment to the CXCL11 Promoter Requires Arg174

Additional ChIP assays were performed to ascertain whether p65

Arg174Lys is recruited to the CXCL11 promoter. Mutation of arginine-to-lysine is a conservative mutation that preserves the positive charge at amino acid 174 but precludes the possibility of methylation by PRMT5. Cells were co-transfected with siRNA targeting the 3´ UTR of p65 along with cDNA encoding either FLAG- tagged wild type p65 or Arg174Lys. DNA-protein complexes were immunoprecipitated with anti-FLAG antibody and probed with primers that amplify the CXCL11 promoter. We detected a significant increase in the association of wild type p65 with the CXCL11 promoter following co-stimulation

(Fig. 5.4A). However, the level of CXCL11 promoter fragments immunoprecipitated from cells reconstituted with p65 Arg174Lys was not statistically different from unstimulated cells. In contrast, CXCL10 (Fig. 5.4B) and

CCL2 (Fig. 5.4C) promoter fragments were enriched equally well in co-stimulated cells expressing either wild type p65 or Arg174Lys p65. We conclude that there is a requirement for p65 Arg174 for the expression of CXCL11, but not CXCL10 or

CCL2.

102

FIGURE 5.4 Mutation of Arg174 to lysine abrogates p65 association with the CXCL11 promoter. Endogenous p65 was depleted with siRNA targeting the 3´ UTR of p65 in EC. p65 was reconstituted in these cells by transfecting cDNA encoding FLAG- tagged wild type p65 or the FLAG-tagged Arg174Lys mutant. Following cell stimulation, the protein-DNA complexes were immunoprecipitated with an anti- FLAG antibody. Enrichment of the CXCL11 (A), CXCL10 (B), and CCL2 (C) promoters by the IP was quantified by real-time PCR. *, p < 0.05; ***, p < 0.005; error bars represent S.E. (n = 3).

103 5.3.6 SDMA at the CXCL11 Promoter is PRMT5-Dependent

In our previous publication we showed that p65 contains SDMA catalyzed by PRMT5 (174). With that information in mind, our next question in the current project asked whether we could detect SDMA-containing proteins associated with the CXCL11 promoter in TNF plus IFN-γ co-stimulated cells. We immunoprecipitated chromatin from PRMT5 siRNA-transfected, co-stimulated cells using an anti-SDMA antibody. We were able to detect a robust enrichment of the CXCL11 promoter following stimulation, but this enrichment was lost in the absence of PRMT5 (Fig. 5.5A). Results were similar when we assayed the immunoprecipitated DNA for the CXCL10 promoter: we were able to detect

SDMA-associated CXCL10 promoter after stimulation in the endogenous condition, but not when PRMT5 was depleted (Fig. 5.5B). We therefore concluded that SDMA-catalyzed by PRMT5 is associated with both the CXCL10 and CXCL11 promoters after simultaneous exposure to TNF and IFN-γ.

104

FIGURE 5.5 PRMT5-catalyzed dimethylation of p65 at Arg174 is necessary for p65 recruitment to the CXCL11 promoter. EC were transfected with PRMT5-specific or control siRNA and stimulated with TNF plus IFN-γ. ChIP was performed using an antibody specific for SDMA. Enrichment of the CXCL11 (A) and CXCL10 (B) promoters was quantified by qRT-PCR. (C-D) ChIP was performed on EC reconstituted with FLAG-tagged wild type p65 or the indicated p65 mutants from TNF plus IFN-γ stimulated EC. Protein-DNA complexes were immunoprecipitated with anti-FLAG antibody. Enrichment of the CXCL11 (C), and CXCL10 (D) promoters was quantified by qRT-PCR. **, p < 0.01; error bars represent S.E. (n = 3).

105 5.3.7 p65 Arg174Lys Reduces SDMA at the CXCL11 Promoter

We next sought to determine whether arginine methylation of p65 amino acid 174 is necessary for CXCL11 induction. We reconstituted EC with wild type,

Arg30Lys, Arg35Lys, and Arg174Lys p65, and performed ChIP with an anti-SDMA antibody. Results indicated that the CXCL11 promoter was pulled down when wild type p65 and the p65 Arg30Lys and Arg35Lys mutants were expressed in co- stimulated EC (Fig. 5.5C). When p65 Arg174Lys was reconstituted, the CXCL11 promoter levels were not statistically different from the unstimulated condition.

This suggests that p65 with lysine at position 174 does not associate with the

CXCL11 promoter.

The ChIP immunoprecipitates were also probed for the presence of the

CXCL10 promoter. We found that the CXCL10 promoter was enriched from both wild type and Arg174Lys reconstituted cells upon co-stimulation (Fig. 5.5D, p <

0.005). When the Arg30Lys and Arg35Lys mutants were expressed, levels of

CXCL10 promoter dropped to ~50% of the levels observed with the wild type and

Arg175Lys mutant (Fig 5.5D, p < 0.005). These results are consistent with our previous finding that methylation of p65 Arg30 and Arg35 are necessary for

CXCL10 induction in EC. Additionally, these results indicate that Arg30 and Arg35 may be methylated independently, as mutation of either Arg30 or Arg35 to lysine does not reduce methylation of the other residue.

106 To confirm that the SDMA antibody is detecting methylation at p65 Arg174 we reconstituted wild type and Arg174Lys mutants in EC, and used the FLAG epitope to immunoprecipitate p65 proteins, which where then probed with anti-

SDMA antibody (SYM10; Fig. 6A). We are able to detect arginine methylation in response to TNF plus IFN-γ only when wild type p65 is present (lane 4), but not when the Arg174Lys was transfected (lane 3). These results indicate that the symmetrical dimethylation of p65 detected by the anti-SDMA antibody is likely modification of Arg174.

107

108 FIGURE 5.6 p65 Arg174 methylation is present on the CXCL11 promoter following co-stimulation. (A) EC were depleted of endogenous p65 and reconstituted with wild type or Arg174Lys mutant p65. Cells were stimulated with TNF plus IFN-γ for 30 minutes and lysed in RIPA buffer. Wild type and mutant p65 was immunoprecipitated using the FLAG epitope or IgG. Immunoprecipitates were immunoblotted for p65 and for the SDMA modification. (B-C) Cells were reconstituted with N-FLAG wild type, Arg30Lys, Arg35Lys, and Arg174Lys p65 constructs, and simultaneously exposed to TNF and IFN-γ. ChIP was performed using anti-FLAG antibody. Sequential ChIP (Re-ChIP) was carried out with addition of normal rabbit serum or anti-SYM10 antibody. Following crosslink reversal, DNA was purified and promoter fragment content was quantitated using qRT-PCR relative to input.

109 We next sought to assess whether p65 and SDMA are detected on the same fragments of the CXCL11 promoter. We utilized a sequential ChIP (Re-

ChIP) approach where we reconstituted wild type and point mutant p65, then immunoprecipitated p65 using the FLAG-tag. After removal of the anti-FLAG antibody, we immunoprecipitated the protein-chromatin complexes a second time using anti-SDMA antibody. The CXCL11 promoter was present at background levels in the absence of cell co-stimulation with TNF and IFN-γ following immunoprecipitation of wild type p65 (Fig. 5.6B, bar 1). We found CXCL11 promoter enrichment when wild type, Arg30Lys, and Arg35Lys were expressed in co-stimulated cells where p65 was immunoprecipitated with the anti-FLAG antibody (Fig. 5.6B, bars 2-4). In contrast, we detected the CXCL11 promoter when Arg174Lys p65 was expressed only at background levels (bar 5).

We included a no antibody control to assess whether the anti-FLAG antibody was successfully dissociated from the chromatin during the Re-ChIP procedure. We did not detect the CXCL11 promoter with this control (Fig. 5.6B, bars 6-9), indicated that we were able to remove the antibody with buffer exchange. Following buffer exchange, we proceeded with the Re-ChIP portion of the experiment, using either the anti-SDMA antibody (SYM10) or normal rabbit serum, as SYM10 is presented in antiserum format (bars 10-13). We did not enrich the CXCL11 promoter in the Re-ChIP stage of the experiment using the serum regardless of whether wild type or any of the mutants were present.

110 Enrichment of the CXCL11 promoter was detected equal to roughly 15% of the input material when wild type, Arg30Lys, and Arg35Lys p65 were present

(bars 14-16). Crucially, we found that the level of CXCL11 was significantly diminished when Arg174Lys was expressed to ~5% of input (p < 0.05; bar 17). In full, these results indicate that the methylation associated with the CXCL11 promoter is predominately symmetrical dimethylation of p65 at Arg174. Arginine methylation of other p65 arginine residues, such as Arg30 or Arg35, is only a minor component of p65 methylation associated with this promoter under TNF plus IFN-

γ co-stimulated conditions.

We also quantitated the presence of the CXCL10 promoter in this experiment (Fig. 5.6C). p65 is not associated with the promoter in the absence of

TNF and IFN-γ (bar 1). A robust increase in promoter association was observed when wild type and Arg174Lys were expressed in stimulated cells and immunoprecipitated with the anti-FLAG antibody (~20% of input). When Arg30Lys and Arg35Lys were present, CXCL10 promoter was enriched to ~7% of input (p <

0.05; bars 2-5). Only background levels of CXCL10 promoter were found following buffer exchange to remove the anti-FLAG antibody (bars 6-9). Levels of

CXCL10 promoter were also at background when rabbit serum was applied in the

Re-ChIP. Quantitation of the promoter showed following Re-ChIP with the

SYM10 antibody showed that wild type and Arg174Lys p65 pulled down CXCL10 promoter equal to ~20% of input, but that Arg30Lys and Arg35Lys mutant

111 expression was associated with lower quantities of the promoter, ~8-10% of input

(p < 0.05; bars 14-17). These results are consistent with our previous findings that methylation of Arg30 and Arg35 is necessary for CXCL10 induction, but that

Arg174 methylation is not required (174). It is interesting to note that mutation of either Arg30 or Arg35 to lysine reduces, but does not eliminate, detection of methylated p65 associated with this promoter, supporting a model where methylation of Arg30 and Arg35 are independent.

Given the sum of our experiments reported here, we conclude that

PRMT5-catalyzed p65 methylation at Arg174, Arg30 and Arg35, is critical for p65 recruitment to the CXCL11 promoter and the subsequent synergistic induction of

CXCL11 in response to TNF plus IFN-γ co-stimulation. This requirement does not exist for the synergistic induction of CXCL10. Our findings are consistent with a mechanism whereby concurrent stimulation of EC by both TNF and IFN-γ activate PRMT5. PRMT5 is then recruited to p65, where it catalyzes dimethylation of Arg174. Arginine methylation of p65 at Arg174 is necessary for p65 recruitment to the CXCL11 promoter. Given the location of Arg174 on the p65 molecule is probable that modification of this residue facilitates a protein-protein interaction that enhances CXCL11 transcription.

We do not currently know the mechanism linking activation of cytokine receptors with p65 methylation by PRMT5. One possible mechanism could

112 involve activation of the JAK kinases by ligation of the IFN-γ receptor by IFN-γ.

PRMT5 was first discovered in human cells as a JAK2 binding protein (221), and it may also interact with JAK1 and JAK3. JAK proteins have been shown to phosphorylate PRMT5 (222). Phosphorylation of PRMT5 by a JAK may increase

PRMT5 methyltransferase activity, or enhance it’s affinity for p65, leading to catalysis of SDMA formation at Arg174. TNF signaling would then mobilize the p65 methylated at Arg174 to the nucleus, where it associates with transcription cofactors and the CXCL11 promoter. It is not clear whether IFN-γ alone is sufficient to trigger methylation at p65 at Arg174, or whether Arg174 methylation is a product of synergism between the TNF and IFN-γ pathways. We also can not rule out contribution of other PRMT enzymes to the methylation of Arg174, including PRMT1 and PRMT4, both of which are found in EC.

Our findings suggest that the PRMT5-mediated methylation imparts an indexing signal to p65 that enables stimulus-specific chemokine expression.

Differential combinations of such modifications enable p65 to interact with DNA or other components of specific transcriptional complexes in a context-specific manner, resulting in unique patterns of gene expression (138,141). Such a PTM code coupling extracellular stimuli to transcriptional outcomes is essential to the regulation of complex physiological and pathological processes such as inflammation. CXCL10 and CXCL11 are both found at high levels in human atheroma, as is TNF, and IFN-γ. CXCL10 and CXCL11 have redundant functions

113 in that both ligate CXCR3 and promote chemotaxis of leukocytic populations and smooth muscle cells, and are angiostatic for EC (8,217,223). Th1-type T-cells recruited by CXCL10 and CXCL11 exacerbate atherosclerosis by producing copious amounts of IFN-γ and proteinases that reduce collagen maturation (224).

However, CXCL10 and CXCL11 differ in their expression in diseases states such as atherosclerosis. Atheroma-associated EC, smooth muscle cells, and macrophages robustly produce CXCL10. CXCL11 is secreted by EC, macrophages, and lesional EC microvessels (35). These cell types are found in discrete regions of the plaque, resulting in the accumulation of T-cells in the shoulder regions and fibrous cap (35,225). Varying levels of expression of both

CXCL10 and CXCL11 by these cell types may be influenced by distinct regulatory and biochemical properties of these ligands. CXCL10 gene expression is elicited by the widest variety of agonists, including IFN-α/β/γ, and is most sensitive to TNF. In contrast, CXCL11 expression is weakly induced by TNF, but is strongly activated by IFN-β/γ (12). CXCL11 has greater potency than CXCL10

(13,126,127) as it is able to trigger receptor internalization, calcium mobilization, and chemotaxis at lower doses. These differences in CXCR3 ligand expression patterns according to cell type, agonist potency, and receptor affinity appear likely to exert fine control of T-cell recruitment within the lesion. Given our findings, examination of the newly discovered specific PRMT5 inhibitors (226-

228) in should be investigated models of chronic inflammation as a means of attenuating disease progression.

114 5.4 Acknowledgements

We thank Chad Braley for isolation of HUVEC used in this study.

115 CHAPTER 6: CONCLUDING DISCUSSION

6.1 Discussion of Key Results

At the outset of this project, we knew that PRMT5 contributes to the TNF- mediated gene induction of the leukocyte adhesion molecules E-selectin and

VCAM-1. PRMT5 contributes to the activation of these promoters by binding and methylating the transcription factor HOXA9 following stimulation with TNF (145).

Results presented in chapter 4 show that PRMT5 also functions to activate gene expression of the chemokine CXCL10 and CX3CL1 in response to TNF.

This mechanism is completely independent of HOXA9. In our model, TNF stimulation results in a slight variant of the canonical NF-κB pathway. Signals from the TNF-receptor activate IKK, which in turn phosphorylates both the IκB complex and p65. A portion of p65 in the sequestered state has already been methylated by PRMT5 at Arg30 and Arg35. This methylation appears to be constitutive at low levels, as we did not detect changes in the level of methylation following treatment with TNF. IκB is subsequently ubiquitinated and degraded by the proteasome, while the active p65-p50 heterodimer translocates to the nucleus and associates with the CXCL10 promoter. Arg30 and Arg35 are part of the p65 RHD DNA-binding domain. Methylation of these residues probably functions to increase p65’s DNA affinity by fostering hydrophobic interactions with the DNA (Fig. 6.1).

116

FIGURE 6.1 Model of SDMA-p65 in CXCL10 and CXCL11 transcription. Left – EC activation with TNF: Ligation of TNF-receptors (TNF-R) leads to activation of the IKK complex, which phosphorylates (P) IκB and p65. IκB is subsequently ubiquitinated (U) and degraded by the proteasome (represented by the X). p65 has already been methylated (M) by PRMT5 at Arg30 and Arg35. This NF-κB complex migrates to the nucleus where it associates with the CXCL10 promoter.

Right – EC co-activation with TNF and IFN-γ: Ligation by IFN-γ of the IFN-γ- receptor increases JAK activity, which phosphorylates the IFN-γ-receptor at tyrosine residues. STAT proteins are recruited to these residues and associate with the phosphotyrosine residues via SH2 domains. The STATs are also phosphorylated by JAK. Phospho-STAT proteins dimerize and translocate to the nucleus, where they associate with the CXCL11 promoter. The TNF component is similar to that shown at left. However, the crucial difference is that signaling from the IFN-γ-receptor increases PRMT5 methyltransferase activity or causes

117 PRMT to catalyze SDMA formation of p65 at Arg174 in some manner. p65 containing SDMA at Arg174 associates with the CXCL11 promoter upon translocation. The SDMA at Arg174 leads to the recruitment and binding of an unknown cofactor with the CXCL11 transcription complex.

118 We also identified a third mechanism whereby PRMT5 participates in pro- inflammatory gene expression. This mechanism is active when we concurrently stimulated EC with both TNF and IFN-γ. We found that PRMT5 is required to achieve full gene induction of the chemokine CXCL11 under these conditions.

Our model hews close to the canonical TNF and Jak-STAT signaling pathways.

Activation of the IFN-γ-receptor increases JAK kinase activity. The JAKs phosphorylate the IFN-γ-receptor at tyrosine residues. STAT proteins are recruited to these residues and associate with the phosphotyrosine residues via

SH2 domains, and are also phosphorylated by the JAKs. Phospho-STAT1 and phospho-STAT3 form heterodimers and translocate to the nucleus and associate with the CXCL11 promoter.

Signals from the TNF-receptor activate the IKK complex, which leads to the degradation of IκB. p65 is basally methylated at Arg30 and Arg35 in this model as well, but methylation of these residues is irrelevant in the activation of

CXCL11 transcription. Crucially, we hypothesize that signals emanating from the

IFN-γ-receptor lead to methylation of p65 at Arg174 by PRMT5. p65 containing

SDMA at Arg174 then associates with the CXCL11 promoter upon translocation.

We hypothesize that SDMA at Arg174 leads to the recruitment and binding of an unknown cofactor with the CXCL11 transcription complex (Fig. 6.1). It is not clear whether IFN-γ alone is sufficient to trigger methylation at p65 at Arg174, or

119 whether Arg174 methylation is a product of synergism between the TNF and IFN-γ pathways.

PRMT5 was first discovered in human cells as a JAK2 binding protein

(221), and there is some evidence that it also interacts with JAK1 and JAK3. It is possible that phosphorylation of PRMT5 by a JAK activated in response to IFN-γ- receptor ligation could increase PRMT5 methyltransferase activity, or enhance it’s affinity for p65, and in such a manner lead to catalysis of SDMA at Arg174. In fact, a mutant form of JAK2, JAK2V617F, has been shown to phosphorylate

PRMT5 (222). JAK2V617F is a common mutant found in patients with myeloproliferative neoplasms. The phosphorylation catalyzed by JAK2V617F of

PRMT5 downregulates PRMT5’s activity, which impairs its ability to methylate histones, and results in neoplastic growth. However, it is possible that phosphorylation of other residues on PRMT5, or the phosphorylation by non- mutant JAKs could be a mechanism of the enhanced p65 methylation at Arg174 that we observed in response to TNF and IFN-γ co-stimulation.

6.2 The Methylarginine PTM Code of NF-κB p65

It is perhaps equally important to note that gene expression of the chemokine CCL2 was not affected by the absence of PRMT5, or by replacement of endogenous p65 with R30K, R35K, or R174K either with TNF alone, or in combination with IFN-γ. PRMT5 is not necessary for CCL2 gene induction, nor is

120 p65 recruitment to the CCL2 promoter significantly different in the presence of any of the reconstituted Arg-to-Lys mutants. Likewise, Arg174 is not necessary for

CXCL10 gene induction by TNF, and Arg30 and Arg35 are dispensable for

CXCL11 mRNA production. Each of these promoters has a distinct requirement for p65 methylarginine, or in the case of CCL2, no such requirement at all. We therefore conclude that arginine methylation catalyzed by PRMT5 imparts indexing signals on p65 that aid gene induction. More work is needed to establish the factors that influence p65-methylarginine requirements at pro-inflammatory gene promoters. These factors could include promoter structure, transcription factor binding site combinations, the presence of cofactors, or specific variants of the κB consensus sequence.

The concept of a PTM code is useful. However, the PTM code is not necessarily a strict code whereby a specific modification definitively encodes a particular physiological readout. The functionality of any particular modification results from the combinatorial outcomes of the modification, the protein’s interactions, and the local environment in the tightly packed cellular environment.

To the extent that a code exists, the local environment provides an “anti-code” that only enables a defined response when the appropriate reading and effecting components are simultaneously available (138).

121 In our work, we show that arginine methylation of various p65 residues contributes to induction of CXCL10 and CXCL11 in a manner in part determined by stimulation with specific agonists or combinations of agonists, and when the appropriate cofactors are also present.

6.3 Implications for Atherosclerosis

The development of atherosclerosis begins as immune cells and lipid droplets are deposited along arterial walls. These deposits may develop into fatty streaks, which are composed primarily of lipid-engorged macrophages, but also include T-cells. Fatty streaks formation is the first stage of atherosclerosis. They may regress over time, or progress into mature plaques (atheromas). As plaques develop EC become increasingly dysfunctional, chronically inflamed, and constitutively express leukocyte adhesion molecules, TNF, and chemokines such as CCL2, CXCL10, and CXCL11. The endothelium becomes increasingly leaky and permeable to macromolecules and leukocytes. Advanced plaques have a more complicated structure consisting of a dense, pro-thrombotic central necrotic core containing foam cells, macrophages, free lipid, cellular debris, and pro- thrombogenic molecules such as tissue factor. Separating the necrotic core from the blood is a fibrous cap comprised of SMC, T-cells, and collagen. The periphery of the fibrous cap where it interfaces the surrounding endothelium is called the shoulder region. This area contains high numbers of T-cells and macrophages and is the site of atheroma growth (8,225).

122 Activated T-cells are estimated to account for ~20% of the total cell population in mature human plaques (225). In combination with other inflammatory and immune cells, T-cells produce inflammatory cytokines such as

TNF and the dominant source of IFN-γ. Expression of these pro-inflammatory molecules promotes endothelial dysfunction, recruitment of additional inflammatory cells, diminishes collagen maturation, and results in the production of substances such as tissue factor (TF) and proteases that can destabilize plaque structure. Weakened fibrous caps may rupture in response to hemodynamic forces, a process that commonly results in immediate thrombotic occlusion of the vessel due to TF exposure with blood components. Blood vessel occlusion may manifest clinically as myocardial infarction, stroke, acute limb ischemia, renal impairment, hypertension, or abdominal aortic aneurysm (8).

Recently, specific small-molecule chemical inhibitors of PRMT5 have been developed (226-228). These molecules should be examined closely in models of chronic pathologies such as atherosclerosis, multiple sclerosis, psoriasis, rheumatoid arthritis, and various cancers in which PRMT5 has been shown to promote disease pathogenesis. Such a strategy could attenuate disease progression by moderating immune responses and may provide therapeutic benefit.

123 6.4 Regulation of PRMT5

The regulation of PRMT5 remains an understudied research area. Our results indicate that PRMT5 gene expression is not induced in EC following stimulation with TNF. Similarly, we have previously reported that PRMT5 protein levels are unchanged following TNF-exposure. We also did not detect migration of PRMT5 between the cytosolic and nuclear compartments as a result of TNF- stimulation (145). In contrast, our results with TNF plus INF-γ co-stimulation indicate that Arg174 methylation is increased following treatment. We did not test whether IFN-γ alone is able to trigger enhanced methylation of this site. We also did not assay whether PRMT5 activity is increased towards other substrates, such as myelin basic protein (MBP) or histones, both of which are commonly used in vitro as methylation acceptors in PRMT-catalyzed reactions. PRMT5 is catalytically active only as a dimer or multimer (221,229). Whether IFN-γ or any other agonist increases formation of higher order PRMT5 oligomers is an unanswered question. It is also possible that PRMT5 activity is enhanced by a

PTM, although that has yet to be demonstrated.

6.4 The Permanence of Arginine Methylation

A general paradigm is that post-translational modifications involved in signal transduction have both a regulated mechanism of addition, and a regulated mechanism of signal termination. This paradigm has been most well-

124 studied in the case of phosphorylation, where hundreds of kinases and phosphatases have been well-characterized.

However, several hundred types of PTM exist, and experiments using radiolabels to track kinetic rates of histone PTM turnover reveal wide differences in lability according to modification type (230). The half-life of most acetylation marks is less than 15 minutes. Phosphorylation half-life is on the scale of hours.

Lysine acetylation is roughly 1-3 days, which is approximately equivalent to the turnover rate of the histones themselves. The variance in these half-life rates suggest that fast signals are encoded by modifications such as phosphorylation, while more durable epigenetic regulatory messages are encoded by modifications with longer half-lives.

Arginine methylation has historically been considered to be a stable, permanent post-translational modification. However, studies since the early

2000s have questioned this characterization, especially as roles for arginine methylation in signal transduction have been discovered. Some ChIP experiments with anti-methylarginine antibodies have indicated that methylarginine levels may decrease certain conditions, such as stimulation with estrogen (231,232).

125 Our results clearly indicate a role for PRMT in response to cytokine signaling necessary for inflammatory gene induction. However, we did not document decreases in p65 arginine methylation. The experiments we performed were concerned with whether arginine methylation increased as a result of EC activation, and we did not assay longer time points to ask whether p65 arginine methylation diminished over time. We did not document loss of arginine methylation using either immunoblotting or mass spectrometry approaches, and only observed an increase in Arg174 dimethylation following EC stimulation with

TNF and IFN-γ.

More importantly, the mechanisms of arginine demethylation that have proposed to date are not sufficiently compelling to support a widespread arginine demethylation in mammalian cells. The two main mechanisms that have been proposed, with some evidence in specific contexts – true arginine demethylation by JMJD6, and deamination/citrullination by PADI4 – are discussed below.

Our current working hypothesis is that p65 arginine methylation is a stable modification, and may serve as a priming function for subsequent waves of NF-

κB methylation-dependent gene expression. In this manner arginine methylation could serve as a sort of epigenetic memory for the lifetime of the protein.

126 6.4.1 JMJD6: An Arginine Demethylase?

The other major alternative to protein turnover is that arginine methylation is transient and enzymatically-removed. Lysine demethylases exist (e.g. LSD1), and there has been significant effort expended to identify arginine demethylases.

A Science report from 2007 indicated that a member of the Jumonji C (JmjC) domain-containing protein family of ferrous iron (Fe2+) and 2-oxoglutarate (2-OG)- dependent dioxygenases, JMJD6, has the ability to demethylate SDMA- containing proteins, including histones (233). Significant disagreement exists in the field as to whether JMJD6 is actually an arginine demethylase. Consensus seems to be building that it is not an arginine demethylase, but is rather a lysine hydroxylase (234-239). It remains possible that true arginine demethylates do exist but remain undiscovered. One avenue of future work would be to explore turnover rates of non-histone, arginine methylated proteins, such as p65 and p53

6.4.2 Arginine Deimination

The enzyme peptidyl arginine deiminase 4 (PADI4) was discovered to convert unmethylated and monomethylarginine histone residues to the non- coded amino acid citrulline (240,241). In this reaction, deimination (also called citrullination), the methyl group and arginine imine group are removed, producing protein-incorporated citrulline (240). Pre-emptive deimination could prevent arginine methylation, and there is some evidence that citrulline levels are transient during transcription (131). However, deamination can not be a primary

127 regulatory mechanism because PADI4 expression is mainly restricted to hematopoietic cells, in contrast to PRMT enzymes, which are ubiquitously expressed. Signaling roles of citrulline are not known, and it is not clear whether citrulline can be regenerated to arginine, as has been postulated.

Conversion of arginine to citrulline results in a molecular mass change of less than 1 dalton (131). The most likely role of deamination, in my view, is in alternating protein conformation due to the conversion of the positive arginine charge to neutrally-charged citrulline. The best example of this is in the formation of neutrophil extracellular traps (NETs), in which the conversion of histone residues to citrulline promotes chromatin decondensation and the formation of extracellular web-like structures that function to trap pathogens (242-245). DNA is subsequently extruded from the neutrophil, where it forms an extracellular web- like structure decorated with antibacterial molecules to trap pathogens. The charge change is critical to this process, as it is thought to provide the mechanical underpinning NET formation. I suspect any role for PADI enzymes in the regulation of arginine methylation – if at all – is secondary to conformational changes due to charge shifts.

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