The Interferon Regulatory Factor 5 (IRF5) Interactome: Investigating the role of co-factors in regulation of inflammation Hayley Eames Supervisors: Prof. Irina A. Udalova Dr. Robin Wait A thesis submitted for the degree of Doctor of Philosophy at the Kennedy Institute of Rheumatology Faculty of Medicine Imperial College London September 2013 Abstract Interferon Regulatory Factor 5 (IRF5) is a key transcription factor that regulates inflammatory responses by acting as a mediator of macrophage plasticity. IRF5 is expressed at high levels in pro-inflammatory (M1) macrophages where it drives expression of characteristic M1 markers, such as IL-12p40, IL-12p35 and IL-23p19, and inhibits expression of typical markers of anti-inflammatory (M2) macrophages, like IL-10. In this way, IRF5 polarises macrophages towards a pro-inflammatory phenotype. There are two modes of IRF5 activity: 1) direct binding to Interferon Stimulated Response Element (ISRE) sites in the DNA, and 2) indirect recruitment via protein- protein interactions. IRF5 can be indirectly recruited via interactions with the RelA subunit of Nuclear Factor kappa B (NFκB) at multiple inflammatory loci genome- wide, including Tnf. This suggests protein-protein interactions between the two factors are of great importance for driving inflammatory gene expression. My research shows the IRF Association Domain (IAD) of IRF5 and the Dimerisation Domain (DD) of RelA form an interaction interface between the two proteins. I have identified a short peptide, a region of IRF5 IAD sequence, that can bind to RelA, and hypothesise this peptide could block IRF5-RelA interactions, potentially dampening expression of inflammatory mediators co-regulated by these two factors. I have also identified a novel co-factor of IRF5: Krüppel Associated Protein 1 (KAP1) by a proteomic screen consisting of affinity purification coupled to mass spectrometry. The IRF5-KAP1 complex is present in M1 macrophages, and absence of KAP1 in this cell type results in prolonged TNF secretion, suggesting that KAP1 is important for ‘switching off’ Tnf gene expression. Further investigation showed KAP1 is important for regulation of a heterochromatin (H3K9me3) environment downstream of the Tnf locus. By understanding the IRF5 interactome, we hope to unravel and potentially manipulate the determinants of IRF5’s key role in inflammation in a cell-type and activity-specific manner. 2 Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or distribution, researchers must make it clear to others the licence terms of this work. All experimental data described in this thesis are original and have been performed by myself, except for the data described in the Figures mentioned below, which were performed by or with great help from others. Mass spectrometry (LC-MS/MS) and trypsin digestion of protein bands in Figures 3.5, 3.15. 3.16 and Supplementary Figure S9.1 were performed either by Dr Robin Wait (Kennedy Institute of Rheumatology) or Dr Mike Deery and his team (Cambridge Centre for Proteomics, University of Cambridge). Dr David Saliba (Udalova Group, Kennedy Institute of Rheumatology) performed the Flag-immunoprecipitation in Figure 5.8, One-Strep affinity purification in Figure 3.16, and the ChIP-seq study referred to in Chapter 5. Dr Pavel Savitsky performed RelA protein purification for Figure 5.18 and the associated bio-layer interferometry was performed with great help from Dr Oleg Fedorov (both from the Structural Genomics Consortium, University of Oxford). This work has been carried out at the Kennedy Institute of Rheumatology and was funded by Arthritis Research UK, Medical Research Council, the European Community Seventh Framework Programme FP7/2007-2013 and the Kennedy Institute trustees. Hayley Eames (September 2013) 3 Acknowledgements First and foremost, I would like to thank my supervisor Prof. Irina Udalova for her fantastic support, guidance and the opportunity to work on a truly interesting project. Thanks also to Dr. Robin Wait for his proteomics expertise in the early stages of this project. Additionally, I would like to say a very big thank you to Dr. David Saliba, who I have considered to be an ‘honorary supervisor’ since day one, and deserves a lot of credit for my improved laboratory ability. He has always been my first point-of-call for technical discussion, and has been a great source of entertainment in the lab, particularly on long days full of many, many ChIP washes! In term of reagents and expertise, I would like to extend my gratitude to Dr. Tim Smallie, Dr. Alexey Ivanov and Prof. Betsy Barnes for their kind gifts of expression constructs, Dr Mike Deery and his team at the Cambridge Centre for Proteomics for mass spectrometry, and Dr. Oleg Fedorov and Dr Pavel Savitsky at the Structural Genomics Consortium for protein purification and bio-layer interferometry. This project would also not have been possible without the help of many people at the Kennedy Institute of Rheumatology, including the trustees, who have funded this research and built us an amazing new building in Oxford. I would like to thank Katrina Blazek – the unofficial boss of the Udalova group – for her incredible organisation, love of tea breaks, and for being a friendly face in the laboratory even at weekends. Katrina has maintained our mouse colonies, is very skilled at bone marrow extraction from femurs, and patiently taught me how to scruff a mouse. Thanks also to Alessandra Lanfrancotti, who has always been a great help to the Udalova group, in particular aiding in elutriation and adenovirus purification. A special mention to Dr. Grigory Ryzhakov, who can suggest an answer to anything, and I’m pretty sure has everything cloned into an expression plasmid! A big thank you to the rest of the Udalova group, past and present – Miriam, Thomas, Adam, Scott, Fergie, Matt, Lynn – and to all the other brilliant members of the London ground floor laboratory for both discussion and giggles. I would like to thank Dr. Will Wood, my undergraduate dissertation supervisor, for giving me the confidence to apply for a PhD project, Sue Boyce at GSK for introducing me to techniques that I routinely use today, and my parents for their constant support and encouragement. To Aaron, thanks for putting up with me when I’m grumpy after a long commute home from London, and it looks like I won the thesis-writing race! Xxx 4 Table of Contents ABSTRACT ..................................................................................................... 2 DECLARATION ............................................................................................... 3 ACKNOWLEDGEMENTS ............................................................................... 4 TABLE OF CONTENTS .................................................................................. 5 LIST OF FIGURES ........................................................................................ 10 LIST OF TABLES .......................................................................................... 12 LIST OF SUPPLEMENTARY FIGURES ....................................................... 12 1.0 – INTRODUCTION .................................................................................. 14 1.1 – THE IMMUNE SYSTEM ............................................................................... 14 1.1.1 – Innate Immune Responses .................................................................... 14 1.1.1.1 – Cellular Innate Immunity ................................................................. 15 1.1.1.2 – Humoral Innate Immunity ................................................................ 16 1.1.2 – Cells of the innate immune system ........................................................ 16 1.1.2.1 – Monocytes ...................................................................................... 16 1.1.2.2 – Macrophages .................................................................................. 17 1.1.2.3 – Dendritic cells ................................................................................. 19 1.1.2.4 – NK cells ........................................................................................... 20 1.1.2.5 – Granulocytes ................................................................................... 20 1.1.3 – Activation of adaptive immunity by innate immunity .............................. 21 1.1.3.1 – T-cell-mediated adaptive immunity ................................................. 21 1.1.3.2 – B-cell-mediated adaptive immunity ................................................. 22 1.2 – REGULATION OF MACROPHAGE GENE EXPRESSION ......................... 24 1.2.1 – Role of PU.1 and epigenetics in macrophage gene expression ............ 25 1.2.2 – Nuclear Factor kappa B (NFκB) family of transcription factors .............. 26 1.3 – TRANSCRIPTIONAL ACTIVITY OF IRFS IN MACROPHAGES ................ 27 1.3.1 - The IRF family of transcription factors .................................................... 28 1.3.2 - IRF1/IRF2: Positive and negative regulators of gene expression .......... 30 1.3.2.1 - IRF1 positively regulates gene expression in macrophages ........... 30 1.3.2.2 - IRF2 can antagonise and co-operate with