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The Role of Histone Modifications in Severe Asthma

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The Role of Histone Modifications in Severe Asthma The Role of Histone Modifications in Severe Asthma A Thesis submitted to Imperial College London for the degree of Doctor of Philosophy by Peter Owen Brook Molecular Cell Biology Airway Disease Section National Heart and Lung Institute The Faculty of Medicine Imperial College London London SW3 6LY 1 2 Abstract Severe asthma is an airways disease that causes restriction of airflow to the lung leading to difficulty breathing, where even high doses of medication does not alleviate the symptoms. There are studies that have demonstrated differentially expressed genes in both CD8+ T-cells and the bronchial epithelium of severe asthmatics, however the causes of the changes in gene expression have not been elucidated. Histones, proteins which store DNA, can be acetylated or methylated which changes their binding of DNA allowing for modified transcription. I hypothesise that modifying the histone modification of Bronchial Epithelium and CD8+ T-cells would be able to limit the inflammatory effects of severe asthma, that measuring changes in histone modifications would show differences between severe and non-serere asthmatics and that by using modern analysis of gene expression and histone modifications it would be possible to discover novel genes and pathways involved in asthma. The Bromodomain and Extra terminal domain mimic JQ1 was able to suppress release of Interleukin-6 and Interleukin-8 and SGC-CBP30 the CBP/p300 inhibitor limited proliferation in bronchial epithelium. CD8+ T-cells from a small group of severe and non-severe asthmatics had gene arrays carried out and were analysed by weighted geneome coexpression network analysis which found modules containing genes involved in intracellular motility and vesicle formation that linked strongly to % predicted Forced Expiratory volume in one second. CD8+ ChIP-Seq studies of T-cell histone modifications were attempted, but not successful. A pilot test on Severe asthmatics CD8+ T-cells treated with SGC-CBP30 showed a reduction in the release of the inflammatory mediator MIP1α and suggested that this was without affecting its mRNA production or cell viability, however higher numbers of patients would be needed to confirm this. 3 Declaration of originality I, Peter Owen Brook declare that I wrote this thesis and the experiments and work described herein, except where appropriately referenced, was performed by myself. Information derived from the published and unpublished work of others has been acknowledged in the text and full references are given. Copyright declaration ‘The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives license. 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 redistribution, researchers must make clear to others the license terms of this work’ 4 Acknowledgements I would like to extend the sincerest thanks to my supervisors Prof. Ian Adcock, Dr Paul Lavender, Dr Andrew Durham and Dr Sharon Mumby. Firstly for giving me the opportunity to work in their fantastic groups, for the academic and intellectual support, but also for their moral support and friendship. I would like to thank Asthma UK/MRC for generously funding my PhD. I would also to thank Prof Fan Chung and Dr Pank Bhavsar for helping gain access to asthmatic blood and airway samples. I’d like to extend a great appreciation to Dr Audrey Kelly for guiding me though the gene array and ChIP-Seq work and to Dr Kate Heesom for carrying out the SILAC mass spectroscopy. The group who I’ve worked with these last few years have had a massive influence on the way I work in science and have been fantastic colleagues including Dr. Christos Rossios, Dr, Coen Wiegman, Dr. Stelios Pavlidis, Dr. Scott Kuo, Elen Jazwari, Dr. Seun Ojo, Dr. Charis Michaeloudes and Dr. Mark Perry. Along with my fellow Students: Kirsty, Younis, Amir, Rebekah, Tank, Lily and Jack and the wonderful masters and undergrad students who I’ve worked with over the last four years. Finally I have to thank Lily, my Mum and Dad, Annabel, and all my friends for helping me through when things were tough and celebrating with when things went well. 5 Table of Contents Chapter 1: Introduction ............................................................................... 20 1.1 Asthma ......................................................................................................... 21 1.1.1 Definition ................................................................................................ 21 1.1.2 Prevalence ............................................................................................. 21 1.1.3 Mechanism of asthma ............................................................................ 21 1.2 Treatments .................................................................................................. 24 1.3 Severe Asthma ........................................................................................... 27 1.3.1 Mechanisms and subtypes of Severe Asthma ....................................... 28 1.4 Bronchial Epithelial Cells .......................................................................... 32 1.5 T-Cells .......................................................................................................... 34 1.5.1 T helper 1 cells ....................................................................................... 35 1.5.2 T helper 2 cells ....................................................................................... 36 1.5.3 T helper 17 Cells .................................................................................... 37 1.5.4 Regulatory T-Cells .................................................................................. 38 1.5.5 Cytotoxic T-Cells .................................................................................... 39 1.5.6 The Role of Cytotoxic T-Cells in Asthma ................................................ 41 1.6 Genetics of Asthma .................................................................................... 44 1.7 Epigenetics ................................................................................................. 45 1.8 Histones Modification ................................................................................ 45 1.8.1 Histone Acetylation ................................................................................. 46 1.8.2 Histone Methylation ................................................................................ 53 1.8.3 H3K4 ...................................................................................................... 53 1.8.4 H3K9 ...................................................................................................... 55 1.8.5 H3K27 .................................................................................................... 55 1.9 Hypothesis and Aims ................................................................................. 60 Chapter 2: Materials & Methods ................................................................. 61 2.1 General Methods ........................................................................................ 62 2.1.1 Enzyme Linked Immunosorbent Assays (ELISA) ................................... 62 2.1.2 Viability Assay ........................................................................................ 62 2.2 Cell proliferation Assay ............................................................................. 63 2.3 Human Bronchial Cell Models ................................................................... 63 2.3.1 BEAS-2B Culture .................................................................................... 63 2.3.2 Normal Human Bronchial Epithelial Cell Culture .................................... 64 6 2.3.3 BEAS-2B Epigenetic Probe/Mimic Treatments ...................................... 64 2.3.4 Treatment of nHBE Cells with Epigenetic Probes/mimics ...................... 66 2.3.5 Effect of Probes/mimics in combination with a corticosteroid in BEAS-2B Cells 66 2.3.6 SILAC Proteomics Methods ................................................................... 66 2.3.7 SILAC labelling and Cell culture ............................................................. 66 2.4 SILAC Methods ........................................................................................... 68 2.4.1 SILAC Protein Extraction ........................................................................ 68 2.4.2 Proteomics using mass spectroscopy .................................................... 68 2.5 Histone Purification and Analysis ............................................................. 70 2.5.1 Global Histone Modification analysis ...................................................... 70 2.5.2 Histone Purification ................................................................................ 70 2.5.3 Histone H3 Post Transcriptional Modification Multiplex ELISA .............. 71 2.6 Isolation of CD8+ T-Cells from Asthmatics .............................................. 73 2.6.1 Patient Selection Criteria for CD8+ T-cells Collection ............................ 73 2.6.2 CD8+ Isolation ........................................................................................ 74 2.6.3 Ficoll-Paque Isolation ............................................................................. 74 2.6.4
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