PROFILING THE EPIGENETIC LANDSCAPE OF THE AIRWAY EPITHELIUM IN ASTHMA by Dorota Stefanowicz B.Sc., Biology, The University of Victoria, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2014 © Dorota Stefanowicz, 2014 Abstract The airway epithelium is the interface between the environment and the submucosa of the lung and thus is the first line of defense against inhaled exogenous agents. In addition to maintaining a structural barrier through homeostasis and repair, the airway epithelium is involved in co- ordination of the mucosal immune response to the inhaled environment. In asthma it is now understood that the airway epithelium is abnormal with dysregulation of genes integral to differentiation, proliferation, and inflammation. Alteration of the chromatin architecture through epigenetic modifications including DNA methylation and histone modifications is reactive to the environment and can establish chromatin states which are permissive or repressive to gene expression. Epigenetic regulation of gene expression is cell specific, as such, it is important to understand epigenetic regulation in cells that are thought to play a central role in asthma. As epigenetic processes are critical to cellular specificity and disease susceptibility the overarching hypothesis of this thesis is that alterations to the epigenome of asthmatic airway epithelial cells contribute to the dysregulation of genes involved in key epithelial functions. To investigate this hypothesis, we performed analysis of DNA methylation, expression of epigenetic modifying genes, and histone acetylation and methylation in airway epithelial cells, airway fibroblasts, and peripheral blood mononuclear cells from asthmatic and healthy subjects. We identified unique signatures of both DNA methylation and expression of epigenetic modifying enzymes in airway epithelial cells and showed that epithelial cells were epigenetically distinct from other cell types. Furthermore, we found that airway epithelial cells from asthmatic subjects displayed changes in DNA methylation, expression of histone kinases, acetylation of ii lysine 18 on histone 3, and occupancy of this histone modification at genes important to epithelial functions. Therefore, epigenetic differences between tissue types were more evident and plentiful than within cell types highlighting the importance of the epigenome to cell specificity, yet subtle differences within each tissue were determined and may play a role in disease pathogenesis. Thus, these findings enhance our understanding of the unique epigenetic landscape which may contribute to the airway epithelial phenotype in health and disease. iii Preface The studies and methods described in this thesis were approved by the University of British Columbia‟s Research Ethics Board (#H13-02173) and by the Providence Research Ethics Committee (#H0-50110), University of British Columbia. This research project originated as a research grant designed by Dr. Peter Paré, Dr. Darryl Knight, and Dr. Micheal Kobor, and the research chapters were further designed by Dr. Tillie- Louise Hackett and myself. Chapter 1: Introduction The introduction contains a portion of the book chapter Hackett TL, Warner SM, Stefanowicz D and Knight DA. (2011) Epithelial Cells, in Inflammation and Allergy Drug Design (eds K. Izuhara, S. T. Holgate and M. Wills-Karp), Wiley-Blackwell, Oxford, UK. doi: 10.1002/9781444346688.ch10. I wrote the genetics section for this book and modified it for use in this chapter. Permission to use this section was obtained from RightsLink. Chapter 3: DNA Methylation Profiles of Airway Epithelial Cells and Peripheral Blood Mononuclear Cells from Healthy, Atopic and Asthmatic Children Chapter 3 is based on the publication Stefanowicz D, Hackett TL, Garmaroudi FS, Günther OP, Neumann S, Sutanto EN, Ling KM, Kobor MS, Kicic A, Stick SM, Paré PD, Knight DA. DNA methylation profiles of airway epithelial cells and PBMCs from healthy, atopic and asthmatic children. PLoS One. 2012;7(9):e44213. PLoS One employs the Creative Commons Attribution (CC BY) license whereby authors maintain ownership of the copyright for their article. I was iv responsible for data analysis strategy, manuscript writing and a portion of the sample preparation. Tillie-Louise Hackett assisted with cell isolation, data analysis and editing the manuscript, and Anthony Kicic and Steve Stick contributed cells for the study. Chapter 4: The Landscape of Epigenetic Modifying Enzymes in Airway Epithelial Cells and Fibroblasts in Health and Disease For Chapter 4, Dr. Hackett and I designed the study. I performed all experiments, data analysis strategy, data interpretation, writing and a portion of the data analysis. Tillie-Louise Hackett contributed epithelial and fibroblast cells, aided with data analysis strategy, and editing of the chapter. Teal Hallstrand also provided epithelial cells. Yunlong Nie contributed to the data analysis. Chapter 5: Alterations in Histone Acetylation and Methylation in Airway Epithelial Cells Derived From Asthmatic and Healthy Subjects For Chapter 5, Dr. Knight, Dr. Hackett, and I designed the study. I performed all data analysis, interpretation, writing, and the greater part of the experiments. Optimization of chromatin immunoprecipitation was performed by Janet Lee and some immunohistochemistry and color segmentation was performed by Furquan Shaheen and Janet Lee. Chapter 6: Discussion I was responsible for interpretation and writing of the summary of data in Chapter 6. Tillie- Louise Hackett assisted with editing of the chapter. v Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ......................................................................................................................... vi List of Tables .............................................................................................................................. xiv List of Figures ...............................................................................................................................xv List of Abbreviations ............................................................................................................... xviii Acknowledgements ................................................................................................................... xxii Dedication ................................................................................................................................. xxiii Chapter 1: Introduction ................................................................................................................1 1.1 Overview of thesis .......................................................................................................... 1 1.2 Asthma definition and prevalence .................................................................................. 1 1.3 Asthma diagnosis ............................................................................................................ 2 1.4 Airway inflammation in asthma...................................................................................... 3 1.5 Allergic sensitization ...................................................................................................... 4 1.6 Early phase allergic reaction ........................................................................................... 6 1.7 Late phase allergic reaction ............................................................................................ 8 1.8 Airway remodeling in asthma ....................................................................................... 10 1.8.1 Goblet cell hyperplasia ............................................................................................. 11 1.8.2 Sub-epithelial fibrosis and thickening of the lamina reticularis ............................... 12 1.8.3 Increased airway smooth muscle (ASM) mass ......................................................... 13 1.8.4 Altered epithelial phenotype ..................................................................................... 13 vi 1.9 Normal epithelial function and wound repair ............................................................... 14 1.10 Abnormal barrier function and wound repair in asthma ............................................... 16 1.10.1 Epidermal growth factor receptor ......................................................................... 16 1.10.2 Transcription factor p63 ........................................................................................ 17 1.10.3 Signal transducer and activator of transcription 6 ................................................ 18 1.11 Epidemiology of asthma ............................................................................................... 18 1.12 Asthma genetics ............................................................................................................ 19 1.13 Epigenetic regulation of gene expression ..................................................................... 21 1.13.1 DNA methylation .................................................................................................