An Inter-Vascular Bed and Inter-Species Investigation of Epigenetic Regulatory Elements in Endothelial Cells by Lina Antounians A thesis submitted in conformity with the requirements for the degree of Master’s of Science Department of Molecular Genetics University of Toronto © Copyright by Lina Antounians 2015 An Inter-Vascular Bed and Inter-Species Investigation of Epigenetic Regulatory Elements in Endothelial Cells Lina Antounians Master’s of Science Molecular Genetics, University of Toronto, 2015 Abstract A major challenge in human genetics is to understand the mechanisms that control gene expression. To identify gene regulatory regions required for vascular homeostasis, we performed chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) for a variety of histone modifications and the JUN transcription factor in primary aortic endothelial cells (ECs) isolated from human, rat (Rattus norvegicus) and bovine (Bos taurus). We generated a chromatin state map for human aortic ECs and found that the vast majority of regulatory regions in aortic ECs were also active in venous ECs. By comparing the genomic occupancy of JUN and a histone modification indicative of active enhancers (H3K27ac) between species, we identified a set of conserved regulatory regions that were enriched for EC-specific pathways and human regulatory disease variants. Overall, we demonstrate that comparative epigenomics is a viable strategy to identify functionally important vascular gene regulatory elements. ii Acknowledgements I would like to express my sincere gratitude to my supervisor Dr. Michael Wilson for his mentorship and encouragement throughout my Master’s degree. I have learned many technical and analytical skills in your lab, but none as important as the passion for learning, teaching, and collaborating with my peers. A heartfelt thanks to Alejandra Medina-Rivera for her insights and hard work she put into our joint project. It has been a pleasure to work alongside you as a team and to become great friends outside of science. I would also like to thank Dr. Johanna Rommens and Dr. Julie Claycomb, my thesis supervisory committee, for their support, enthusiasm, and critical feedback in both exciting and difficult times. To Liis, I am thankful for the long conversations about science and life, and for your constant encouragement. I had the pleasure to work alongside other brilliant minds in the Wilson lab, both past and present, especially Huayun, Minggao, Xuefei, Maisam, and Azad. Thank you for listening and critiquing my long practice talks and teaching me everything I know about computational biology (including how to get over the fear of using Terminal). Thank you to Xiaoli, Lindsy, and Ted, who were instrumental in helping me set up my project. I am forever grateful to have shared my graduate school experience with wonderful colleagues, including many past and current members in the Gagnon lab, Pearson lab, Minassian lab, Bazett-Jones lab, Rommens lab, Weksberg lab, Justice lab, and the Fish lab. Thank you to all my peers and mentors in the Genetics and Genome Biology Program at SickKids and the Department of Molecular Genetics at University of Toronto for the memorable experience. I am fortunate that support from my family has been a key to my success in graduate school; this thesis is dedicated to you. To my parents, who have always supported me and pushed me to become a better version of myself every day, I endeavor to make you proud. To Leo, my little big brother, thank you for helping me realize and focus on what is most important in life. I am sincerely grateful to my extended family, for their love and for always encouraging me to continue striving and working hard in different aspects of my life. And lastly, to my other half, Keon, thank you for being my inspiration and my motivation, we will continue climbing together. iii Data Attribution The data presented here was acquired and analyzed in collaboration with post-doctoral fellow Dr. Alejandra Medina-Rivera. I conducted all experimental procedures, including 1) expanding primary ECs in cell culture, 2) conducting immunofluorescence and quality control assays, and 3) performing ChIP-seq and ChIP-qPCR experiments. Using scripts written by Dr. Medina-Rivera and others in the lab, I analyzed experimental data. This included 1) aligning sequencing reads, conducting quality control, and calling peaks, 2) building chromatin states, 3) conducting enrichment analysis for biological pathway, human disease variants, and transcription factor binding sites, and 4) defining biochemically conserved sites across multiple species. I interpreted the results together with Dr. Medina-Rivera. iv Table of Contents Abstract .......................................................................................................................................... ii Acknowledgements ....................................................................................................................... iii Data Attribution ............................................................................................................................ iv List of Figures .............................................................................................................................. vii List of Tables .............................................................................................................................. viii List of Supplementary Figures ...................................................................................................... ix List of Supplementary Tables ....................................................................................................... ix List of Abbreviations ..................................................................................................................... x List of Genes ................................................................................................................................. xi Chapter 1 Introduction ................................................................................................................... 1 Endothelial cells play prominent roles in cardiovascular health and disease .......................................... 1 ECs retain their vascular-bed specific gene expression profiles in tissue culture ................................... 1 Ex vivo EC models are essential for understanding EC biology .............................................................. 2 Arterial and venous specification is controlled in part by sets of transcription factors ........................... 4 Integrating multiple ChIP-seq datasets is a powerful way to characterize the epigenome ...................... 7 Thesis Rationale for Chapter 1 .............................................................................................................. 10 Chapter 1 Materials and Methods ................................................................................................ 11 Chapter 1 Results ......................................................................................................................... 19 1.1 Characterizing the epigenetic landscape of human aortic endothelial cells .................................... 19 1.2 Active enhancer and promoter regions can distinguish biological pathways between HAEC and HUVEC .................................................................................................................................................. 25 1.3 Searching for functional regulatory elements in HAEC-only and HUVEC-only regions ............... 30 1.4 Prominent regulatory regions private to endothelial cells highlight endothelial cell-specific biology ............................................................................................................................................................... 34 Chapter 1 Summary ..................................................................................................................... 37 Chapter 2 Introduction ................................................................................................................. 38 Identifying functional cis-regulatory elements using comparative genomics ....................................... 38 Non-coding regions carry crucial information related to human disease gene regulation .................... 39 Epigenetic constraint between species reveals biologically relevant pathways and diseases ............... 39 v Cell models from multiple species are commonly used to study vascular biology ............................... 40 Thesis Rationale for Chapter 2 .............................................................................................................. 41 Chapter 2 Materials and Methods ................................................................................................ 42 Chapter 2 Results ......................................................................................................................... 45 2.1 A minority of human aortic EC H3K27ac and JUN binding sites are shared with rat and bovine aortic ECs ............................................................................................................................................... 45 2.2 Determining the distribution of biochemically-conserved sites in EC-private and pan-tissue regulatory regions .................................................................................................................................. 48 2.3 Using H3K27ac as a baseline