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Defining the Molecular and Cellular Mechanisms of EZH2's Dynamic Role In Defining the Molecular and Cellular Mechanisms of EZH2's Dynamic Role in Tumourigenesis Thomas Mortimer University College London and The Francis Crick Institute PhD Supervisor: Dr Paola Scaffidi A thesis submitted for the degree of Doctor of Philosophy University College London July 2019 Declaration I, Thomas Mortimer confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Epigenetic regulators are recurrently hijacked by cancer cells to enhance and maintain their malignant properties. Remarkably, epigenetic regulators are often co-opted by cancer cells in their wild-type unmutated form without any discernible changes to their intrinsic properties. However, the molecular mechanisms that underlie the co-option of these key regulators of cellular identity remain largely unclear. To generate a mechanistic paradigm for this phenomenon, I investigate how the Polycomb component EZH2 transitions from its physiological role in regulating normal development and tissue maintenance, to its pathological role as a tumour promoter. Focussing on the role of EZH2 in the central nervous system, I show that oncogenic signalling redistributes EZH2 on chromatin, leading to transcriptional misregulation of key neural homeotic genes and a malignant rewiring of normal development programmes. This is achieved by de-repression of spinal cord-specifying HOX genes and repression of the forebrain and neural stem cell transcription factor EMX2, an expression switch observed in both glioblastoma cell lines and patient samples. By in vivo assays, I show that EMX2 is a potent tumour suppressor in glioblastoma, suggesting that EMX2 repression by EZH2 is important for glioblastoma maintenance. Beyond its chromatin distribution, I also show that transformation alters the binding partners of EZH2, suggesting that dynamic changes to the composition of Polycomb complexes may promote tumour formation. Thus, by performing a detailed multi-omics characterisation of how neoplastic transformation alters the distribution, activity and binding partners of EZH2, I provide a unique insight into the molecular and cellular mechanisms underlying wild-type EZH2's dynamic role in tumourigenesis. 3 Impact Statement Therapeutic targeting of epigenetic regulators is emerging as an effective treatment strategy in many cancer types. In particular, EZH2, a transcriptional repressor crucial in development and tissue maintenance, acts as a tumour promoter in various malignancies and is also a clinically-relevant therapeutic target. However, the full range of cancer types for which EZH2-targeted therapy may be an effective option remains unclear and there exists no means to identify the specific patients who may benefit. Moreover, by undetermined mechanisms, EZH2 acts as both a tumour suppressor and promoter in cancer, potentially confounding the use of EZH2-targeted therapy. To address these important issues, I performed a detailed characterisation of molecular mechanisms which enable EZH2 to acts as a tumour promoter in cancer, leading to discoveries that I believe are of both academic and clinical interest. In the first part of this study, I reveal that in glioblastoma, transformation- induced redistribution of EZH2 causes gene expression changes at key neural transcription factors. This finding provides a clinically-relevant insight into the molecular mechanisms which drive glioblastoma growth, a pertinent finding given glioblastomas poor prognosis and our limited knowledge of its underlying mechanisms. Furthermore, based on EZH2’s mechanism of action in glioblastoma, I propose a stage-specific model to describe EZH2’s dichotomous tumour suppressive and promoting roles, presenting a theoretical framework which may help others research and interpret the tumour promoting roles of more epigenetic regulators. In addition, the extensive ChIP-seq, RNA- seq and proteomics data sets generated in this study will provide a valuable resource to others in the epigenetics community, potentially stimulating and complementing future research. In the second part of this study, I describe how transformation alters the composition of PRC2 through changing the binding of specific accessory proteins. The role of PRC2 accessory proteins remains poorly defined, and hence this provides an important insight to how these proteins function in both normal and cancer cells. Moreover, I present preliminary evidence that PRC2 accessory proteins in their wild-type form can play tumour suppressive and promoting roles in cancer, providing a starting point for future research to 4 characterise in greater detail the function of these proteins in cancer - research which may have important clinical implications. In the final part of this study, I present a detailed comparative analysis of genetic and pharmacological loss-of-function approaches for determining the genes regulated by EZH2. This controlled comparison will provide a reference point for others considering on embarking on similar studies, enabling them to make a rational and informed decision as to the experimental loss-of-function approach to employ. 5 Acknowledgements I would like to begin first and foremost by thanking my Ph.D. supervisor Paola Scaffidi. Paola, your phenomenal support, superlative critiques and boundless creativity have made my Ph.D. not only scientifically formative and engaging, but also an incredible journey. I am immensely appreciative for the countless hours you have spent diligently proof-reading work, patiently explaining concepts and dishing out sage advice. I have been unbelievably fortunate to have shared my Ph.D. with a cast of fantastic lab mates/friends. Josep, you have been as much part of my Ph.D. as my pipettes, indispensable but only good with small amounts of liquid… In all seriousness, I am lucky to have had someone with your sense of humour and drive to do my Ph.D. alongside. Cristina, you are the bedrock of this lab. Your support and advice has enabled me to overcome so many of my Ph.D.’s twists and turns I think I’ve lost count... Not only that, but working with you has been amazing fun! Elanor, you are a consummate scientist, and if I can achieve just a fraction of your professionalism and skill I will surely retire a happy man. Thanks for everything you’ve helped me with and all the great times along the way! Giannis, Puay, Marina and Thomas, it’s been a pleasure spending days, nights, weekends and everything in-between in the lab with you all. You are top notch scientists and are going to achieve incredible things. Of course a huge thanks to all past lab members, especially to Matt Burney whose tutoring in all things R and command line was invaluable for this project’s success. I want to thank everyone in the FACS, Advanced sequencing, Genomics, Histopathology and Bioinformatics STPs, without whom none of this would have been possible. In particular, Harshil Patel for imparting his computational wisdom to help initiate me into the world of bioinformatics. Claire, you may never know what EZH2 is, but you have helped me through every stage of this journey. I am enormously indebted to you. 6 Table of contents Declaration…. .............................................................................................. 2 Abstract..........................................................................................................3 Impact Statement ........................................................................................ 4 Acknowledgements .................................................................................... 6 Table of contents ........................................................................................ 7 Table of figures ......................................................................................... 11 Abbreviations ............................................................................................ 14 Chapter 1. Introduction ............................................................................. 17 1.1 Architecture and mechanisms of Polycomb repressive complexes ....................................................................................... 18 1.1.1 PRC2 composition and function ................................................. 19 1.1.2 PRC1 composition and activity ................................................... 24 1.1.3 PRC2 targeting to chromatin ...................................................... 25 1.1.4 Synergistic activity of PRC1 and PRC2 ...................................... 26 1.1.5 Complex interplay between PRC2 and DNA methylation ........... 28 1.2 PRC2 in development and tissue maintenance ............................ 29 1.2.1 PRC2 in mammalian development ............................................. 29 1.2.2 PRC2 in tissue maintenance ...................................................... 32 1.3 Epigenetic mechanisms and cancer .............................................. 34 1.3.1 Mutagenic disruption of epigenetic mechanisms ........................ 34 1.3.2 Tumour promotion by wild-type epigenetic regulators ................. 37 1.4 PRC2 in cancer ............................................................................... 38 1.4.1 Tumour promotion by wild-type PRC2 ........................................ 38 1.4.2 Oncogenic PRC2 mutations ....................................................... 40 1.4.3 Tumour suppression by PRC2 ..................................................
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