Dedicated to my father 1952 – 2013 Abstract While the aetiology and pathogenesis of cancer is variable and dependent on the initial cellular and carcinogenic environment, all manifestations of the disease are unified by a gross dysfunction of normal epigenetic control. Recent technological advances have revealed epigenomic disruption of large domains that encompass multiple genes, as well as a deregulation of spatial and temporal control of DNA in the nucleus. Here I consolidate these findings by identifying large genomic domains that are epigenetically and transcriptionally activated in prostate tumourigenesis, a phenomenon we termed Long Range Epigenetic Activation (LREA). These regions contain oncogenes, miRNAs and multiple prostate cancer biomarkers, such as prostate cancer antigen 3 (PCA3) and the prostate specific antigen (PSA). LREA regions are characterised by an increase of histone modifications H3K4me3 and H3K9ac with a simultaneous depletion of H3K27me3 at gene promoters. While I found little evidence of CpG island hypomethylation causing gene activation, I identified hypermethylation of CpG-islands associated with gene activation and differential promoter usage in prostate cancer. The presence of both epigenetically activated and repressed domains in prostate cancer indicates the deregulation of superior, “long-range” acting processes such as chromatin looping or the timing of DNA replication. Using the Kallikrein gene family locus, I describe the presence of chromatin loops anchored by the CTCF protein that commonly demarcate the limits of this cancer-specific regional epigenetic modulation. To investigate how replication timing can influence the cancer epigenome I optimised and carried out the high-resolution “Repli-seq” technique, which details the time of replication for all genomic loci. I show that epigenetic gene activation and repression at both individual genes and at domains is associated with a shift in replication timing between normal and cancer cells. I identify the presence of a cancer-specific signature of replication timing, as well as a replication timing shift at the common prostate oncogene, ERG. Genome-wide hypomethylation which commonly affects cancer DNA is shown to exclusively occur at DNA that replicates late in both normal and cancer cells. Together, the work presented here provides a significant and novel contribution in understanding the nature of epigenetic regulation and the consequences of its deregulation in carcinogenesis. Acknowledgements To begin, I would like to thank my supervisor Prof Sue Clark. The entirety of the research this thesis represents, from its conception and realisation, to its documentation here, all exist due to her unfailing support and guidance. Moreover, while it may have indulged my idle temperament, her friendship and collegiality are amongst my most valued memories of the Garvan. I would also like to thank my co-supervisor Dr Clare Stirzaker, for always taking the time to care for me, even when I didn’t realise I needed it. I am grateful to the Garvan Institute for creating such a unique and welcoming research environment for young scientists, and Dr Alessandra Bray for her many efforts to mitigate the consequences of my failing to properly read guidelines. The research presented here relied on the expertise and generosity of many people at the Garvan. Specifically, I would like to thank: Dr Marcel Coolen and Dr Liz Caldon for their assistance in experimental design; Dr Mark Robinson, Dr Nicola Armstrong and Dario Strbenac for their critical bioinformatics support and analyses, as well as their patience; Jenny Song, Dr Fatima Valdes and Dr Phillippa Taberlay for all of their experimental data which contributed to this thesis. I would like to acknowledge all of the Clark lab, past and present, for creating a welcoming and stimulating environment away from home. To all my fellow PhD students, I felt grateful and elevated by your company. I would like to specifically thank Dr Warick Locke for his undying and beloved enmity, and Zena Kassir for her treasured and undeserved friendship. It would be amiss to not thank Aaron Statham, for his contributions to this thesis, which span bioinformatics support, experimental guidance and youthful mentorship, and for his valued friendship during my years at the Garvan. I am thankful to my friends, for their generous hearts and continual affections that my many and varied flaws should never have warranted. I am most grateful to Giselle and Aurélie, who honoured me with their company and whose love and understanding I will always cherish. I would like to thank my brother for his life-long rivalry and for his persisting successes that have always spurned me to higher grounds. I would like to thank my mother for her unqualified love, for giving me the audacity to never shy from myself or from life’s challenges. Finally, I would like to thank my father, for his unshakable belief in a rational universe, in spite of all the evidence that can be borne; for his grace and quiet insight; and for his unwavering support of all my studies, even when they mattered the least. Financial Support and Publications I gratefully acknowledgement the following scholarships, prizes and financial support received during my PhD: 2009 - 2012: Australian Postgraduate Award, University of New South Wales 2009 – 2012: Rising Star Award, Faculty of Medicine, University of New South Wales 2009 – 2011: Cancer Institute NSW Research Scholar Award 2009: The 19th St Vincent’s and Mater Health Sydney Research Symposium Oral Presentation Prize 2009 – 2012: The Garvan Institute Postgraduate Supplementary Scholarship Publications: Bert, S.A., Robinson, M.D., Strbenac, D., Statham, A.L., Song, J.Z., Hulf, T., Sutherland, R.L., Coolen, M.W., Stirzaker, C., and Clark, S.J. (2013). Regional activation of the cancer genome by long-range epigenetic remodeling. Cancer Cell 23, 9-22 Conference Publications Bert, S.A., Robinson, M.D., Strbenac, D., Statham, A.L., Song, J.Z., Coolen, M.W., Stirzaker, C., and Clark, S.J. Gene Activation Occurs In Epigenetically Controlled Domains In Cancer, presented at: 19th St Vincent’s and Mater Health Sydney Research Symposium, Sydney, Australia 2009. Oral presentation. 4th Asian Epigenomics Meeting, Genome Institute Singapore, Singapore 2009. Poster presentation. Lorne Genome Conference, Lorne, Australia 2010. Poster presentation. 5th PACRIM Breast and Prostate Cancer Meeting, Gold Coast, Australia 2011. Poster presentation. CONTENTS CONTENTS CHAPTER 1: Introduction 1 1.1 The regulated genome ............................................................................................ 2 1.2 The epigenetic code ................................................................................................ 3 1.2.1 Histone modifications ...................................................................................... 3 1.2.2 DNA methylation .............................................................................................. 6 1.2.2.1 CpG Island Methylation ............................................................................ 7 1.2.2.2 Low CpG Density Methylation .................................................................. 8 1.3 Domains of regulation ............................................................................................. 8 1.3.1 Domains of sequence composition ................................................................ 10 1.3.2 Histone modification domains ....................................................................... 11 1.3.3 CpG methylation domains.............................................................................. 11 1.3.4 Nuclear domains ............................................................................................ 12 1.3.4.1 Lamina associated domains .................................................................... 12 1.3.4.2 Active domains........................................................................................ 13 1.3.4.3 Globular structure of the genome .......................................................... 14 1.3.5 DNA replication domains ............................................................................... 15 1.4 Cancer, the diseased genome ............................................................................... 17 1.4.1 DNA methylation in tumourigenesis .............................................................. 18 1.4.2 Chromatin remodelling in tumourigenesis .................................................... 20 1.4.2.1 Histone methylation and cancer............................................................. 20 1.4.2.2 Histone acetylation and cancer .............................................................. 21 CONTENTS 1.4.3 Domain level epigenomic remodelling in cancer ........................................... 22 1.5 Justification & Aims ............................................................................................... 24 CHAPTER 2: Methods 26 2.1 Cell lines and culture ............................................................................................. 27 2.1.1 LNCaP cell line: Lymph Node Cancer of the Prostate .................................... 27 2.1.2 PrEC cell line: Prostate Epithelial Cells ........................................................... 27 2.2 Nucleic acid extraction .......................................................................................... 28 2.2.1 DNA extraction ..............................................................................................