Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. Aberrant DNA methylation in general occurred more often within H3K27me3 stem cell domains. We found a striking association between enrichment of H3K9me3 stem cell domains and toxicant-induced agglomerative DNA methylation. Global gene expression profiling of the arsenite-transformed prostate epithelial cells showed that gene expression changes and DNA methylation changes were negatively correlated, but less than 10% of the hypermethylated genes were down-regulated. These studies confirm that a majority of the DNA hypermethylation events occur at transcriptionally repressed, H3K27me3 marked genes. In contrast to aberrant DNA methylation targeting H3K27me3 pre-marked silent genes, we found that actively expressed ZNF genes marked with H3K9me3 on their 3' ends, are preferred targets of DNA methylation linked gene silencing. H3K9me3 mediated gene silencing of ZNF genes was widespread, occurring at individual ZNF genes on multiple chromosomes and across ZNF gene family clusters. At ZNF gene promoters, H3K9me3 and DNA hypermethylation replaced H3K4me3, resulting in a widespread down-regulation of ZNF gene expression which accounted for 8% of all the down-regulated genes in the arsenical-transformed cells. In summary, these studies associate arsenical exposure with agglomerative DNA methylation of gene family clusters and widespread silencing of ZNF genes by DNA hypermethylation-linked H3K9me3 spreading, further implicating epigenetic dysfunction as a driver of arsenical-induced carcinogenesis.

The degree of chromatin compaction plays a fundamental role in controlling the accessibility of DNA to the transcription machinery as well as other DNA-dependent biological pathways. The mammalian HP1 (Heterochromatin protein 1) protein family consists of three members: HP1α, β and γ. Each paralogue regulates formation and maintenance of heterochromatin by binding to the repressive chromatin marks H3K9me2/3 with their chromodomains (CDs). Despite high sequence conservation, each HP1 paralogue possesses specific functions, which are likely to be cell type specific. The aim of my thesis was to find novel functions for HP1γ in mouse embryonic stem cells (mESCs) and breast cancer cells. Mass spectrometry analysis identified citrullination of residues R38 and R39 within the CD of HP1γ. I show that these residues are citrullinated by peptidyl arginine deiminase 4 (PADI4) in vitro and in vivo. Mutations in HP1γ (R38/9A), designed to mimic the loss of charge accompanied with citrullination, affect HP1γ’s binding to H3K9me3 peptides and reduce its residence time on chromatin in differentiated mESCs, indicating a role for citrullination in regulating HP1γ binding to chromatin during differentiation. Furthermore, I studied the phenotype of HP1γ depletion in two human breast cancer models and found that HP1γ is essential for cell proliferation and viability of cancer, but not of normal epithelial cells. I performed whole transcriptome analysis in breast cancer cells depleted of HP1γ and cross-referenced it with its genomic localisation, which identified increased expression of interferon/antiviral defense genes and activation of pro-apoptotic pathways. Whilst genes involved in these pathways were not directly bound by HP1γ, this analysis also identified HP1γ as a novel regulator of zinc finger (ZNF) genes. In summary, I identified novel post-translational modifications in HP1γ and characterised them in mESCs. I further demonstrated a role for HP1γ regulating breast cancer cell viability and identified HP1γ as a novel regulator of ZNF genes. My findings highlight HP1γ as a potential target for breast cancer therapy.