Trimethylation of histone H3 lysine 9 (H3K9me3) and DNA methylation mark heterochromatin, contributing to gene silencing and normal cellular functions. My research investigated the control of H3K9me3 and DNA methylation in the filamentous fungus Neurospora crassa. The H3K9 methyltransferase complex, DCDC, consists of DIM-5, DIM-7, DIM-9, DDB1, and CUL4. Each component of DCDC is required for H3K9me3. The DIM-9/DDB1/CUL4 subunits are reminiscent of known cullin E3 ubiquitin ligases. I showed that core features of CUL4-based E3 ubiquitin ligases are not required for H3K9me3 and DNA methylation in Neurospora. H3K9me3 is bound by heterochromatin protein 1 (HP1) to recruit the DIM-2 DNA methyltransferase and the HCHC histone deacetylase complex. HCHC consists of HP1, CDP-2, HDA-1, and CHAP. Both HP1 and CDP-2 harbor conserved chromodomains that bind H3K9me3, and CHAP contains two putative AT-hook domains that bind A:T-rich DNA. To test the contributions of these domains to HCHC function, I deleted the chromodomains of HP1 and CDP-2. Deletion of the HP1 chromodomain resulted in a reduction of DNA methylation, which was not exacerbated by deletion of the CDP-2 chromodomain. A strain with deletions of chap and the HP1 chromodomain showed a DNA methylation phenotype comparable to the loss of the HDA-1 catalytic subunit. These findings support a model in which recognition of H3K9me3 and A:T-rich DNA by HP1 and CHAP, respectively, are required for proper HCHC function. To examine the relationships between H3K9me3, DNA methylation, and histone acetylation, I utilized in vivo protein tethering of core heterochromatin components. The requirement of DIM-7 for native heterochromatin, previously implicated in localizing the H3K9 methyltransferase DIM-5, was not bypassed by DIM-5 tethering, indicating that DIM-7 has additional roles within the DCDC. Artificial localization of the HCHC histone deacetylase, by tethering HP1 or HDA-1, resulted in induction of H3K9me3, DNA methylation, and gene silencing, but silencing did not require H3K9me3 or DNA methylation. HCHC-mediated establishment of H3K9me3 was not required for de novo heterochromatin formation at native heterochromatic loci suggesting a role in heterochromatin spreading. Together, this work implicates HDA-1 activity as a key driver of heterochromatin spreading and silencing. This dissertation includes previously published co-authored material.

Since the discovery of DNA packaging into chromatin, and McClintock's (1951) work on position-effect variegation providing evidence of non-mendelian inheritance, the principal of a genome maintaining 'on' and 'off' states has been widely adopted. However, the underlying mechanisms that regulate these dynamic chromatin states and their effect on disease are still poorly understood. DNA methylation and histone trimethylation at H3K9 and H4K20 are the core hallmarks of the heterochromatic constitutively 'off' state. Constitutive heterochromatin is predominantly comprised of repetitive satellite containing pericentromeric regions and telomeres and in mouse heterochromatin clusters into large chromocenters. These regions are cytologically more compact and generally transcriptionally silent across embryonic and differentiated mouse cell types. However, in addition to increased genomic instability, mouse tumour cells sustain increased satellite expression suggesting constitutive heterochromatin is disrupted. Therefore how constitutive heterochromatin is maintained has important implications for genome regulation and disease, and remains poorly understood. While satellite DNA sequences are not evolutionarily conserved, pericentromeric and telomeric heterochromatin occurs across species. Heterochromatin formation is therefore independent of the underlying DNA sequence, supporting the hypothesis that epigenetic components can regulate chromatin structure. DNA methylation is generally thought to be associated with transcriptional silencing and chromatin compaction. However, Gilbert et al (2007) showed that the complete loss of DNA methylation did not affect the compaction at heterochromatin or global genome compaction. The role of H3K9me3 in regulating heterochromatin has also been an area of keen interest. H3K9me3 patterns are established by suppressor of variegation 3-9 homologues and provide the binding site for heterochromatic protein 1 [HP1] which can in turn recruit Suv39h1. This Suv3-9h-HP1-H3K9 axis enables its propagation throughout heterochromatin. Peters et al (2001) demonstrated that in mice loss of suv39 homologues 1 and 2 caused a loss of H3K9me3 at constitutive heterochromatic domains. These Suv39h null mice demonstrated decreased genome stability, and an increased prevalence of oncogenesis. However cytological chromocenters are still present in the absence of H3K9me3. Therefore the function of H3K9me3 as a causative agent in heterochromatin formation is still debated. Broadly the aim was to investigate the phenotypic role of heterochromatic epigenetic components in cancer progression, and address whether H3K9me3 effects large scale chromatin structure. To identify heterochromatic gene silencing components, an inhibitor screen was performed in an artificial silenced reporter system. The reporter fluorophore was silenced by the presence of centromeric arrays from yeast/bacterial artificial chromosomes and human alpha satellite repeats enriched for H3K9me3. To address the function of the de-silencing components identified in cancer, the fitness of colon cancer cells [HCT116] was investigated before and after the development of resistance to the MEK inhibitor trametinib. The most intriguing result was that BET protein inhibition resulted in derepression of the reporter construct and trametinib resistant HCT116 cells were more sensitive to BET inhibitors, while subsequent investigation showed HP1 protein levels were altered. Analysis of publically available datasets of tumour drug resistance, showed elevated BET protein binding at HP1 promoters in resistant cell lines suggesting an indirect role in gene silencing. To investigate the consequence of H3K9me3 loss on chromatin structure, mouse embryonic stem cells that lacked both Suv39 homologues were used. Microccocal nuclease digestion and sucrose sedimentation demonstrated a global decompaction of large-scale chromatin fibres whilst re-expression of suv39h1 rescued H3K9me3 at chromocenters and global chromatin decompaction. Loss of Suv39h also increased chromatin associated RNA levels that were also rescued by Suv39h1 re-expression. This suggests that H3K9me3 has a role chromatin fibre compaction globally as well as at constitutive heterochromatin, potentially mediated by chromatin associated RNA. To conclude, multiple components were identified that are involved in transcriptional silencing. Evaluating their function in tumour progression demonstrated a possible role of BET proteins in the development of MEKi resistance that may be mediated through HP1 proteins. H3K9me3 and its binding partner HP1 affect global chromatin compaction. The global decompaction after Suv39h loss correlates with an increase in chromatin associated RNA, suggesting a possible mechanism for changes in chromatin compaction beyond H3K9me3.