| EZH2 (Enhancer of zeste 2) is an important developmental regulator that plays an important role in numerous biological processes including multi-cellular development, stem cell biology, and cancer development. As an H3K27 histone methyltransferase, EZH2 is an epigenetic regulator that functions to repress transcription. It is often upregulated in human cancers and its oncogenic potential is thought to stem from its role in cell proliferation and repression of tumor suppressor genes. Hence, regulation of EZH2 function is important for normal cellular growth. In this dissertation, the molecular function and regulation of EZH2 are investigated. I demonstrate that genomic targets of EZH2 not only include proteincoding genes, but also encompasses lncRNAs (long non-coding RNAs), a class of RNAs that account for the vast majority of the mammalian transcriptome. Using a custom-designed microarray, I show that lncRNAs exhibit cell type specific expression patterns and that their expression levels correlate with the epigenetic marks, H3K4me3 and H3K27me3. Consistent with this finding, knockdown of EZH2 results in the derepression of many lncRNAs. With regard to EZH2 regulation, I provide evidence that EZH2 contains numerous phosphorylation sites although their functions are currently unknown. Characterization of two phosphorylation sites, threonines 345 and 487, has revealed important aspects of EZH2 regulation. Through in vitro and in vivo experiments, I show that phosphorylation of these two residues can be mediated by the cyclin dependent kinase, CDK1. Consistent with the cell cycle phase that exhibits peak CDK1 activity, mitotic cell extracts are enriched for phospho-EZH2. Importantly, when EZH2 is phosphorylated at T345 and T487, the stability of the protein is compromised as EZH2 is targeted for ubiquitination and degradation by the proteasome. Collectively, this work reveals a novel class of EZH2 targets and provides a mechanism by which EZH2 protein levels are regulated in the cell. |
