Epigenetic gene silencing has become an area of intensive study due to its critical functions in regulating multiple fundamental biological processes, including heterochromatin formation, transcriptional regulation, genomic imprinting, and dosage compensation. One of the best systems in understanding epigenetic silencing is the Polycomb group (PcG) gene silencing system, which is conserved from Drosophila to human. Genetics and biochemical studies indicate that PcG proteins exist in at least two distinct protein complexes, PRC1 and PRC2. One of the most significant breakthroughs in the studies of PcG gene silencing is the demonstration that PRC1 and PRC2 possess both H2A ubiquitin E3 ligase and H3K27 methyltransferase activities. Our group discovered the two enzymatic activities and also reconstituted both enzymatic complexes and dissected the function of individual components. Our results suggest a hierarchical model by which the PRC2 is recruited to PRE through interactions with DNA binding proteins. PRC2-mediated H3K27 methylation then facilitates recruitment of PRC1, which mediates H2A ubiquitylation and transcriptional silencing. Building on these studies, we have established the following specific aims for this proposal: 1. Understand the PRC2 substrate specificity, processivity, and methyl-H3K27 inheritance. This will be accomplished by using recombinant PRC2 and reconstituted nucleosomes containing H3 that is methylated to various levels on H3K27. 2. Identification and characterization of a putative H3K27 demethylase. This will be accomplished by using a candidate approach and an unbiased biochemical activity-based approach. 3. Understand the functional significance of EZH2 phosphorylation. Our preliminary studies indicated that EZH2, the catalytic subunit of PRC2, is subject to phosphorylation. We will try to understand the functional significance of EZH2 phosphorylation by mapping the phosphorylation site, determining the temporal and spatial relationship of EZH2 phosphorylation with PRC2 enzymatic activity, and identifying the responsible kinase. 4. Determine the genome-wide location of ubH2A and identify ubH2A-binding protein(s). A ChIP coupled genomic microarray using a cell line with tagged ubiquitinated H2A will be used to determine the genome- wide location of ubH2A. A 2D gel electrophoresis approach comparing affinity purified samples from paired cell lines with or without H2A ubiquitylation will be used to identify the ubH2A binding protein(s). 5. .Determine the transcriptional effect and understand the mechanism of H3K27 methylation and H2A ubiquitylation using a reconstituted chromatin transcription system. More description in proposal.
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