| Summary: | Polycomb proteins are a group of repressor proteins that form multiprotein complexes, Polycomb repressor complexes (PRC) that mediate gene silencing. Two major complexes that have been widely studied are PRC1 and PRC2.Both complexes have enzymatic activities that modify specific histone residues, monoubiquitination of H2AK119 (H2AK119u1) and methylation of H3K27 (H3K27me3) respectively, and it is thought that this is the basis for their silencing activity. The basis for recruitment of PcG proteins in mammalian systems remains to be fully elucidated. A key concept is the hierarchical model explaining co-occupancy of PRC1 and PRC2 at target loci. Here it is proposed that a chromodomain in the protein polycomb (Pc), a core PRC1 subunit, recognises the H3K27me3 histone modification catalysed by PRC2. Although some observations appear inconsistent with this idea it is nevertheless widely accepted. In this study the Ink4b-Arf-Ink4a locus was used as a model to study PcG recruitment both by examining PcG localization and by setting up a reporter system that could be used to screen for novel regulators of PcG recruitment. The former analysis revealed that Exon E2, which is shared between Ink4a and Arf is strongly bound by PcG proteins, suggesting it as a good candidate for a recruitment centre. However, deletion of exon E2 does not inhibit the recruitment of PcG to other regulatory regions within the locus. In the latter study P1-derived artificial chromosome (PAC) recombineering was used to produce a modified PAC in which fluorescent marker proteins replaced endogenous Ink4a and Arf open reading frames. This will provide a useful tool for screening for factors that contribute to Ink4a-Arf activation including PcG recruitment factors. Preliminary data from the lab indicated that global levels of PRC1 mediated H2AK119u1 are largely unaffected in cells lacking H3K27me3, an unexpected outcome based on the hierarchical recruitment model. Chromatin immunoprecipitation (ChIP) was carried out on Eed mutant (H3K27me3 depleted) ESCs and consistent with the hierarchical model, occupancy of the core PRC1 proteins RING1B and MEL18 was strongly reduced. However, H2AK119u1 was clearly detectable at target loci, suggesting PRC1 recruitment independent of H3K27me3. Genome wide analysis of RING1B occupancy substantiated this conclusion, demonstrating significant RING1B levels at polycomb target loci in Eed<sup>-/-</sup> ESCs. From this study it can be concluded that PRC1 and PRC2 are recruited independently to sites that they co-occupy. Additionally evidence is provided that the primary function of H3K27me3 is to increase the residency of PRC1 at target loci and thereby to contribute to the stability of PRC1 mediated silencing.
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