Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.
Bottom Line: Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development.Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain.This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development.
Affiliation: Laboratory of Chromatin Biology and Transcription, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.Show MeSH
Mentions: Surprisingly, PCGF proteins that form canonical PRC1 complexes appeared less competent at H2AK119ub1 placement in tethering assays (Figure 1D). This lack of activity could be inherent to canonical PRC1 complexes or possibly result from their covalent fusion to TetR. To circumvent the necessity to fuse canonical complexes to TetR, PRC2 was recruited to the TetO via a TetR-EED fusion (Figure 2A) (Hansen et al., 2008). This led to deposition of H3K27me3 and recruitment of endogenous PCGF2 and CBX7, but not PCGF1, suggesting PRC2-dependent recruitment of canonical PRC1 complexes (Figure 2B). As was the case with direct tethering of PCGF2 or 4, native canonical PRC1 complex nucleation failed to deposit H2AK119ub1, suggesting the lack of activity in canonical PRC1 tethering experiments does not result from TetR fusion (Figure 2B). Interestingly, the binding profiles for canonical PRC1 components were not completely coincident with H3K27me3, as might be expected if occupancy was entirely CBX dependent. It remains unclear why this disparity in profiles existed, but it may result from secondary structural effects driven by exclusive canonical PRC1 complex recruitment (Isono et al., 2013) or other undefined mechanisms involved in canonical PRC1 recruitment to regions containing PRC2 and H3K27me3. Interestingly, a similar discordance between CBX (PC) protein binding and H3K27me3 was observed at polycomb target sites in Drosophila cell culture models (Schwartz et al., 2006). Nevertheless, this apparent failure of PRC2 and H3K27me3 to direct H2AK119ub1 parallels observations in mouse ESC lines devoid of H3K27me3 where levels of H2AK119ub1 at polycomb target sites are largely unaffected (Schoeftner et al., 2006; Tavares et al., 2012). Together, these observations strongly suggest that PRC2-mediated recruitment of canonical PRC1 complexes fails to catalyze significant levels of H2AK119ub1.
Affiliation: Laboratory of Chromatin Biology and Transcription, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.