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
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Mentions: Deletion of RING1A/B in mouse ESCs supports a model whereby H2AK119ub1 contributes to the occupancy of PRC2 at natural target sites in vivo. However, removal of RING1A/B disrupts both canonical and variant PRC1 complex activity. Understanding if variant PRC1 complexes can drive this process at natural target sites is challenging, as variant PRC1 complex targeting mechanisms remain poorly defined. An exception is the PCGF1/PRC1 complex which contains a histone lysine demethylase protein, KDM2B, which binds to nonmethylated DNA via a ZF-CxxC DNA-binding domain (Farcas et al., 2012; He et al., 2013; Long et al., 2013; Wu et al., 2013). Nonmethylated DNA is generally concentrated in vertebrate regulatory elements called CpG islands, and most mammalian polycomb target sites are associated with CpG islands (Ku et al., 2008). KDM2B may therefore represent a direct molecular link between recognition of CpG island target sites and occupancy of both PRC1 and PRC2. To determine if KDM2B binding is sufficient to recruit the PCGF1/PRC1 complex and establish a polycomb domain de novo, a TetR-KDM2B fusion protein was stably expressed in the TetO cell line (Figure 5A). TetR-KDM2B led to RING1B, PCGF1, and H2AK119ub1 deposition (Figure 5A). This was not observed with the related KDM2A protein which does not interact with PRC1 (Figure 5A) (Blackledge et al., 2010). Importantly, PCGF1/PRC1 recruitment by KDM2B resulted in binding of PRC2 and H3K27me3 (Figures 5A and S4C). This activity was dependent on recruitment of PCGF1/PRC1, as depletion of PCGF1 in the TetR-KDM2B line caused a clear reduction in both PRC1 and PRC2 (Figures 5B and 5C). Interestingly, polycomb domain formation did not rely on KDM2B demethylase activity, as a catalytic mutant of KDM2B or a short form of the protein that lacks the demethylase domain recruited PRC1 and PRC2 to similar levels (Figures S4A–S4C). Therefore, de novo targeting of the PCGF1/PRC1 complex by KDM2B leads to polycomb domain formation in a manner similar to TetR-PCGF1 (Figure 1).
Affiliation: Laboratory of Chromatin Biology and Transcription, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.