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: The observation that KDM2B can nucleate PRC1 and PRC2 provided an opportunity to examine whether targeting of the PCGF1/PRC1 variant complex to natural CpG island target sites is important for polycomb domain formation. To achieve this, a novel genetic system was designed in which an exon that encodes most of the KDM2B ZF-CxxC domain and is shared by both the long and short form of the protein (Figures S4 and S5A) (Fukuda et al., 2011) was flanked by loxP sites (Kdm2bfl/fl) (Figure 5D). Homozygous Kdm2bfl/fl mouse ESC lines were then derived that also stably express a tamoxifen-inducible form of Cre-recombinase. Addition of tamoxifen rapidly yielded KDM2B long and short form proteins that lack the ZF-CxxC domain (Figures 5E and S5B), but remain associated with the PCGF1/PRC1 variant complex (Figure S5D). Importantly, cellular levels of PRC1 and PRC2 components were unaffected (Figure S5C). ChIP-seq for KDM2B in the Kdm2bfl/fl cells revealed KDM2B occupancy at CpG islands as previously described (Figure 5F) (Farcas et al., 2012; He et al., 2013; Wu et al., 2013). However, tamoxifen-mediated deletion of the ZF-CxxC domain caused a near complete loss of KDM2B chromatin occupancy and removal of PCGF1 from CpG islands (Figures 5F and 5G). Therefore, deletion of the KDM2B ZF-CxxC domain is sufficient to ablate normal targeting of the PCGF1/PRC1 complex in vivo.
Affiliation: Laboratory of Chromatin Biology and Transcription, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.