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: The hierarchical recruitment model posits that PRC2-dependent H3K27me3 is required to recruit canonical PRC1 complexes to chromatin. The recent demonstration that variant PRC1 complexes bind to many target sites, albeit at lower levels, independently of H3K27me3 suggests that PRC1 function may be more complex than previously envisaged (Tavares et al., 2012). This new insight necessitates a more detailed examination of mammalian PRC1 complex function and targeting in vivo. Therefore, we sought to design a system in which individual PRC1 complexes could be targeted de novo to a chromatin environment free from the complexities and regulatory influences of natural polycomb target sites. To achieve this, a large bacterial artificial chromosome (BAC) containing human DNA that lacks identifiable genes and shows no evidence for gene-, enhancer-, or polycomb-associated chromatin modifications was selected and bacterial Tet operator (TetO) DNA-binding sites were centrally inserted (Figure 1B). Importantly, the TetO lacks CpG dinucleotides and has no resemblance to natural polycomb targets which are CpG-rich (Ku et al., 2008). The TetO BAC was transposed into mouse ES cells at a site on chromosome 8, effectively flanking the TetO array with long stretches of inert chromatin (Figures 1B, S1A, and S1B). Fusion of PRC1 components to the bacterial Tet repressor (TetR) DNA-binding domain would permit de novo recruitment to the TetO array (Figure 1C) and the direct consequences of fusion protein occupancy could be examined by chromatin immunoprecipitation (ChIP).
Affiliation: Laboratory of Chromatin Biology and Transcription, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.