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Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Blackledge NP, Farcas AM, Kondo T, King HW, McGouran JF, Hanssen LL, Ito S, Cooper S, Kondo K, Koseki Y, Ishikura T, Long HK, Sheahan TW, Brockdorff N, Kessler BM, Koseki H, Klose RJ - Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Biology and Transcription, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.

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Related in: MedlinePlus

Variant PRC1 Complex-Dependent PRC2 Targeting Occurs on an Inert TetO Array Template and at a Single Natural TetO Site in Mouse ESCs, Related to Figure 1(A) Bisulfite sequencing reveals that CpG dinucleotides flanking the engineered TetO array in the Human BAC DNA remain methylated, as they are in human tissue, when stably inserted into the mouse genome. This indicates that normal DNA methylation features are recapitulated on the integrated DNA sequence.(B) ChIP-qPCR analysis of the human BAC/TetO array stably integrated into mouse ESCs. ChIP was performed with antibodies specific for permissive chromatin marks and features of active transcription (left: H3K4me3, H3K9ac, H3K36me3, and RNAPII) and repressive chromatin features (right: H3K27me3, H2AK119ub1, H4K20me3, and H3K9me3). For each antibody, qPCR enrichment at a known target site is shown as a positive control. For gene-associated chromatin modifications this includes promoter (prom) and body amplicons for the indicated genes, while H3K9me3 and H4K20me3 were analyzed at sites in the repetitive major satellite regions. Together these observations indicate that the human BAC DNA, when inserted into mouse ESCs, retains its inert features and remains suitable for studying polycomb domain formation in tethering assays.(C) A position on mouse chromosome 1, distant from surrounding genes, contains a single site that has a high degree of homology to a consensus bacterial TetO-binding site. This provides a “natural” site in the genome at which to analyze TetR fusion protein binding and function.(D) ChIP-qPCR analysis of TetR, TetR-PCGF1 (variant PRC1), and TetR-PCGF4 (canonical PRC1) binding at the single natural TetO and flanking regions (Top). In each case, occupancy of PRC1 leads to RING1B recruitment (second panel). However, as observed at the engineered TetO array EZH2, SUZ12, and H3K27me3 are only recruited to the endogenous TetO site when a variant PRC1 complex occupies the site (bottom three panels). Together these observations demonstrate that variant PRC1 complex-dependent PRC2 recruitment and de novo polycomb domain formation is observed at a “natural” sequence in the mouse genome, and is not the result of copy number or site specific features inherent to the engineered TetO array. All ChIP experiments in (B and D) were performed in biological duplicate with error bars showing SEM.
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figs1: Variant PRC1 Complex-Dependent PRC2 Targeting Occurs on an Inert TetO Array Template and at a Single Natural TetO Site in Mouse ESCs, Related to Figure 1(A) Bisulfite sequencing reveals that CpG dinucleotides flanking the engineered TetO array in the Human BAC DNA remain methylated, as they are in human tissue, when stably inserted into the mouse genome. This indicates that normal DNA methylation features are recapitulated on the integrated DNA sequence.(B) ChIP-qPCR analysis of the human BAC/TetO array stably integrated into mouse ESCs. ChIP was performed with antibodies specific for permissive chromatin marks and features of active transcription (left: H3K4me3, H3K9ac, H3K36me3, and RNAPII) and repressive chromatin features (right: H3K27me3, H2AK119ub1, H4K20me3, and H3K9me3). For each antibody, qPCR enrichment at a known target site is shown as a positive control. For gene-associated chromatin modifications this includes promoter (prom) and body amplicons for the indicated genes, while H3K9me3 and H4K20me3 were analyzed at sites in the repetitive major satellite regions. Together these observations indicate that the human BAC DNA, when inserted into mouse ESCs, retains its inert features and remains suitable for studying polycomb domain formation in tethering assays.(C) A position on mouse chromosome 1, distant from surrounding genes, contains a single site that has a high degree of homology to a consensus bacterial TetO-binding site. This provides a “natural” site in the genome at which to analyze TetR fusion protein binding and function.(D) ChIP-qPCR analysis of TetR, TetR-PCGF1 (variant PRC1), and TetR-PCGF4 (canonical PRC1) binding at the single natural TetO and flanking regions (Top). In each case, occupancy of PRC1 leads to RING1B recruitment (second panel). However, as observed at the engineered TetO array EZH2, SUZ12, and H3K27me3 are only recruited to the endogenous TetO site when a variant PRC1 complex occupies the site (bottom three panels). Together these observations demonstrate that variant PRC1 complex-dependent PRC2 recruitment and de novo polycomb domain formation is observed at a “natural” sequence in the mouse genome, and is not the result of copy number or site specific features inherent to the engineered TetO array. All ChIP experiments in (B and D) were performed in biological duplicate with error bars showing SEM.

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).


Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Blackledge NP, Farcas AM, Kondo T, King HW, McGouran JF, Hanssen LL, Ito S, Cooper S, Kondo K, Koseki Y, Ishikura T, Long HK, Sheahan TW, Brockdorff N, Kessler BM, Koseki H, Klose RJ - Cell (2014)

Variant PRC1 Complex-Dependent PRC2 Targeting Occurs on an Inert TetO Array Template and at a Single Natural TetO Site in Mouse ESCs, Related to Figure 1(A) Bisulfite sequencing reveals that CpG dinucleotides flanking the engineered TetO array in the Human BAC DNA remain methylated, as they are in human tissue, when stably inserted into the mouse genome. This indicates that normal DNA methylation features are recapitulated on the integrated DNA sequence.(B) ChIP-qPCR analysis of the human BAC/TetO array stably integrated into mouse ESCs. ChIP was performed with antibodies specific for permissive chromatin marks and features of active transcription (left: H3K4me3, H3K9ac, H3K36me3, and RNAPII) and repressive chromatin features (right: H3K27me3, H2AK119ub1, H4K20me3, and H3K9me3). For each antibody, qPCR enrichment at a known target site is shown as a positive control. For gene-associated chromatin modifications this includes promoter (prom) and body amplicons for the indicated genes, while H3K9me3 and H4K20me3 were analyzed at sites in the repetitive major satellite regions. Together these observations indicate that the human BAC DNA, when inserted into mouse ESCs, retains its inert features and remains suitable for studying polycomb domain formation in tethering assays.(C) A position on mouse chromosome 1, distant from surrounding genes, contains a single site that has a high degree of homology to a consensus bacterial TetO-binding site. This provides a “natural” site in the genome at which to analyze TetR fusion protein binding and function.(D) ChIP-qPCR analysis of TetR, TetR-PCGF1 (variant PRC1), and TetR-PCGF4 (canonical PRC1) binding at the single natural TetO and flanking regions (Top). In each case, occupancy of PRC1 leads to RING1B recruitment (second panel). However, as observed at the engineered TetO array EZH2, SUZ12, and H3K27me3 are only recruited to the endogenous TetO site when a variant PRC1 complex occupies the site (bottom three panels). Together these observations demonstrate that variant PRC1 complex-dependent PRC2 recruitment and de novo polycomb domain formation is observed at a “natural” sequence in the mouse genome, and is not the result of copy number or site specific features inherent to the engineered TetO array. All ChIP experiments in (B and D) were performed in biological duplicate with error bars showing SEM.
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Related In: Results  -  Collection

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figs1: Variant PRC1 Complex-Dependent PRC2 Targeting Occurs on an Inert TetO Array Template and at a Single Natural TetO Site in Mouse ESCs, Related to Figure 1(A) Bisulfite sequencing reveals that CpG dinucleotides flanking the engineered TetO array in the Human BAC DNA remain methylated, as they are in human tissue, when stably inserted into the mouse genome. This indicates that normal DNA methylation features are recapitulated on the integrated DNA sequence.(B) ChIP-qPCR analysis of the human BAC/TetO array stably integrated into mouse ESCs. ChIP was performed with antibodies specific for permissive chromatin marks and features of active transcription (left: H3K4me3, H3K9ac, H3K36me3, and RNAPII) and repressive chromatin features (right: H3K27me3, H2AK119ub1, H4K20me3, and H3K9me3). For each antibody, qPCR enrichment at a known target site is shown as a positive control. For gene-associated chromatin modifications this includes promoter (prom) and body amplicons for the indicated genes, while H3K9me3 and H4K20me3 were analyzed at sites in the repetitive major satellite regions. Together these observations indicate that the human BAC DNA, when inserted into mouse ESCs, retains its inert features and remains suitable for studying polycomb domain formation in tethering assays.(C) A position on mouse chromosome 1, distant from surrounding genes, contains a single site that has a high degree of homology to a consensus bacterial TetO-binding site. This provides a “natural” site in the genome at which to analyze TetR fusion protein binding and function.(D) ChIP-qPCR analysis of TetR, TetR-PCGF1 (variant PRC1), and TetR-PCGF4 (canonical PRC1) binding at the single natural TetO and flanking regions (Top). In each case, occupancy of PRC1 leads to RING1B recruitment (second panel). However, as observed at the engineered TetO array EZH2, SUZ12, and H3K27me3 are only recruited to the endogenous TetO site when a variant PRC1 complex occupies the site (bottom three panels). Together these observations demonstrate that variant PRC1 complex-dependent PRC2 recruitment and de novo polycomb domain formation is observed at a “natural” sequence in the mouse genome, and is not the result of copy number or site specific features inherent to the engineered TetO array. All ChIP experiments in (B and D) were performed in biological duplicate with error bars showing SEM.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Biology and Transcription, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.

Show MeSH
Related in: MedlinePlus