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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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Conditional Deletion of RING1A/B Results in Depletion of H2AK119ub1, Loss of PRC2 Occupancy, and H3K27me3 at Target Genes Independently of the Magnitude of Associated Gene Expression Change, Related to Figure 4(A) Snapshots of ChIP-seq traces for RING1B, SUZ12, EZH2 and H3K27me3 in the Ring1a−/−Ring1bfl/fl cells prior to (−OHT) and following 48 hr (+OHT) of tamoxifen treatment. Three representative genes are depicted illustrating the reduction in RING1B, SUZ12, EZH2, and H3K27me3 following removal of the RING1A/B. A Bio-CAP sequencing trace is shown to indicate the location of nonmethylated DNA and a CpG Island (CGI) prediction annotation track is show as green bars under the traces. This complements examples already shown in main Figure 4.(B) ChIP-qPCR analysis for PRC2 components SUZ12, EZH2, and H3K27me3 at a series of sites showing loss of these factors in the ChIP-seq analysis in main Figure 4 after tamoxifen treatment. All ChIP experiments in (C-D) were performed in biological triplicate with error bars showing SEM.(C) Gene expression analysis for the polycomb target genes analyzed by ChIP in (B). There is no correlation between gene expression change and scale of polycomb group protein loss, suggesting that altered gene expression is not driving PRC2 loss. RT-PCR was performed in biological triplicate and is normalized to Hprt1 expression. Error bars show SEM.
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figs2: Conditional Deletion of RING1A/B Results in Depletion of H2AK119ub1, Loss of PRC2 Occupancy, and H3K27me3 at Target Genes Independently of the Magnitude of Associated Gene Expression Change, Related to Figure 4(A) Snapshots of ChIP-seq traces for RING1B, SUZ12, EZH2 and H3K27me3 in the Ring1a−/−Ring1bfl/fl cells prior to (−OHT) and following 48 hr (+OHT) of tamoxifen treatment. Three representative genes are depicted illustrating the reduction in RING1B, SUZ12, EZH2, and H3K27me3 following removal of the RING1A/B. A Bio-CAP sequencing trace is shown to indicate the location of nonmethylated DNA and a CpG Island (CGI) prediction annotation track is show as green bars under the traces. This complements examples already shown in main Figure 4.(B) ChIP-qPCR analysis for PRC2 components SUZ12, EZH2, and H3K27me3 at a series of sites showing loss of these factors in the ChIP-seq analysis in main Figure 4 after tamoxifen treatment. All ChIP experiments in (C-D) were performed in biological triplicate with error bars showing SEM.(C) Gene expression analysis for the polycomb target genes analyzed by ChIP in (B). There is no correlation between gene expression change and scale of polycomb group protein loss, suggesting that altered gene expression is not driving PRC2 loss. RT-PCR was performed in biological triplicate and is normalized to Hprt1 expression. Error bars show SEM.

Mentions: To examine the possibility that H2AK119ub1 may play a general role in PRC2 localization and activity at normal polycomb target sites, we exploited a Ring1a−/−Ring1bfl/fl mouse ESC system, in which H2AK119ub1 can be rapidly depleted by removing the catalytic core of all PRC1 complexes (RING1A/B) through addition of the drug tamoxifen, without disrupting the cellular protein levels of PRC2 components (Endoh et al., 2008) (Figures 4A–4C). Following RING1A/B deletion, ChIP-sequencing revealed a clear loss of SUZ12, EZH2, and H3K27me3 at individual genes (Figures 4D and S2A) and at target sites genome-wide (Figure 4E and 4F). Indeed, 85% of SUZ12 and 83% of EZH2 sites showed a greater than 1.5-fold reduction in occupancy after PRC1 removal (Figures 4G and S3A). A closer inspection of SUZ12 sites defined as having a less than 1.5-fold change in PRC2, revealed that these sites do exhibit an observable loss in PRC2 binding (Figure S3B, S3C, and S3D) suggesting that most PRC2 sites are affected by loss of PRC1 activity. These effects on PRC2 occupancy were seemingly independent of high-level gene reactivation, as PRC2 reductions occurred at genes displaying small or large fluctuations in gene expression (Figures S2B and S2C).


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)

Conditional Deletion of RING1A/B Results in Depletion of H2AK119ub1, Loss of PRC2 Occupancy, and H3K27me3 at Target Genes Independently of the Magnitude of Associated Gene Expression Change, Related to Figure 4(A) Snapshots of ChIP-seq traces for RING1B, SUZ12, EZH2 and H3K27me3 in the Ring1a−/−Ring1bfl/fl cells prior to (−OHT) and following 48 hr (+OHT) of tamoxifen treatment. Three representative genes are depicted illustrating the reduction in RING1B, SUZ12, EZH2, and H3K27me3 following removal of the RING1A/B. A Bio-CAP sequencing trace is shown to indicate the location of nonmethylated DNA and a CpG Island (CGI) prediction annotation track is show as green bars under the traces. This complements examples already shown in main Figure 4.(B) ChIP-qPCR analysis for PRC2 components SUZ12, EZH2, and H3K27me3 at a series of sites showing loss of these factors in the ChIP-seq analysis in main Figure 4 after tamoxifen treatment. All ChIP experiments in (C-D) were performed in biological triplicate with error bars showing SEM.(C) Gene expression analysis for the polycomb target genes analyzed by ChIP in (B). There is no correlation between gene expression change and scale of polycomb group protein loss, suggesting that altered gene expression is not driving PRC2 loss. RT-PCR was performed in biological triplicate and is normalized to Hprt1 expression. Error bars show SEM.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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figs2: Conditional Deletion of RING1A/B Results in Depletion of H2AK119ub1, Loss of PRC2 Occupancy, and H3K27me3 at Target Genes Independently of the Magnitude of Associated Gene Expression Change, Related to Figure 4(A) Snapshots of ChIP-seq traces for RING1B, SUZ12, EZH2 and H3K27me3 in the Ring1a−/−Ring1bfl/fl cells prior to (−OHT) and following 48 hr (+OHT) of tamoxifen treatment. Three representative genes are depicted illustrating the reduction in RING1B, SUZ12, EZH2, and H3K27me3 following removal of the RING1A/B. A Bio-CAP sequencing trace is shown to indicate the location of nonmethylated DNA and a CpG Island (CGI) prediction annotation track is show as green bars under the traces. This complements examples already shown in main Figure 4.(B) ChIP-qPCR analysis for PRC2 components SUZ12, EZH2, and H3K27me3 at a series of sites showing loss of these factors in the ChIP-seq analysis in main Figure 4 after tamoxifen treatment. All ChIP experiments in (C-D) were performed in biological triplicate with error bars showing SEM.(C) Gene expression analysis for the polycomb target genes analyzed by ChIP in (B). There is no correlation between gene expression change and scale of polycomb group protein loss, suggesting that altered gene expression is not driving PRC2 loss. RT-PCR was performed in biological triplicate and is normalized to Hprt1 expression. Error bars show SEM.
Mentions: To examine the possibility that H2AK119ub1 may play a general role in PRC2 localization and activity at normal polycomb target sites, we exploited a Ring1a−/−Ring1bfl/fl mouse ESC system, in which H2AK119ub1 can be rapidly depleted by removing the catalytic core of all PRC1 complexes (RING1A/B) through addition of the drug tamoxifen, without disrupting the cellular protein levels of PRC2 components (Endoh et al., 2008) (Figures 4A–4C). Following RING1A/B deletion, ChIP-sequencing revealed a clear loss of SUZ12, EZH2, and H3K27me3 at individual genes (Figures 4D and S2A) and at target sites genome-wide (Figure 4E and 4F). Indeed, 85% of SUZ12 and 83% of EZH2 sites showed a greater than 1.5-fold reduction in occupancy after PRC1 removal (Figures 4G and S3A). A closer inspection of SUZ12 sites defined as having a less than 1.5-fold change in PRC2, revealed that these sites do exhibit an observable loss in PRC2 binding (Figure S3B, S3C, and S3D) suggesting that most PRC2 sites are affected by loss of PRC1 activity. These effects on PRC2 occupancy were seemingly independent of high-level gene reactivation, as PRC2 reductions occurred at genes displaying small or large fluctuations in gene expression (Figures S2B and S2C).

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