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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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PRC1 Has a Genome-wide-Role in PRC2 Recruitment and Polycomb Domain Formation at Target Sites in Mouse ESCs, Related to Figure 4(A) A Venn diagram showing the overlap of RING1B (light blue) and EZH2 (light pink) peaks including a further segregation of EZH2-bound regions that show a greater than 1.5-fold change in EZH2 occupancy (ΔEZH2, dark pink) after RING1A/B deletion. The majority of EZH2-bound locations show changes in EZH2 signal following removal of RING1A/B, and most of the changes are restricted to sites associated with RING1B peaks.(B) A metaplot of RING1B and SUZ12 ChIP-seq data at RING1B peaks that do not overlap with SUZ12 sites identified by peak calling. This indicates that these regions exhibit SUZ12 enrichment but are likely below the enrichment level required for peak detection.(C) A metaplot of RING1B and SUZ12 ChIP-seq data at SUZ12 peaks that do not overlap with RING1B sites identified by peak calling. This indicates that these regions exhibit RING1B enrichment but are likely below the enrichment level required for peak detection.(D) A metaplot of SUZ12 ChIP-seq data in the Ring1a−/−Ring1bfl/flb cells at SUZ12 peaks exhibiting a less than 1.5-fold reduction in SUZ12 signal following tamoxifen treatment. Importantly these sites still show reduction in SUZ12 signal suggesting that loss of RING1A/B affects PRC2 occupancy at most sites.(E) A box and whisker plot indicating the Log2 fold change in polycomb factors at SUZ12-bound sites with and without REST following loss of RING1A/B. This indicates that loss of PRC2 occurs at REST-bound sites in the absence of PRC1.(F) 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 at sites previously reported to rely on the Meg3 long noncoding RNA for PRC2 targeting. In all cases we observe appreciable loss of PRC2 following RING1A/B deletion indicating that Meg3-dependent targeting is not sufficient to maintain normal levels of PRC2 at these sites.(G) A box and whisker plot indicating the Log2 fold change in PRC2 factors and H3K27me3 at sites considered to be bivalent. Bivalent sites appear to have slightly larger fold changes in PRC2 occupancy following RING1A/B deletion.(H) A scatter plot comparing the fold change of RING1B and EZH2 at EZH2 peaks. This indicates a clear correlation between the magnitude in RING1B and EZH2 change suggesting these changes may be mechanistically linked.
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figs3: PRC1 Has a Genome-wide-Role in PRC2 Recruitment and Polycomb Domain Formation at Target Sites in Mouse ESCs, Related to Figure 4(A) A Venn diagram showing the overlap of RING1B (light blue) and EZH2 (light pink) peaks including a further segregation of EZH2-bound regions that show a greater than 1.5-fold change in EZH2 occupancy (ΔEZH2, dark pink) after RING1A/B deletion. The majority of EZH2-bound locations show changes in EZH2 signal following removal of RING1A/B, and most of the changes are restricted to sites associated with RING1B peaks.(B) A metaplot of RING1B and SUZ12 ChIP-seq data at RING1B peaks that do not overlap with SUZ12 sites identified by peak calling. This indicates that these regions exhibit SUZ12 enrichment but are likely below the enrichment level required for peak detection.(C) A metaplot of RING1B and SUZ12 ChIP-seq data at SUZ12 peaks that do not overlap with RING1B sites identified by peak calling. This indicates that these regions exhibit RING1B enrichment but are likely below the enrichment level required for peak detection.(D) A metaplot of SUZ12 ChIP-seq data in the Ring1a−/−Ring1bfl/flb cells at SUZ12 peaks exhibiting a less than 1.5-fold reduction in SUZ12 signal following tamoxifen treatment. Importantly these sites still show reduction in SUZ12 signal suggesting that loss of RING1A/B affects PRC2 occupancy at most sites.(E) A box and whisker plot indicating the Log2 fold change in polycomb factors at SUZ12-bound sites with and without REST following loss of RING1A/B. This indicates that loss of PRC2 occurs at REST-bound sites in the absence of PRC1.(F) 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 at sites previously reported to rely on the Meg3 long noncoding RNA for PRC2 targeting. In all cases we observe appreciable loss of PRC2 following RING1A/B deletion indicating that Meg3-dependent targeting is not sufficient to maintain normal levels of PRC2 at these sites.(G) A box and whisker plot indicating the Log2 fold change in PRC2 factors and H3K27me3 at sites considered to be bivalent. Bivalent sites appear to have slightly larger fold changes in PRC2 occupancy following RING1A/B deletion.(H) A scatter plot comparing the fold change of RING1B and EZH2 at EZH2 peaks. This indicates a clear correlation between the magnitude in RING1B and EZH2 change suggesting these changes may be mechanistically linked.

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)

PRC1 Has a Genome-wide-Role in PRC2 Recruitment and Polycomb Domain Formation at Target Sites in Mouse ESCs, Related to Figure 4(A) A Venn diagram showing the overlap of RING1B (light blue) and EZH2 (light pink) peaks including a further segregation of EZH2-bound regions that show a greater than 1.5-fold change in EZH2 occupancy (ΔEZH2, dark pink) after RING1A/B deletion. The majority of EZH2-bound locations show changes in EZH2 signal following removal of RING1A/B, and most of the changes are restricted to sites associated with RING1B peaks.(B) A metaplot of RING1B and SUZ12 ChIP-seq data at RING1B peaks that do not overlap with SUZ12 sites identified by peak calling. This indicates that these regions exhibit SUZ12 enrichment but are likely below the enrichment level required for peak detection.(C) A metaplot of RING1B and SUZ12 ChIP-seq data at SUZ12 peaks that do not overlap with RING1B sites identified by peak calling. This indicates that these regions exhibit RING1B enrichment but are likely below the enrichment level required for peak detection.(D) A metaplot of SUZ12 ChIP-seq data in the Ring1a−/−Ring1bfl/flb cells at SUZ12 peaks exhibiting a less than 1.5-fold reduction in SUZ12 signal following tamoxifen treatment. Importantly these sites still show reduction in SUZ12 signal suggesting that loss of RING1A/B affects PRC2 occupancy at most sites.(E) A box and whisker plot indicating the Log2 fold change in polycomb factors at SUZ12-bound sites with and without REST following loss of RING1A/B. This indicates that loss of PRC2 occurs at REST-bound sites in the absence of PRC1.(F) 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 at sites previously reported to rely on the Meg3 long noncoding RNA for PRC2 targeting. In all cases we observe appreciable loss of PRC2 following RING1A/B deletion indicating that Meg3-dependent targeting is not sufficient to maintain normal levels of PRC2 at these sites.(G) A box and whisker plot indicating the Log2 fold change in PRC2 factors and H3K27me3 at sites considered to be bivalent. Bivalent sites appear to have slightly larger fold changes in PRC2 occupancy following RING1A/B deletion.(H) A scatter plot comparing the fold change of RING1B and EZH2 at EZH2 peaks. This indicates a clear correlation between the magnitude in RING1B and EZH2 change suggesting these changes may be mechanistically linked.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048464&req=5

figs3: PRC1 Has a Genome-wide-Role in PRC2 Recruitment and Polycomb Domain Formation at Target Sites in Mouse ESCs, Related to Figure 4(A) A Venn diagram showing the overlap of RING1B (light blue) and EZH2 (light pink) peaks including a further segregation of EZH2-bound regions that show a greater than 1.5-fold change in EZH2 occupancy (ΔEZH2, dark pink) after RING1A/B deletion. The majority of EZH2-bound locations show changes in EZH2 signal following removal of RING1A/B, and most of the changes are restricted to sites associated with RING1B peaks.(B) A metaplot of RING1B and SUZ12 ChIP-seq data at RING1B peaks that do not overlap with SUZ12 sites identified by peak calling. This indicates that these regions exhibit SUZ12 enrichment but are likely below the enrichment level required for peak detection.(C) A metaplot of RING1B and SUZ12 ChIP-seq data at SUZ12 peaks that do not overlap with RING1B sites identified by peak calling. This indicates that these regions exhibit RING1B enrichment but are likely below the enrichment level required for peak detection.(D) A metaplot of SUZ12 ChIP-seq data in the Ring1a−/−Ring1bfl/flb cells at SUZ12 peaks exhibiting a less than 1.5-fold reduction in SUZ12 signal following tamoxifen treatment. Importantly these sites still show reduction in SUZ12 signal suggesting that loss of RING1A/B affects PRC2 occupancy at most sites.(E) A box and whisker plot indicating the Log2 fold change in polycomb factors at SUZ12-bound sites with and without REST following loss of RING1A/B. This indicates that loss of PRC2 occurs at REST-bound sites in the absence of PRC1.(F) 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 at sites previously reported to rely on the Meg3 long noncoding RNA for PRC2 targeting. In all cases we observe appreciable loss of PRC2 following RING1A/B deletion indicating that Meg3-dependent targeting is not sufficient to maintain normal levels of PRC2 at these sites.(G) A box and whisker plot indicating the Log2 fold change in PRC2 factors and H3K27me3 at sites considered to be bivalent. Bivalent sites appear to have slightly larger fold changes in PRC2 occupancy following RING1A/B deletion.(H) A scatter plot comparing the fold change of RING1B and EZH2 at EZH2 peaks. This indicates a clear correlation between the magnitude in RING1B and EZH2 change suggesting these changes may be mechanistically linked.
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
Related in: MedlinePlus