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A Cbx8-containing polycomb complex facilitates the transition to gene activation during ES cell differentiation.

Creppe C, Palau A, Malinverni R, Valero V, Buschbeck M - PLoS Genet. (2014)

Bottom Line: Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination.We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes.Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state.

View Article: PubMed Central - PubMed

Affiliation: Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain.

ABSTRACT
Polycomb proteins play an essential role in maintaining the repression of developmental genes in self-renewing embryonic stem cells. The exact mechanism allowing the derepression of polycomb target genes during cell differentiation remains unclear. Our project aimed to identify Cbx8 binding sites in differentiating mouse embryonic stem cells. Therefore, we used a genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq). Our analysis identified 171 high confidence peaks. By crossing our data with previously published microarray analysis, we show that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 partially impairs the transcriptional activation of these genes. Both interaction analysis, as well as chromatin immunoprecipitation experiments support the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modular and changes when embryonic stem cells commit to differentiation. We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state. As this affects several key regulatory differentiation genes this mechanism is likely to contribute to the robust execution of differentiation programs.

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Cbx8 binds to persisting K27 methylation during differentiation.(A) Trimethylation of lysine 27 of Histone H3 was analyzed by ChIP during a time course of RA-induced differentiation of ES cells. Control ChIPs with IgG are shown in the top panel on the same scale. Error bars denote s.d.; n≥3; n.d.  =  not done. (B) ChIP of H3K27me3 (+) and IgG (-). For each Cbx8 target gene values are plotted relatively to control cells. Enrichments on-target genes are plotted relative to the average enrichment on all six target genes in control cells. Error bars denote s.d.; n = 3. (C) ReChIPs using anti-K27me3 antibody and IgG as control were performed on material enriched in a primary ChIP with anti-Cbx8 antibody. Data is plotted relative to IgG. Error bars denote the variation of the mean of two independent experiments.
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pgen-1004851-g007: Cbx8 binds to persisting K27 methylation during differentiation.(A) Trimethylation of lysine 27 of Histone H3 was analyzed by ChIP during a time course of RA-induced differentiation of ES cells. Control ChIPs with IgG are shown in the top panel on the same scale. Error bars denote s.d.; n≥3; n.d.  =  not done. (B) ChIP of H3K27me3 (+) and IgG (-). For each Cbx8 target gene values are plotted relatively to control cells. Enrichments on-target genes are plotted relative to the average enrichment on all six target genes in control cells. Error bars denote s.d.; n = 3. (C) ReChIPs using anti-K27me3 antibody and IgG as control were performed on material enriched in a primary ChIP with anti-Cbx8 antibody. Data is plotted relative to IgG. Error bars denote the variation of the mean of two independent experiments.

Mentions: We then focused our attention on the mechanism that recruits Cbx8 to its target genes. An obvious possibility is that it binds directly to H3K27me3. Although activated Cbx8 target genes progressively reduced their H3K27me3 levels, in contrast to Ring1b, this reduction occurred only very slowly and even after five days of differentiation genes still retained half-maximal or even higher levels of H3K27me3 (Fig. 7A). Depletion of Cbx8 did not affect the amount of H3K27me3 detectable 3 days after RA treatment (Fig. 7B). We then analyzed by ChIP-ReChIP if Cbx8 and H3K27me3 co-occur on target genes in ES cells treated for 3 days with RA. Indeed, we found that Cbx8 and H3K27me3 co-existed on Cbx8 target genes (Fig. 7C). This suggests that also in the context of gene activation the interaction of Cbx8 with H3K27me3 is the most likely mechanism of recruitment to chromatin.


A Cbx8-containing polycomb complex facilitates the transition to gene activation during ES cell differentiation.

Creppe C, Palau A, Malinverni R, Valero V, Buschbeck M - PLoS Genet. (2014)

Cbx8 binds to persisting K27 methylation during differentiation.(A) Trimethylation of lysine 27 of Histone H3 was analyzed by ChIP during a time course of RA-induced differentiation of ES cells. Control ChIPs with IgG are shown in the top panel on the same scale. Error bars denote s.d.; n≥3; n.d.  =  not done. (B) ChIP of H3K27me3 (+) and IgG (-). For each Cbx8 target gene values are plotted relatively to control cells. Enrichments on-target genes are plotted relative to the average enrichment on all six target genes in control cells. Error bars denote s.d.; n = 3. (C) ReChIPs using anti-K27me3 antibody and IgG as control were performed on material enriched in a primary ChIP with anti-Cbx8 antibody. Data is plotted relative to IgG. Error bars denote the variation of the mean of two independent experiments.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263398&req=5

pgen-1004851-g007: Cbx8 binds to persisting K27 methylation during differentiation.(A) Trimethylation of lysine 27 of Histone H3 was analyzed by ChIP during a time course of RA-induced differentiation of ES cells. Control ChIPs with IgG are shown in the top panel on the same scale. Error bars denote s.d.; n≥3; n.d.  =  not done. (B) ChIP of H3K27me3 (+) and IgG (-). For each Cbx8 target gene values are plotted relatively to control cells. Enrichments on-target genes are plotted relative to the average enrichment on all six target genes in control cells. Error bars denote s.d.; n = 3. (C) ReChIPs using anti-K27me3 antibody and IgG as control were performed on material enriched in a primary ChIP with anti-Cbx8 antibody. Data is plotted relative to IgG. Error bars denote the variation of the mean of two independent experiments.
Mentions: We then focused our attention on the mechanism that recruits Cbx8 to its target genes. An obvious possibility is that it binds directly to H3K27me3. Although activated Cbx8 target genes progressively reduced their H3K27me3 levels, in contrast to Ring1b, this reduction occurred only very slowly and even after five days of differentiation genes still retained half-maximal or even higher levels of H3K27me3 (Fig. 7A). Depletion of Cbx8 did not affect the amount of H3K27me3 detectable 3 days after RA treatment (Fig. 7B). We then analyzed by ChIP-ReChIP if Cbx8 and H3K27me3 co-occur on target genes in ES cells treated for 3 days with RA. Indeed, we found that Cbx8 and H3K27me3 co-existed on Cbx8 target genes (Fig. 7C). This suggests that also in the context of gene activation the interaction of Cbx8 with H3K27me3 is the most likely mechanism of recruitment to chromatin.

Bottom Line: Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination.We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes.Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state.

View Article: PubMed Central - PubMed

Affiliation: Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain.

ABSTRACT
Polycomb proteins play an essential role in maintaining the repression of developmental genes in self-renewing embryonic stem cells. The exact mechanism allowing the derepression of polycomb target genes during cell differentiation remains unclear. Our project aimed to identify Cbx8 binding sites in differentiating mouse embryonic stem cells. Therefore, we used a genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq). Our analysis identified 171 high confidence peaks. By crossing our data with previously published microarray analysis, we show that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 partially impairs the transcriptional activation of these genes. Both interaction analysis, as well as chromatin immunoprecipitation experiments support the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modular and changes when embryonic stem cells commit to differentiation. We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state. As this affects several key regulatory differentiation genes this mechanism is likely to contribute to the robust execution of differentiation programs.

Show MeSH