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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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Loss of Cbx8 is partially compensated by Cbx7.The presence of Ring1b, Cbx7 and H2A ubiquitination (H2Aub1) in control cells (sh Scr) and after shRNA-mediated knockdown of Cbx8 using two independent hairpins was analyzed by ChIP after 3 days RA treatment. IgG was used as background control and is indicated by minus signs. 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; *, p<0.05. (compared to control cells).
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pgen-1004851-g006: Loss of Cbx8 is partially compensated by Cbx7.The presence of Ring1b, Cbx7 and H2A ubiquitination (H2Aub1) in control cells (sh Scr) and after shRNA-mediated knockdown of Cbx8 using two independent hairpins was analyzed by ChIP after 3 days RA treatment. IgG was used as background control and is indicated by minus signs. 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; *, p<0.05. (compared to control cells).

Mentions: To further support this, we have analyzed the occupancy of Cbx8 target genes by Ring1b, Cbx7 and H2A ubiquitination in RA-treated cells after Cbx8 knockdown. The resulting reduction of Cbx8 occupancy (Fig. 3D) correlated with a small but consistent reduction of Ring1b on several Cbx8 target genes without affecting non-target genes (Fig. 6). Notably, global Ring1b protein levels were not affected (S4B Figure). However, we observed an increased incorporation of Cbx7 that could partially compensate for Cbx8 loss (Fig. 6). This was not the consequence of an upregulation of Cbx7 expression as knockdown of Cbx8 did not affect the mRNA levels of Cbx7 or other Cbx proteins (S4C Figure). Moreover we found that Cbx8 loss did not affect the levels of H2A ubiquitination on its target genes (Fig. 6). In order to address the question whether Cbx8-containing PRC1 on activated genes is repressive or activating, we stably interfered with the expression of Ring1b, the least variant component of PRC1. When analyzing Ring1b knockdown cell after 3 days of RA treatment we did not observe any compensation by Ring1a but a slight increase in Cbx8 mRNA (S4D Figure). Under these conditions the Cbx8 target gene Gata2 was further upregulated while other target genes such as Nkx6-1 and Sox17 were less activated (S4D Figure). These results are difficult to interpret as knockdown of Ring1b interferes with all PRC1 complexes present prior and after RA treatment.


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)

Loss of Cbx8 is partially compensated by Cbx7.The presence of Ring1b, Cbx7 and H2A ubiquitination (H2Aub1) in control cells (sh Scr) and after shRNA-mediated knockdown of Cbx8 using two independent hairpins was analyzed by ChIP after 3 days RA treatment. IgG was used as background control and is indicated by minus signs. 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; *, p<0.05. (compared to control cells).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004851-g006: Loss of Cbx8 is partially compensated by Cbx7.The presence of Ring1b, Cbx7 and H2A ubiquitination (H2Aub1) in control cells (sh Scr) and after shRNA-mediated knockdown of Cbx8 using two independent hairpins was analyzed by ChIP after 3 days RA treatment. IgG was used as background control and is indicated by minus signs. 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; *, p<0.05. (compared to control cells).
Mentions: To further support this, we have analyzed the occupancy of Cbx8 target genes by Ring1b, Cbx7 and H2A ubiquitination in RA-treated cells after Cbx8 knockdown. The resulting reduction of Cbx8 occupancy (Fig. 3D) correlated with a small but consistent reduction of Ring1b on several Cbx8 target genes without affecting non-target genes (Fig. 6). Notably, global Ring1b protein levels were not affected (S4B Figure). However, we observed an increased incorporation of Cbx7 that could partially compensate for Cbx8 loss (Fig. 6). This was not the consequence of an upregulation of Cbx7 expression as knockdown of Cbx8 did not affect the mRNA levels of Cbx7 or other Cbx proteins (S4C Figure). Moreover we found that Cbx8 loss did not affect the levels of H2A ubiquitination on its target genes (Fig. 6). In order to address the question whether Cbx8-containing PRC1 on activated genes is repressive or activating, we stably interfered with the expression of Ring1b, the least variant component of PRC1. When analyzing Ring1b knockdown cell after 3 days of RA treatment we did not observe any compensation by Ring1a but a slight increase in Cbx8 mRNA (S4D Figure). Under these conditions the Cbx8 target gene Gata2 was further upregulated while other target genes such as Nkx6-1 and Sox17 were less activated (S4D Figure). These results are difficult to interpret as knockdown of Ring1b interferes with all PRC1 complexes present prior and after RA treatment.

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