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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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The exchange of Cbx7 for Cbx8 is required but not sufficient to promote the differentiation of mES cells.(A-D) Parental ES cells treated with RA for three days or left untreated were compared to untreated ES cells expressing exogenous Cbx8 and differentiating ES treated with RA for three days and expressing exogenous Cbx7. Consistent color coding of cells and treatments is as indicated in A. (A) The expression levels of endogenous and exogenous epitope (e)-tagged Cbx proteins is shown by Western blot of lysates from stable transfected ES cells (e-Cbx7 and e-Cbx8, respectively) and parental control cells. Exogenous and endogenous protein bands are marked by one or two asterisks, respectively. Cells were treated with RA for three days as indicated. Histone H3 was used as loading control. (B) The occupancy of Cbx7 and Cbx8 on selected genes was analyzed by ChIP in untreated ES cells overexpressing Cbx8 (red bars) and parental control cells treated with RA for three days (black bars) or left untreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximal enrichment. IgG is shown relative to Cbx8 ChIP. Error bars denote s.d.; n = 3; * = p<0.05. (C) Relative mRNA levels of the same genes as in B measured by qRT-PCR are shown. Error bars denote s.d.; n = 3; * = p<0.05; n.s.  =  not significant. (D) ChIP of Cbx8 or Cbx7 in Cbx7-overexpressing cells treated with RA for three days (blue bars) and control cells treated with RA (black bars) or left untreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximal enrichment. IgG is shown relative to Cbx8 ChIP. Error bars denote s.d.; n = 3; * = p<0.05. (E) Cartoon illustrating our finding that Cbx8-containing PRC1 complexes replace Cbx7-containing PRC1 complexes during the initial activation of differentiation genes. During a metastable transition state binding of Cbx8-containing PRC1 co-occurs with the presence of both repressive marks such as H3K27me3 and active marks such as H3K36me3. Prolonged activation results in loss of Cbx8-containing PRC1 that precedes the removal of H3K27me3.
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pgen-1004851-g008: The exchange of Cbx7 for Cbx8 is required but not sufficient to promote the differentiation of mES cells.(A-D) Parental ES cells treated with RA for three days or left untreated were compared to untreated ES cells expressing exogenous Cbx8 and differentiating ES treated with RA for three days and expressing exogenous Cbx7. Consistent color coding of cells and treatments is as indicated in A. (A) The expression levels of endogenous and exogenous epitope (e)-tagged Cbx proteins is shown by Western blot of lysates from stable transfected ES cells (e-Cbx7 and e-Cbx8, respectively) and parental control cells. Exogenous and endogenous protein bands are marked by one or two asterisks, respectively. Cells were treated with RA for three days as indicated. Histone H3 was used as loading control. (B) The occupancy of Cbx7 and Cbx8 on selected genes was analyzed by ChIP in untreated ES cells overexpressing Cbx8 (red bars) and parental control cells treated with RA for three days (black bars) or left untreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximal enrichment. IgG is shown relative to Cbx8 ChIP. Error bars denote s.d.; n = 3; * = p<0.05. (C) Relative mRNA levels of the same genes as in B measured by qRT-PCR are shown. Error bars denote s.d.; n = 3; * = p<0.05; n.s.  =  not significant. (D) ChIP of Cbx8 or Cbx7 in Cbx7-overexpressing cells treated with RA for three days (blue bars) and control cells treated with RA (black bars) or left untreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximal enrichment. IgG is shown relative to Cbx8 ChIP. Error bars denote s.d.; n = 3; * = p<0.05. (E) Cartoon illustrating our finding that Cbx8-containing PRC1 complexes replace Cbx7-containing PRC1 complexes during the initial activation of differentiation genes. During a metastable transition state binding of Cbx8-containing PRC1 co-occurs with the presence of both repressive marks such as H3K27me3 and active marks such as H3K36me3. Prolonged activation results in loss of Cbx8-containing PRC1 that precedes the removal of H3K27me3.

Mentions: Cbx7 and Cbx8 are expressed in an almost mutually exclusive manner in self-renewing and differentiating ES cells, respectively [16], [17]. To further gain additional insight into the functional relevance of the switch from Cbx7 to Cbx8 on target genes, we generated mouse embryonic stem cells that stably express exogenous Cbx8 and analyzed them in self-renewing conditions while cells expressing exogenous Cbx7 were analyzed in differentiating cells after 3 days of retinoic acid induction (Fig. 8A). When expressed in self-renewing cells, exogenous Cbx8 was able to efficiently outcompete Cbx7 for its target genes (Fig. 8B). The enforced recruitment of exogenous Cbx8 achieved under these conditions was two-to-three-fold higher than that observed in differentiating cells for the endogenous protein (Fig. 8B), although, this did not affect the low expression level of these genes (Fig. 8C). In the converse experiment during differentiation, Cbx7 overexpression significantly reduced the activation of Cbx8 target genes (Fig. 8C). Although enrichment of overexpressed Cbx7 on target genes did not reach the levels of the endogenous protein in self-renewing cells, it resulted in an efficient displacement of Cbx8 (Fig. 8D).


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)

The exchange of Cbx7 for Cbx8 is required but not sufficient to promote the differentiation of mES cells.(A-D) Parental ES cells treated with RA for three days or left untreated were compared to untreated ES cells expressing exogenous Cbx8 and differentiating ES treated with RA for three days and expressing exogenous Cbx7. Consistent color coding of cells and treatments is as indicated in A. (A) The expression levels of endogenous and exogenous epitope (e)-tagged Cbx proteins is shown by Western blot of lysates from stable transfected ES cells (e-Cbx7 and e-Cbx8, respectively) and parental control cells. Exogenous and endogenous protein bands are marked by one or two asterisks, respectively. Cells were treated with RA for three days as indicated. Histone H3 was used as loading control. (B) The occupancy of Cbx7 and Cbx8 on selected genes was analyzed by ChIP in untreated ES cells overexpressing Cbx8 (red bars) and parental control cells treated with RA for three days (black bars) or left untreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximal enrichment. IgG is shown relative to Cbx8 ChIP. Error bars denote s.d.; n = 3; * = p<0.05. (C) Relative mRNA levels of the same genes as in B measured by qRT-PCR are shown. Error bars denote s.d.; n = 3; * = p<0.05; n.s.  =  not significant. (D) ChIP of Cbx8 or Cbx7 in Cbx7-overexpressing cells treated with RA for three days (blue bars) and control cells treated with RA (black bars) or left untreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximal enrichment. IgG is shown relative to Cbx8 ChIP. Error bars denote s.d.; n = 3; * = p<0.05. (E) Cartoon illustrating our finding that Cbx8-containing PRC1 complexes replace Cbx7-containing PRC1 complexes during the initial activation of differentiation genes. During a metastable transition state binding of Cbx8-containing PRC1 co-occurs with the presence of both repressive marks such as H3K27me3 and active marks such as H3K36me3. Prolonged activation results in loss of Cbx8-containing PRC1 that precedes the removal of H3K27me3.
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pgen-1004851-g008: The exchange of Cbx7 for Cbx8 is required but not sufficient to promote the differentiation of mES cells.(A-D) Parental ES cells treated with RA for three days or left untreated were compared to untreated ES cells expressing exogenous Cbx8 and differentiating ES treated with RA for three days and expressing exogenous Cbx7. Consistent color coding of cells and treatments is as indicated in A. (A) The expression levels of endogenous and exogenous epitope (e)-tagged Cbx proteins is shown by Western blot of lysates from stable transfected ES cells (e-Cbx7 and e-Cbx8, respectively) and parental control cells. Exogenous and endogenous protein bands are marked by one or two asterisks, respectively. Cells were treated with RA for three days as indicated. Histone H3 was used as loading control. (B) The occupancy of Cbx7 and Cbx8 on selected genes was analyzed by ChIP in untreated ES cells overexpressing Cbx8 (red bars) and parental control cells treated with RA for three days (black bars) or left untreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximal enrichment. IgG is shown relative to Cbx8 ChIP. Error bars denote s.d.; n = 3; * = p<0.05. (C) Relative mRNA levels of the same genes as in B measured by qRT-PCR are shown. Error bars denote s.d.; n = 3; * = p<0.05; n.s.  =  not significant. (D) ChIP of Cbx8 or Cbx7 in Cbx7-overexpressing cells treated with RA for three days (blue bars) and control cells treated with RA (black bars) or left untreated (grey bars). ChIP-Input ratios for each gene and antibody are shown relative to the maximal enrichment. IgG is shown relative to Cbx8 ChIP. Error bars denote s.d.; n = 3; * = p<0.05. (E) Cartoon illustrating our finding that Cbx8-containing PRC1 complexes replace Cbx7-containing PRC1 complexes during the initial activation of differentiation genes. During a metastable transition state binding of Cbx8-containing PRC1 co-occurs with the presence of both repressive marks such as H3K27me3 and active marks such as H3K36me3. Prolonged activation results in loss of Cbx8-containing PRC1 that precedes the removal of H3K27me3.
Mentions: Cbx7 and Cbx8 are expressed in an almost mutually exclusive manner in self-renewing and differentiating ES cells, respectively [16], [17]. To further gain additional insight into the functional relevance of the switch from Cbx7 to Cbx8 on target genes, we generated mouse embryonic stem cells that stably express exogenous Cbx8 and analyzed them in self-renewing conditions while cells expressing exogenous Cbx7 were analyzed in differentiating cells after 3 days of retinoic acid induction (Fig. 8A). When expressed in self-renewing cells, exogenous Cbx8 was able to efficiently outcompete Cbx7 for its target genes (Fig. 8B). The enforced recruitment of exogenous Cbx8 achieved under these conditions was two-to-three-fold higher than that observed in differentiating cells for the endogenous protein (Fig. 8B), although, this did not affect the low expression level of these genes (Fig. 8C). In the converse experiment during differentiation, Cbx7 overexpression significantly reduced the activation of Cbx8 target genes (Fig. 8C). Although enrichment of overexpressed Cbx7 on target genes did not reach the levels of the endogenous protein in self-renewing cells, it resulted in an efficient displacement of Cbx8 (Fig. 8D).

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
Related in: MedlinePlus