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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 target genes are PRC1 target genes in self-renewing ES cells.(A) Cbx8-enriched GO categories are visualized using REVIGO [48] which allows to cluster GO according to their similarity in a semantic space. Only GO categories with an adjusted P-value of 0.001 or less are shown using “medium” for the allowed semantic similarity. (B) ChIP-seq profiles of H3K27me3, Ring1b, Cbx7 and Cbx8 in ES cells in self-renewing conditions and of Cbx8 and IgG in RA-treated ES cells are shown using the UCSC genome browser. (C) Venn diagram showing the overlap of Cbx8 target genes in RA-treated cells with those of Cbx7 in untreated, self-renewing ES cells.
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pgen-1004851-g004: Cbx8 target genes are PRC1 target genes in self-renewing ES cells.(A) Cbx8-enriched GO categories are visualized using REVIGO [48] which allows to cluster GO according to their similarity in a semantic space. Only GO categories with an adjusted P-value of 0.001 or less are shown using “medium” for the allowed semantic similarity. (B) ChIP-seq profiles of H3K27me3, Ring1b, Cbx7 and Cbx8 in ES cells in self-renewing conditions and of Cbx8 and IgG in RA-treated ES cells are shown using the UCSC genome browser. (C) Venn diagram showing the overlap of Cbx8 target genes in RA-treated cells with those of Cbx7 in untreated, self-renewing ES cells.

Mentions: To study the functional importance of Cbx8 for the activation of differentiation genes, we used lentiviral vectors expressing two different short hairpin RNAs (shRNA) directed specifically against the Cbx8 transcript. After selection stably transduced cells were used for the study. Both shRNAs efficiently repressed the expression of Cbx8 on both the mRNA and protein levels in ES cells treated with RA (Fig. 3A,B). The activation of upregulated Cbx8 target genes was significantly decreased in cells depleted for Cbx8 (Fig. 3C). Among the Cbx8-sensitive genes were several pivotal regulators of differentiation processes such as Sox9 and Nkx6-1, that have been shown to be important transcription factors required for normal brain development [21], [22]. The reduction of Cbx8 occupancy was similarly efficient on up- as well as downregulated genes (Fig. 3D), however, the reduction in Cbx8 levels didn't affect the repression of its target genes Prdm14 and Otx2 (Fig. 3C). Importantly, the non-target genes Oct4 and Nanog, which encode the regulators of pluripotency, were similarly repressed in Cbx8 deficient and control cells (Fig. 3C,D). Plotting enriched gene ontologies according to their similarity in a semantic space illustrates a clear overrepresentation of both transcriptional and differentiation regulators (Fig. 4A), which are the classical categories of Polycomb target genes in self-renewing ES cells [12]. Gene ontologies related to neuronal development were preferentially enriched in the subgroup of activated Cbx8 genes but not those target genes that did not show any change in gene expression (S3A Figure). Downregulated genes were not sufficient in number to yield a result in gene ontology analysis. We compared our genome wide Cbx8 binding profile in differentiating ES cells with published ChIP-seq data obtained from self-renewing ES cells [17]. The binding of Cbx8 in RA-treated differentiating ES cells mirrors the binding of PRC1 proteins Ring1b and Cbx7 within H3K27me3 domains in untreated self-renewing ES cells (Fig. 4B). As shown in Fig. 4C, this holds true for the vast majority of Cbx8 binding sites in RA-treated ES cells as 133/171 overlapped with sites bound by Cbx7 in self-renewing ES cells. Similar overlaps were observed with Ring1b and H3K27me3 (S3B Figure).


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 target genes are PRC1 target genes in self-renewing ES cells.(A) Cbx8-enriched GO categories are visualized using REVIGO [48] which allows to cluster GO according to their similarity in a semantic space. Only GO categories with an adjusted P-value of 0.001 or less are shown using “medium” for the allowed semantic similarity. (B) ChIP-seq profiles of H3K27me3, Ring1b, Cbx7 and Cbx8 in ES cells in self-renewing conditions and of Cbx8 and IgG in RA-treated ES cells are shown using the UCSC genome browser. (C) Venn diagram showing the overlap of Cbx8 target genes in RA-treated cells with those of Cbx7 in untreated, self-renewing ES cells.
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pgen-1004851-g004: Cbx8 target genes are PRC1 target genes in self-renewing ES cells.(A) Cbx8-enriched GO categories are visualized using REVIGO [48] which allows to cluster GO according to their similarity in a semantic space. Only GO categories with an adjusted P-value of 0.001 or less are shown using “medium” for the allowed semantic similarity. (B) ChIP-seq profiles of H3K27me3, Ring1b, Cbx7 and Cbx8 in ES cells in self-renewing conditions and of Cbx8 and IgG in RA-treated ES cells are shown using the UCSC genome browser. (C) Venn diagram showing the overlap of Cbx8 target genes in RA-treated cells with those of Cbx7 in untreated, self-renewing ES cells.
Mentions: To study the functional importance of Cbx8 for the activation of differentiation genes, we used lentiviral vectors expressing two different short hairpin RNAs (shRNA) directed specifically against the Cbx8 transcript. After selection stably transduced cells were used for the study. Both shRNAs efficiently repressed the expression of Cbx8 on both the mRNA and protein levels in ES cells treated with RA (Fig. 3A,B). The activation of upregulated Cbx8 target genes was significantly decreased in cells depleted for Cbx8 (Fig. 3C). Among the Cbx8-sensitive genes were several pivotal regulators of differentiation processes such as Sox9 and Nkx6-1, that have been shown to be important transcription factors required for normal brain development [21], [22]. The reduction of Cbx8 occupancy was similarly efficient on up- as well as downregulated genes (Fig. 3D), however, the reduction in Cbx8 levels didn't affect the repression of its target genes Prdm14 and Otx2 (Fig. 3C). Importantly, the non-target genes Oct4 and Nanog, which encode the regulators of pluripotency, were similarly repressed in Cbx8 deficient and control cells (Fig. 3C,D). Plotting enriched gene ontologies according to their similarity in a semantic space illustrates a clear overrepresentation of both transcriptional and differentiation regulators (Fig. 4A), which are the classical categories of Polycomb target genes in self-renewing ES cells [12]. Gene ontologies related to neuronal development were preferentially enriched in the subgroup of activated Cbx8 genes but not those target genes that did not show any change in gene expression (S3A Figure). Downregulated genes were not sufficient in number to yield a result in gene ontology analysis. We compared our genome wide Cbx8 binding profile in differentiating ES cells with published ChIP-seq data obtained from self-renewing ES cells [17]. The binding of Cbx8 in RA-treated differentiating ES cells mirrors the binding of PRC1 proteins Ring1b and Cbx7 within H3K27me3 domains in untreated self-renewing ES cells (Fig. 4B). As shown in Fig. 4C, this holds true for the vast majority of Cbx8 binding sites in RA-treated ES cells as 133/171 overlapped with sites bound by Cbx7 in self-renewing ES cells. Similar overlaps were observed with Ring1b and H3K27me3 (S3B Figure).

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