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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 is part of a PRC1 complex and transiently bound to activated genes.(A) Time course of RA-induced differentiation of ES cells. mRNA levels and occupancy by Cbx8, Ring1b and H2A K117 mono-ubiquitination (H2Aub1) were analyzed by qRT-PCR and ChIP, respectively. Error bars denote s.d.; n≥3. (B) FLAG-affinity purifications from mock and FLAG epitope-tagged (e)-Cbx8 expressing cells treated with RA for 3 days, analyzed by LC-MS/MS. Enriched proteins were ranked according to abundance (see also S2 Table). Relative abundance to bait is indicated with +, 1–10%; ++, 10–30%; +++, 30–50%; and ++++, >50%. (C) ReChIPs using anti-Ring1b, anti-H2Aub1 antibodies 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-g005: Cbx8 is part of a PRC1 complex and transiently bound to activated genes.(A) Time course of RA-induced differentiation of ES cells. mRNA levels and occupancy by Cbx8, Ring1b and H2A K117 mono-ubiquitination (H2Aub1) were analyzed by qRT-PCR and ChIP, respectively. Error bars denote s.d.; n≥3. (B) FLAG-affinity purifications from mock and FLAG epitope-tagged (e)-Cbx8 expressing cells treated with RA for 3 days, analyzed by LC-MS/MS. Enriched proteins were ranked according to abundance (see also S2 Table). Relative abundance to bait is indicated with +, 1–10%; ++, 10–30%; +++, 30–50%; and ++++, >50%. (C) ReChIPs using anti-Ring1b, anti-H2Aub1 antibodies 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: The unexpected link between Cbx8 recruitment and gene activation prompted us to analyze whether Cbx8 acted alone, as part of a PRC1 or part of a different complex. First we compared the dynamics in occupancy of Cbx8 and Ring1b, which is the least variant component of PRC1. As already suggested by the ChIP-seq data (Fig. 4B), Ring1b was strongly enriched on target genes in self-renewing ES cells. While the recruitment of Cbx8 initially increased and peaked after three days of retinoic acid induction, the binding of Ring1b decreased progressively reaching background levels at day five of differentiation (Fig. 5A). At day five of retinoic acid induced differentiation, the Cbx8 occupancy dropped to very low levels similar to Ring1b. In contrast, H2A ubiquitination, mark set by Ring1b [23], persisted over the entire time course (Fig. 5A). In order to understand whether Cbx8 and Ring1b are acting together, we decided to identify the proteins that bind Cbx8 during ES cell differentiation. Therefore, we generated ES cells stably expressing epitope-tagged Cbx8 (Fig. 5B). Exogenous Cbx8 expressed in untreated ES cells bound to the same target genes as endogenous Cbx8 in RA-treated ES cells suggesting that the epitope does not affect its function (S4A Figure). We harvested cells at day three of differentiation which corresponds to the time point with maximal recruitment of endogenous Cbx8 to target genes (Fig. 5A). Cbx8 and interacting proteins were enriched by affinity purification, analyzed by mass spectrometry and significantly enriched proteins were ranked according to their abundance (S2 Table). After the bait protein Cbx8, the top five ranked proteins were the PRC1 subunits Ring1a/b, Phc2 and the Pcgf proteins Mel18 and Bmi1. Three additional PRC1 subunits were found in lower abundance (Fig.5B). Next, we tested whether Ring1b and H2A ubiquitination co-occur with Cbx8 on genes. Therefore, we have used anti-Cbx8 antibody-enriched chromatin as input material for a secondary ChIP with IgG, anti-Ring1b and anti-ubiquitinated H2A antibody. As shown in Fig. 5C we could detect co-enrichment on the target genes Sox9, Nkx6-1, Lhx2 and Gata2 but not on the non-target genes Oct4, Rpo or Gapdh. Taken together, these results suggested that Cbx8 acts as part of a PRC1 complex.


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 is part of a PRC1 complex and transiently bound to activated genes.(A) Time course of RA-induced differentiation of ES cells. mRNA levels and occupancy by Cbx8, Ring1b and H2A K117 mono-ubiquitination (H2Aub1) were analyzed by qRT-PCR and ChIP, respectively. Error bars denote s.d.; n≥3. (B) FLAG-affinity purifications from mock and FLAG epitope-tagged (e)-Cbx8 expressing cells treated with RA for 3 days, analyzed by LC-MS/MS. Enriched proteins were ranked according to abundance (see also S2 Table). Relative abundance to bait is indicated with +, 1–10%; ++, 10–30%; +++, 30–50%; and ++++, >50%. (C) ReChIPs using anti-Ring1b, anti-H2Aub1 antibodies 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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getmorefigures.php?uid=PMC4263398&req=5

pgen-1004851-g005: Cbx8 is part of a PRC1 complex and transiently bound to activated genes.(A) Time course of RA-induced differentiation of ES cells. mRNA levels and occupancy by Cbx8, Ring1b and H2A K117 mono-ubiquitination (H2Aub1) were analyzed by qRT-PCR and ChIP, respectively. Error bars denote s.d.; n≥3. (B) FLAG-affinity purifications from mock and FLAG epitope-tagged (e)-Cbx8 expressing cells treated with RA for 3 days, analyzed by LC-MS/MS. Enriched proteins were ranked according to abundance (see also S2 Table). Relative abundance to bait is indicated with +, 1–10%; ++, 10–30%; +++, 30–50%; and ++++, >50%. (C) ReChIPs using anti-Ring1b, anti-H2Aub1 antibodies 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: The unexpected link between Cbx8 recruitment and gene activation prompted us to analyze whether Cbx8 acted alone, as part of a PRC1 or part of a different complex. First we compared the dynamics in occupancy of Cbx8 and Ring1b, which is the least variant component of PRC1. As already suggested by the ChIP-seq data (Fig. 4B), Ring1b was strongly enriched on target genes in self-renewing ES cells. While the recruitment of Cbx8 initially increased and peaked after three days of retinoic acid induction, the binding of Ring1b decreased progressively reaching background levels at day five of differentiation (Fig. 5A). At day five of retinoic acid induced differentiation, the Cbx8 occupancy dropped to very low levels similar to Ring1b. In contrast, H2A ubiquitination, mark set by Ring1b [23], persisted over the entire time course (Fig. 5A). In order to understand whether Cbx8 and Ring1b are acting together, we decided to identify the proteins that bind Cbx8 during ES cell differentiation. Therefore, we generated ES cells stably expressing epitope-tagged Cbx8 (Fig. 5B). Exogenous Cbx8 expressed in untreated ES cells bound to the same target genes as endogenous Cbx8 in RA-treated ES cells suggesting that the epitope does not affect its function (S4A Figure). We harvested cells at day three of differentiation which corresponds to the time point with maximal recruitment of endogenous Cbx8 to target genes (Fig. 5A). Cbx8 and interacting proteins were enriched by affinity purification, analyzed by mass spectrometry and significantly enriched proteins were ranked according to their abundance (S2 Table). After the bait protein Cbx8, the top five ranked proteins were the PRC1 subunits Ring1a/b, Phc2 and the Pcgf proteins Mel18 and Bmi1. Three additional PRC1 subunits were found in lower abundance (Fig.5B). Next, we tested whether Ring1b and H2A ubiquitination co-occur with Cbx8 on genes. Therefore, we have used anti-Cbx8 antibody-enriched chromatin as input material for a secondary ChIP with IgG, anti-Ring1b and anti-ubiquitinated H2A antibody. As shown in Fig. 5C we could detect co-enrichment on the target genes Sox9, Nkx6-1, Lhx2 and Gata2 but not on the non-target genes Oct4, Rpo or Gapdh. Taken together, these results suggested that Cbx8 acts as part of a PRC1 complex.

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