H2A.Z.1 mono-ubiquitylation antagonizes BRD2 to maintain poised chromatin in ESCs
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Laurie Boyer
PROVIDER: E-GEOD-53208 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in ESCs to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 mono-ubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1K3R3). We show that H2A.Z.1K3R3 is properly incorporated at target promoters in murine ESCs (mESCs), however, loss of mono-ubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and to faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member Brd2, are enriched in H2A.Z.1 chromatin. We further show that Brd2 is gained at de-repressed promoters in H2A.Z.1K3R3 mESCs whereas Brd2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and Brd2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs. RNA-Seq analysis on mouse embryonic stem cells harboring H2A.Z or H2A.Z.K3R3 (3 C-terminal lysines mutated to arginines) tagged with YFP, in the presence of a knockdown hairpin targeting the endogenous H2A.Z transcript.
2016-01-21 | E-GEOD-53207 | biostudies-arrayexpress
Project description:The H2A variant H2AZ is essential for embryonic development and for proper execution of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions in H2AZ are likely key for its functional specialization, but we know little about how these differences contribute to chromatin regulation. Here, we show that the extended acidic patch, specifically the three divergent residues in the C-terminal docking domain, is necessary for lineage commitment during ESC differentiation and proper execution of gene expression programs during ESC differentiation. Surprisingly, disruption of the acidic patch domain has a distinct consequence on cellular specification compared to H2AZ depletion. This is consistent with differences in gene expression profiles of H2AZ M-bM-^@M-^Sdepleted and acidic patch (AP) mutant ESCs during early lineage commitment. Interestingly, the distinct consequence of AP mutant expression on gene regulation is coincidence with an altered destabilized chromatin state and high chromatin mobility dependent on active transcription. Collectively, our data shows that the divergent residues within the acidic patch domain are key structural determinants of H2AZ function and links chromatin structure and dynamics with gene regulation and cell fate specification. H2AZ extended acidic patch was mutated, or H2AZ was KD in mouse embryonic stem cells and RNA-Seq analysis was performed on the resulting cultures. Characterization of H2AZ-WT and -AP3-mutant binding specificities were performed by ChIP-Seq.
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Project description:The H2A variant H2AZ is essential for embryonic development and for proper execution of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions in H2AZ are likely key for its functional specialization, but we know little about how these differences contribute to chromatin regulation. Here, we show that the extended acidic patch, specifically the three divergent residues in the C-terminal docking domain, is necessary for lineage commitment during ESC differentiation and proper execution of gene expression programs during ESC differentiation. Surprisingly, disruption of the acidic patch domain has a distinct consequence on cellular specification compared to H2AZ depletion. This is consistent with differences in gene expression profiles of H2AZ M-bM-^@M-^Sdepleted and acidic patch (AP) mutant ESCs during early lineage commitment. Interestingly, the distinct consequence of AP mutant expression on gene regulation is coincidence with an altered destabilized chromatin state and high chromatin mobility dependent on active transcription. Collectively, our data shows that the divergent residues within the acidic patch domain are key structural determinants of H2AZ function and links chromatin structure and dynamics with gene regulation and cell fate specification. H2AZ extended acidic patch was mutated, or H2AZ was KD in mouse embryonic stem cells and RNA-Seq analysis was performed on the resulting cultures. Characterization of H2AZ-WT and -AP3-mutant binding specificities were performed by ChIP-Seq.
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Project description:Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in ESCs to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 mono-ubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1K3R3). We show that H2A.Z.1K3R3 is properly incorporated at target promoters in murine ESCs (mESCs), however, loss of mono-ubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and to faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member Brd2, are enriched in H2A.Z.1 chromatin. We further show that Brd2 is gained at de-repressed promoters in H2A.Z.1K3R3 mESCs whereas Brd2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and Brd2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs. ChIP-Seq analysis on mouse embryonic stem cells harboring H2A.Z or H2A.Z.K3R3 (3 C-terminal lysines mutated to arginines) tagged with YFP, in the presence of a knockdown hairpin targeting the endogenous H2A.Z transcript.
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Project description:Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated. Two ESC lines mutant in genes of core components of Polycomb Repressive Complex 2 were assessed for H3K27me3 by ChIP-seq, as compared to a wild type ESC line.
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2014-11-17 | E-GEOD-61248 | biostudies-arrayexpress
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