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Denaturing purifications demonstrate that PRC2 and other widely-reported chromatin proteins do not appear to bind directly to RNA in vivo [ChIP-seq]

ABSTRACT: The Polycomb Repressive Complex 2 (PRC2) has been reported to bind to many RNAs and has become a central player in reports describing the mechanisms of how long non-coding RNAs (lncRNAs) regulate gene expression. Based largely on these observations of PRC2, many additional chromatin proteins have similarly been reported to bind RNA to enact their functions. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here we revisit the evidence supporting broad RNA binding by PRC2 and other chromatin proteins and show that many previously reported RNA-protein interactions do not represent in vivo interactions. We find that denaturing purification of in vivo crosslinked RNA-protein complexes leads to loss of detectable PRC2-RNA interactions. Similarly, we fail to detect in vivo RNA binding of other chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, WDR5, and others), despite accurately mapping bona fide RNA-protein binding sites across a range of proteins including several specific chromatin-associated proteins (SPEN, CHTOP, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE253475 | GEO | 2024/02/20

REPOSITORIES: GEO

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Denaturing purifications demonstrate that PRC2 and other widely-reported chromatin proteins do not appear to bind directly to RNA in vivo [CLIP and CLAP]

Project description:The Polycomb Repressive Complex 2 (PRC2) has been reported to bind to many RNAs and has become a central player in reports describing the mechanisms of how long non-coding RNAs (lncRNAs) regulate gene expression. Based largely on these observations of PRC2, many additional chromatin proteins have similarly been reported to bind RNA to enact their functions. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here we revisit the evidence supporting broad RNA binding by PRC2 and other chromatin proteins and show that many previously reported RNA-protein interactions do not represent in vivo interactions. We find that denaturing purification of in vivo crosslinked RNA-protein complexes leads to loss of detectable PRC2-RNA interactions. Similarly, we fail to detect in vivo RNA binding of other chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, WDR5, and others), despite accurately mapping bona fide RNA-protein binding sites across a range of proteins including several specific chromatin-associated proteins (SPEN, CHTOP, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.

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RNA is essential for PRC2 chromatin occupancy and function in human pluripotent stem cells

Project description:Many chromatin-binding proteins and protein complexes that regulate transcription also bind RNA. One of these, Polycomb repressive complex 2 (PRC2), deposits the H3K27me3 mark of facultative heterochromatin and is required for stem cell differentiation. PRC2 binds RNAs broadly in vivo and in vitro. Yet, whether this RNA binding has any biological importance remains unsettled. Here we tackle this question in human induced pluripotent stem cells using multiple complementary approaches. Perturbation of RNA-PRC2 interaction by RNaseA, by a chemical inhibitor of transcription, or by an RNA-binding-defective mutant all disrupted PRC2 chromatin occupancy and localization genome-wide. The physiological relevance of PRC2-RNA interactions is further underscored by the dramatic cardiomyocyte differentiation defect upon genetic disruption. We conclude that PRC2 requires RNA binding for chromatin localization in human pluripotent stem cells and in turn for defining cellular state.

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The interaction of PRC2 with RNA or chromatin is mutually antagonistic

Project description:Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain genes in a repressed state during development. PRC2 is primarily associated with CpG islands at repressed genes and also possesses RNA binding activity. However, the RNAs that bind PRC2 in cells, the subunits that mediate these interactions, and the role of RNA in PRC2 recruitment to chromatin all remain unclear. By performing iCLIP for PRC2 in comparison with other RNA binding proteins, we show here that PRC2 binds nascent RNA at essentially all active genes. Although interacting with RNA promiscuously, PRC2 binding is enriched at specific locations within RNAs, primarily exon-intron boundaries and the 3’UTR. Deletion of other PRC2 subunits reveals that SUZ12 is sufficient to establish this RNA binding profile. Contrary to prevailing models, we also demonstrate that the interaction of PRC2 with RNA or chromatin is mutually antagonistic in cells and in vitro. RNA degradation in cells triggers PRC2 recruitment to CpG islands at active genes. Correspondingly, release of PRC2 from chromatin in cells increases RNA binding. Consistent with this, RNA and nucleosomes compete for PRC2 binding in vitro. We propose that RNA prevents PRC2 recruitment to chromatin at active genes and that mutual antagonism between RNA and chromatin underlies the pattern of PRC2 chromatin association across the genome.

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The interaction of PRC2 with RNA or chromatin is mutually antagonistic [iCLIP-Seq]

Project description:Polycomb repressive complex 2 (PRC2) maintains developmental regulator genes in a repressed state through methylation of histone H3 at lysine 27 (H3K27me3) and is necessary for cell differentiation. We and others have previously found that the PRC2 subunit Suz12 interacts with RNA in vitro and other studies have shown that Ezh2 and Jarid2 also possess RNA binding function. The interaction of PRC2 with RNA has been suggested to regulate PRC2 targeting or enzymatic activity, but the RNAs directly bound by PRC2 in cells, and the role of each PRC2 RNA binding subunit, remain unclear. We have used different CLIP techniques, which use UV-crosslinking to allow detection of direct Suz12-RNA interactions as they occur in living mouse ES cells. Suz12 binds nascent RNA and has a preference for interaction with the 3’UTR, showing it does have binding specificity in cells. RNAs bound by Suz12 at the 3’UTR encode developmental regulator genes. Suz12 remains bound to RNA upon deletion of Ezh2 or Jarid2 showing that it binds RNA independently of other PRC2 subunits. We also show that binding of Suz12 to RNA or chromatin is mutually inhibitory. Although Ezh2 and Jarid2 also bind RNA, Ezh2 and Jarid2 deletion causes an increase in Suz12 RNA binding, without changing its specificity, which reflects the loss of Suz12 from chromatin. Similarly, disruption of Suz12-RNA interactions by RNA polymerase II inhibition or RNase treatment increases Suz12 binding to chromatin. These results therefore suggest that Suz12 acts as an RNA sensor, binding to the 3’UTR of nascent RNAs and modulating the interaction of PRC2 with chromatin.

2016-05-06 | GSE66829 | GEO

The interaction of PRC2 with RNA or chromatin is mutually antagonistic [RNA-Seq]

2016-05-06 | GSE66814 | GEO

The interaction of PRC2 with RNA or chromatin is mutually antagonistic [ChIP-Seq]

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Denaturing purifications demonstrate that PRC2 and other widely-reported chromatin proteins do not appear to bind directly to RNA in vivo

Project description:Denaturing purifications demonstrate that PRC2 and other widely-reported chromatin proteins do not appear to bind directly to RNA in vivo

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Mapping RNA interactions of human ATRX with RepA fragment of Xist

Project description:Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes.

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Denaturing purifications demonstrate that PRC2 and other widely-reported chromatin proteins do not appear to bind directly to RNA in vivo [ChIP-seq]

Project description:Denaturing purifications demonstrate that PRC2 and other widely-reported chromatin proteins do not appear to bind directly to RNA in vivo [ChIP-seq]

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Denaturing purifications demonstrate that PRC2 and other widely-reported chromatin proteins do not appear to bind directly to RNA in vivo [CLIP and CLAP]

Project description:Denaturing purifications demonstrate that PRC2 and other widely-reported chromatin proteins do not appear to bind directly to RNA in vivo [CLIP and CLAP]

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