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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]

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 Mus Homo sapiens

PROVIDER: GSE253466 | 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 [ChIP-seq]

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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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 [RNA-Seq]

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