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Apparent RNA bridging between PRC2 and chromatin is an artifact of non-specific chromatin precipitation upon RNA degradation

ABSTRACT: Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain repression of genes specific for other cell lineages and is essential for development. In vitro, RNA inhibits PRC2 catalytic activity and interaction with nucleosomes but the effect of RNA on PRC2 in cells has been unclear, with studies concluding that RNA either antagonises or promotes the association of PRC2 with chromatin. Suggesting the existence of an RNA bridge that connects PRC2 to chromatin, treatment of formaldehyde-crosslinked, sonicated nuclear extracts with RNase A reduces co-precipitation of polycomb target sites with PRC2. Here, we show that this apparent loss of PRC2 chromatin association after RNase A treatment is due to non-specific chromatin precipitation. RNA degradation causes chromatin to precipitate out of solution, thereby reducing the enrichment of specific DNA sequences in chromatin immunoprecipitation reactions. Maintaining chromatin solubility by the addition of poly-L-glutamic acid (PGA) rescues detection of PRC2 chromatin association upon RNA degradation. These findings undermine support for the model that RNA bridges PRC2 and chromatin in cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE240380 | GEO | 2024/02/22

REPOSITORIES: GEO

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Apparent loss of PRC2 chromatin occupancy as an artefact of RNA depletion

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Apparent RNA bridging between PRC2 and chromatin is an artifact of non-specific chromatin precipitation upon RNA degradation

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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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Chip-chip from WT and Polycomb Component Knock Out Mouse ES cells for H2AZ, H3K27me3, EZH2 and Ring1B.

2012-04-16 | GSE36999 | GEO

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