Apparent RNA bridging between PRC2 and chromatin is an artifact of non-specific chromatin precipitation upon RNA degradation
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. Overall design: Chromatin Immunoprecipitation and DNA-sequencing (ChIP-seq) for histone mark H3K27me3 and chromatin binding proteins RPB1 and SUZ12, in mESCs and following addition of RNaseA and or poly-glutamic acid.