Apparent loss of PRC2 chromatin occupancy as an artefact of RNA depletion
RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favour and against this idea. RNase treatment of chromatin is a prevalent tool for the study of RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. One of the most studied chromatin modifiers in the context of RNA-mediated regulation is the H3K27me3 methyltransferase Polycomb Repressive Complex 2 (PRC2). RNase A treatment during chromatin immunoprecipitation (rChIP) reduces the occupancy of PRC2 on chromatin. This led to suggestions of an “RNA bridge" between PRC2 and chromatin. Here we show that RNase A treatment during chromatin immunoprecipitation leads to the apparent loss of all facultative heterochromatin, including both PRC2 and its H3K27me3 mark genome-wide. This phenomenon persists in mouse embryonic stem cells, human cancer cells and human-induced pluripotent stem cells. We track this observation to a global gain of chromatin that artificially reduces ChIP signals from facultative heterochromatin. Our results point to substantial limitations in RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay. Overall design: ChIP sequecning for EZH2, SUZ12, RPB1, H3K27me3 and H3K27Ac in the presence or absence of RNase A treatment performed in mouse embryonic stem cells, K562 cells and human induced pluripotent stem cells. Independent replicates included.