The apparent loss of PRC2 chromatin occupancy as an artifact of RNA depletion.

RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favor and against this idea. RNase treatment of chromatin is commonly used to study RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (ChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase Polycomb repressive complex 2 (PRC2). This led to suggestions of an "RNA bridge" between PRC2 and chromatin. Here, we show that RNase A treatment during ChIP causes the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome-wide. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results emphasize substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay.

PALI1 facilitates DNA and nucleosome binding by PRC2 and triggers an allosteric activation of catalysis.

The polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identities. JARID2 is the only accessory subunit of PRC2 that known to trigger an allosteric activation of methyltransferase. Yet, this mechanism cannot be generalised to all PRC2 variants as, in vertebrates, JARID2 is mutually exclusive with most of the accessory subunits of PRC2. Here we provide functional and structural evidence that the vertebrate-specific PRC2 accessory subunit PALI1 emerged through a convergent evolution to mimic JARID2 at the molecular level. Mechanistically, PRC2 methylates PALI1 K1241, which then binds to the PRC2-regulatory subunit EED to allosterically activate PRC2. PALI1 K1241 is methylated in mouse and human cell lines and is essential for PALI1-induced allosteric activation of PRC2. High-resolution crystal structures revealed that PALI1 mimics the regulatory interactions formed between JARID2 and EED. Independently, PALI1 also facilitates DNA and nucleosome binding by PRC2. In acute myelogenous leukemia cells, overexpression of PALI1 leads to cell differentiation, with the phenotype altered by a separation-of-function PALI1 mutation, defective in allosteric activation and active in DNA binding. Collectively, we show that PALI1 facilitates catalysis and substrate binding by PRC2 and provide evidence that subunit-induced allosteric activation is a general property of holo-PRC2 complexes.