RNA is essential for PRC2 chromatin occupancy and function in human pluripotent stem cells

Many chromatin-binding proteins and protein complexes that regulate transcription also bind RNA. One of these, Polycomb repressive complex 2 (PRC2), deposits the H3K27me3 mark of facultative heterochromatin and is required for stem cell differentiation. PRC2 binds RNAs broadly in vivo and in vitro. Yet, whether this RNA binding has any biological importance remains unsettled. Here we tackle this question in human induced pluripotent stem cells using multiple complementary approaches. Perturbation of RNA-PRC2 interaction by RNaseA, by a chemical inhibitor of transcription, or by an RNA-binding-defective mutant all disrupted PRC2 chromatin occupancy and localization genome-wide. The physiological relevance of PRC2-RNA interactions is further underscored by the dramatic cardiomyocyte differentiation defect upon genetic disruption. We conclude that PRC2 requires RNA binding for chromatin localization in human pluripotent stem cells and in turn for defining cellular state. Two independent CRSIPR clones of each of the two edited cell lines (WT or mutant EZH2) were analyzed. For ChIP-seq and fRIP-seq, two or three technical replicates of the pulldown were sequenced on each lysate; For RNA-seq on undifferentiated cells, two technical replicates of libary construction were sequenced for each clone; For RNA-seq on differentiated cells, three biological replicates generated from independent differentiation experiments were sequenced for each clone.