PROJECT SUMMARY Polycomb repressive complex 2 (PRC2) is a histone methyltransferase (HMTase) that sequentially deposits three methyl groups onto lysine 27 of histone H3 (H3K27me1/2/3), and its activity is crucial for epigenetic silencing during development and cancer. How PRC2 is targeted to genetic loci is of considerable interest, given its critical function and abundance of target genes. It’s well accepted that PRC2 interacts broadly with RNA, and that these interactions regulate PRC2’s localization at genomic sites destined for epigenetic silencing. However, the details about the nature and mechanism(s) of PRC2’s regulation by RNA remain quite controversial. While some studies have proposed a role for RNA in PRC2’s recruitment to chromatin, more have suggested roles in PRC2 eviction from chromatin and/or inhibition of PRC2 activity. Notably, a recent work has demonstrated that the PRC2-RNA interaction is critical in vivo for maintaining H3K27me3 levels and chromatin occupancy at PRC2 target genes. Thus, while there’s evidence RNA plays a role in evicting PRC2 from chromatin under certain conditions, it also seems it must facilitate chromatin binding and H3K27me3 deposition in at least some circumstances. To date, the PRC2 literature lacks direct evidence for a mechanism that can reconcile these prior data and explain RNA-mediated chromatin-binding and HMTase activity by PRC2. My preliminary data unexpectedly imply PRC2 has the intrinsic capacity to transfer directly between polynucleotides, and this phenomenon could be broadly relevant to RNA-mediated regulation of chromatin-modifying enzymes. Based on these and other data, I hypothesize a ‘hand-off’ model where PRC2 can be directly transferred between nascent RNA and spatially proximal chromatin to facilitate H3K27me3 deposition or eviction from chromatin. Studies proposed herein will quantify PRC2’s binding and competition kinetics for RNA and DNA/nucleosomes, determine the polynucleotide species PRC2 could be transferred between, characterize the prevalence and biophysical requisites of this phenomenon in other proteins, and interrogate the effect of RNA-nucleosome spatial relationships on PRC2’s HMTase activity. Findings from these studies will expand our understanding of how chromatin-modifying enzymes are regulated, which could have profound implications in our understanding of cell differentiation, embryonic development, and cancer.

PROJECT NARRATIVE Prior research has convincingly demonstrated a role for RNA in regulating PRC2’s suppression of target gene expression, but the nature and mechanism of this regulation remains unclear. The proposed studies will biophysically interrogate the mechanism(s) by which RNA regulates PRC2 activity. A mechanistic understanding of the PRC2-RNA relationship may be broadly relevant to enzymes regulating gene expression, and it could have profound implications in embryonic development and cancer.