PRC2 direct transfer from G-quadruplex RNA to dsDNA: Implications for RNA-binding chromatin modifiers

Abstract

AbstractThe chromatin-modifying enzyme, Polycomb Repressive Complex 2 (PRC2), deposits the H3K27me3 epigenetic mark to negatively regulate expression at numerous target genes, and this activity has been implicated in embryonic development, cell differentiation, and various cancers. A biological role for RNA binding in regulating PRC2 histone methyltransferase activity is generally accepted, but the nature and mechanism of this relationship remains an area of active investigation. Notably, manyin vitrostudies demonstrate that RNA inhibits PRC2 activity on nucleosomes through mutually antagonistic binding, while somein vivostudies indicate that PRC2’s RNA-binding activity is critical for facilitating its biological function(s). Here we use biochemical, biophysical, and computational approaches to interrogate PRC2’s RNA and DNA binding kinetics. Our findings demonstrate that PRC2-polynucleotide dissociation rates are dependent on the concentration of free ligand, indicating the potential for direct transfer between ligands without a free-enzyme intermediate. Direct transfer explains the variation in dissociation kinetics reported previously, allows reconciliation of priorin vitroandin vivostudies, and expands the potential mechanisms of RNA-mediated PRC2 regulation. Moreover, simulations indicate that such a direct transfer mechanism could be obligatory for RNA to recruit proteins to chromatin.SignificanceStudies of PRC2in vitroindicate that RNA inhibits its histone methyltransferase (HMTase) activity through mutually antagonistic binding with nucleosomes, but somein vivostudies paradoxically suggest that RNA binding is necessary to facilitate chromatin occupancy and HMTase activity. Our findings unveil a protein-intrinsic mechanism for directly exchanging RNA and DNA/nucleosome in PRC2’s binding site(s), which reconciles these prior findings by allowing antagonistic or synergistic RNA-mediated regulation dependent on RNA-nucleosome proximity. Furthermore, there is an increasing awareness that multiple chromatin-associated proteins exhibit regulatory RNA binding activity, and our findings indicate this “direct transfer” mechanism may be generally required for such regulation.