PROJECT SUMMARY Our research program is unified by an effort to understand the mechanisms underlying X-chromosome inactivation (XCI) and how functional interactions between Polycomb repressive complex 2 (PRC2) and long noncoding RNA (lncRNA) spread the silencing process through the X-chromosome. XCI is the dosage compensation process in mammals that leads to silencing of one X-chromosome in the peri-implantation embryo. The X-inactivation center (Xic) — the X-linked region that controls the initiation, spread, and maintenance of silencing — harbors a large number of genes encoding functional lncRNAs, including the prototype, Xist, a 17-kb RNA that “coats” the inactive X (Xi) to initiate silencing. Towards understanding mechanisms, in 2008 we identified Polycomb repressive complex 2 (PRC2) as an interacting protein partner for Xist RNA. PRC2 is the epigenetic complex responsible for trimethylating H3 lysine27 (H3K27me3). Under this NIH award, we have identified of a “nucleation center” required for loading of Xist-Polycomb complexes onto the Xi prior to spreading, defined YY1 as the tether, and demonstrated that Xist RNA is but one of thousands of RNA partners for EZH2, the catalytic subunit of PRC2. We have hypothesized that regulatory interactions between PRC2 and RNA would become a recurrent theme in epigenetic regulation. Two major unsolved problems in the field are how the interaction specificity between PRC2 and RNA is achieved and how they regulate gene expression in a locus-specific manner. In the past funding cycle, we have made significant progress towards addressing these problems. We have: (i) mapped Xi-binding sites for Xist RNA and PRC2, (ii) performed single-molecule imaging of the Xist-PRC2 relationship on the Xi, (iii) defined the interaction specificity and regulatory interactions between Xist's Repeat A motif and various subunits of PRC2, (iv) identified an in vivo chaperone that helps determine the interaction specificity between Xist and PRC2, and (v) revealed a role for SMCHD1 (a chromosome architectural protein) in the regional spreading of Xist-PRC2 complexes. We have furthermore discovered a surprising non-canonical activity of EZH2 — an activity independent of its histone methyltransferase activity and that triggers a site-specific cleavage of a SINE repeat RNA (B2) during the stress response. In the next five years, we will address key follow-up questions. First, we aim to define specific motifs for the Polycomb-RNA interaction and understand how ATRX influences the interaction. Second, we will investigate underlying mechanisms for the Xist-PRC2 "spreading" function on the Xi through mutagenesis of Xist and perturbation of factors required for higher order Xi structures. Finally, we will examine how EZH2 triggers site-specific cleavage of RNA and determine whether this activity impacts XCI.