EPIGENETIC REGULATION BY LONG NONCODING RNAS AND THEIR PROTEIN COFACTORS

Long noncoding RNAs (lncRNAs) silence genes across megabase-scale chromosomal domainsthrough recruitment of Polycomb Repressive Complexes (PRCs). The lncRNA Xist silences theentire X chromosome, while the imprinted lncRNAs Airn and Kcnq1ot1 repress multi-megabasedomains in the mouse placenta. Despite shared requirements for PRCs and the RNA-binding protein(RBP) HNRNPK, fundamental questions remain about how lncRNAs recruit PRCs to chromatin.Additionally, while RBP associations with Xist are well characterized, the RBPs that associate withAirn and Kcnq1ot1 and their functional roles remain largely unknown.In this dissertation, we profiled RBP and PRC associations across all three lncRNAs to uncovershared mechanisms of lncRNA-mediated gene silencing. Through formaldehyde crosslinked RNAimmunoprecipitation, we found that PRC1—not PRC2—components were consistently enrichedand colocalized with HNRNPK. HNRNPK depletion reduced PRC1 associations, supporting aprotein-bridged recruitment model.Using an isogenic ectopic expression system, we demonstrated that Airn recruits PRCs withpotency rivaling Xist. Yet strikingly, Airn failed to silence genes unless fused to Xist’s Repeat Adomain, indicating that Repeat A provides a silencing function distinct from PRC recruitment.We next systematically profiled the RBP landscape of Airn and Kcnq1ot1 through RIP-seq of27 RBPs, revealing novel highly enriched protein associations. Remarkably, Airn and Kcnq1ot1exhibited shared RBP enrichment patterns with Xist, suggesting conserved mechanisms acrosslncRNAs. Most importantly, we identified HNRNPU as universally required for PRC-directed chromatin modifications by all three lncRNAs, yet dispensable for Airn and Kcnq1ot1 chromatinlocalization.Together, this work establishes that lncRNAs recruit PRCs through protein bridges, revealsRBP associations mediating Airn and Kcnq1ot1 function, and identifies HNRNPU as essential forPolycomb deposition across all three lncRNAs.