Xist Deletional Analysis Reveals an Interdependency between Xist RNA and Polycomb Complexes for Spreading along the Inactive X

During X-inactivation, Xist RNA spreads along an entire chromosome to establish silencing. However, the mechanism and functional RNA elements involved in spreading remain undefined. By performing a comprehensive endogenous Xist deletion screen, we identify Repeat B as crucial for spreading Xist and maintaining Polycomb repressive complexes 1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unexpectedly, spreading of these three factors is inextricably linked. Deleting Repeat B or its direct binding partner, HNRNPK, compromises recruitment of PRC1 and PRC2. In turn, ablating PRC1 or PRC2 impairs Xist spreading. Therefore, Xist and Polycomb complexes require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading causes failure of de novo Xi silencing, with partial compensatory downregulation of the active X, and also disrupts topological Xi reconfiguration. Thus, Repeat B is a multifunctional element that integrates interdependent Xist/ Polycomb spreading, silencing, and changes in chromosome architecture. Xist CHART-seq, H3K27me3 ChIP-seq, H2AK119ub ChIP-seq in WT, Xist RepB, EED KO, RING1A/B KO MEF cells. RNA-seq in WT MEF, Xist RepB mutant MEF, WT ES and two RepB mutant ES cells. in situ HiC in WT MEF, Xist RepB mutant MEF, WT ES, and RepB mutant ES cells