Xist RNA has been established as the master regulator of X‐chromosome inactivation (XCI) in female eutherian mammals, but its mechanism of action remains unclear. By creating novel Xist‐inducible mutants at the endogenous locus in male mouse embryonic stem (ES) cells, we dissect the role of the conserved A‐B‐C‐F repeats in the initiation of XCI. We find that transcriptional silencing can be largely uncoupled from Polycomb repressive complex 1 and complex 2 (PRC1/2) recruitment, which requires B and C repeats. Xist ΔB+C RNA specifically loses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners is largely unaffected. However, a slight relaxation of transcriptional silencing in Xist ΔB+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways. In this context, Polycomb recruitment seems to be of moderate relevance in the initiation of silencing.

Highlights d RNase H2 efficiently processes R-loops and rNMPs postreplicatively d rNMPs processed by RNase H2 in S phase require Rad52mediated repair d RNase H1 functions independently of the cell cycle d RNase H1 responds to R-loop-induced stress

Xist RNA has been established as the master regulator of X-chromosome inactivation (XCI) in female eutherian mammals but its mechanism of action remains unclear. By creating novel Xist mutants at the endogenous locus in mouse embryonic stem (ES) cells, we dissect the role of the conserved A-B-C-F repeats. We find that transcriptional silencing can be largely uncoupled from Polycomb repressive complex 1 and 2 (PRC1/2) recruitment, which requires repeats B and C. Xist ΔB+C RNA specifically loses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners is largely unaffected. However, a slight relaxation of transcriptional silencing in Xist ΔB+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways. In this context, Polycomb recruitment seems to be of moderate relevance in the initiation of silencing.