Xist Repeats A and B account for two distinct phases of X-inactivation establishment

X-chromosome inactivation (XCI) is a global silencing mechanism by which XX and XY mammals equalize X-linked gene dosages. XCI begins with an establishment phase during which Xist RNA spreads and induces de novo heterochromatinization across a female X chromosome, and is followed by a maintenance phase when multiple epigenetic pathways lock down the inactive X (Xi) state. Involvement of Polycomb repressive complexes 1 and 2 in XCI has been intensively studied, but with conflicting conclusions regarding their recruitment and role in Xi silencing. Here we reveal that establishment of XCI has two phases and reconcile the roles that Xist Repeats A and B play in gene silencing and Polycomb recruitment. Repeat A initiates both processes, whereas Repeat B bolsters or stabilizes them thereafter. Once established, XCI no longer requires Repeat A during maintenance. These findings integrate disparate studies and present a unified view of Xist?s role in Polycomb-mediated silencing. Overall design: RNAseq, H3K27me3 ChIP-seq, H2AK119ub ChIP-seq in WT and RepB ES cells during differentiation timecourse; H3K27me3, H2AK119ub ChIP-seq in RepA MEF