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. [Display omitted] •X inactivation establishment is a biphasic process requiring Xist repeats A and B•Polycomb complexes can initially be recruited without repeat B•Repeat A initiates Polycomb recruitment and X silencing, whereas B stabilizes them•Frequency of X chromosome loss (XO state) is heightened by deleting repeat A Colognori et al. show that X inactivation establishment is a biphasic process with distinct genetic requirements. Repeat A initiates Polycomb recruitment and gene silencing, whereas repeat B stabilizes them. Surprisingly, X inactivation can initiate without repeat B. Without repeat A, however, differentiating female cells lose one X chromosome to overcome loss of silencing.