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.

X 染色体失活 (XCI) 是一种全局沉默机制，XX 和 XY 哺乳动物通过该机制平衡 X 连锁基因剂量。XCI 从建立阶段开始，在此期间 Xist RNA 传播并诱导从头跨女性 X 染色体的异染色质化，然后是多个表观遗传途径锁定非活性 X (Xi) 状态时的维持阶段。Polycomb 抑制复合物 1 和 2 在 XCI 中的参与已得到深入研究，但关于它们在 Xi 沉默中的招募和作用的结论相互矛盾。在这里，我们揭示 XCI 的建立有两个阶段，并协调 Xist 重复 A 和 B 在基因沉默和 Polycomb 募集中的作用。重复 A 启动这两个过程，而重复 B 在此后加强或稳定它们。一旦建立，XCI 在维护期间不再需要重复 A。这些发现整合了不同的研究，并对 Xist 在 Polycomb 介导的沉默中的作用提出了统一的看法。