The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes1-3. During XCI, Xist coats the future inactive X (Xi)4 and recruits Polycomb Repressive Complex 2 (PRC2) to the X-inactivation center (Xic)5. How Xist spreads silencing on a 150 Mb scale is unclear. Here we generate high-resolution maps of Xist binding on the X chromosome across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism, initially targeting gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), suggesting co-migration of Xist and PRC2. Interestingly, when the Xi is acutely stripped off Xist in post-XCI cells, Xist recovers quickly within both gene-rich and -poor domains on a time-scale of hours instead of days, suggesting a previously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms: During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and -poor regions.

Pluripotency of embryonic stem (ES) cells is controlled by defined transcription factors1,2. During differentiation, mouse ES cells undergo global epigenetic reprogramming, as exemplified by X-chromosome inactivation (XCI) whereby one female X-chromosome is silenced to achieve gene dosage parity between the sexes3-5. Somatic XCI is regulated by homologous X-chromosome pairing6,7, counting8-10, and random choice of future active X (Xa) and inactive X’s. XCI and cell differentiation are tightly coupled11, as blocking one process compromises the other8,12 and dedifferentiation of somatic cells to induced pluripotent stem (iPS) cells is accompanied by X-reactivation2. Recent evidence suggests coupling of Xist expression to pluripotency factors13, but how the two are interconnected remains unknown. Here, we show that the Oct414 lies at the top of the XCI hierarchy and regulates XCI by triggering X-chromosome pairing and counting. Oct4 directly binds Tsix and Xite, two regulatory ncRNA genes of the X-inactivation center15,16, and also complexes with XCI trans-factors, Ctcf and Yy117, through protein-protein interactions. Depletion of Oct4 blocks homologous X-chromosome pairing and results in inactivation of both Xs in female cells. Thus, we have identified the first trans-factor that regulates counting and ascribed novel functions to Oct4 during X-chromosome reprogramming.