X Chromosome Reactivation Dynamics Reveal Stages of Reprogramming to Pluripotency

Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming. [Display omitted] •Sequential epigenetic changes occur on the Xi and define reprogramming stages•XCR requires pluripotency gene induction, DNA demethylation, and Xist loss•Tet1, Tet2, and global 5hmC are dispensable for XCR•The reprogramming stage of any cell can be determined by epigenetic states Tracking the steps of reactivation of the inactive X chromosome during reprogramming to pluripotency defines discrete stages of the reprogramming process and reveals that some aspects of pluripotency induction follow developmental sequences in reverse, but others, such as removal DNA methylation, occur in a different sequence.