Transcription-dependent heterochromatin at the Xist promoter shapes the random choice of the inactive X chromosome [Xist-Tsix-perturbations_CutnTag]

In female mammals, Xist, the master regulator of X-chromosome inactivation (XCI), is expressed monoallelically. This pattern is established during early embryonic development, when the active Xist allele is chosen at random in each cell. How this choice is made remains incompletely understood. Here we identify a role for the repressive chromatin mark H3K9me3 in the initiation of XCI by combining knock-down and overexpression strategies in differentiating mouse embryonic stem cells, which recapitulate the onset of random XCI. We show that H3K9me3 accumulates at the promoter-proximal region of the silent Xist allele in female cells at the same time when the monoallelic Xist expression is established. Unexpectedly, we find that H3K9me3 accumulation requires prior transcription of Xist itself. This is likely to occur during the initial phase of Xist upregulation, when Xist is frequently transcribed also in male cells and from both X chromosomes in females. A repressive function of Xist-dependent H3K9me3 accumulation is supported by our finding that premature, transient Xist overexpression primes an allele for future silencing and thereby skews the choice of the inactive X. Xist-dependent H3K9me3 recruitment does not require transcription of its antisense transcript Tsix, which can however enhance subsequent maintenance of the mark. In addition, the X-linked Xist activator RNF12 counteracts H3K9me3 formation at the locus independently of its known target REX1. Our results thus suggest an important role of facultative heterochromatin formation in the choice process at the onset of XCI. Activation and repression mechanisms are intertwined, to ensure the silencing of one Xist allele and the activation of the other within each female cell.