Differential methylation of Xite and CTCF sites in Tsix mirrors the pattern of X-inactivation choice in mice

During mammalian dosage compensation, one of two X-chromosomes in female cells is inactivated. The choice of which X is silenced can be imprinted or stochastic. Although genetic loci influencing the choice decision have been identified, the primary marks for imprinting and random selection remain undefined. Here, we examined the role of DNA methylation, a mechanism known to regulate imprinting in autosomal loci, and sought to determine whether differential methylation on the two Xs might predict their fates. To identify differentially methylated domains (DMDs) at the X-inactivation center, we used bisulfite sequencing and methylation-sensitive restriction enzyme analyses. We found DMDs in Tsix and Xite, two genes previously shown to influence choice. Interestingly, the DMDs in Tsix lie within CTCF binding sites. Allelic methylation differences occur in gametes and are erased in embryonic stem cells carrying two active Xs. Because the pattern of DNA methylation mirrors events of X-inactivation, we propose that differential methylation of DMDs in Tsix and Xite constitute a primary mark for epigenetic regulation. The discovery of DMDs in CTCF sites draws further parallels between X-inactivation and autosomal imprinting.