A Primary Role for the Tsix lncRNA in Maintaining Random X-Chromosome Inactivation

Differentiating pluripotent epiblast cells in eutherians undergo random X-inactivation, which equalizes X-linked gene expression between the sexes by silencing one of the two X-chromosomes in females. Tsix RNA is believed to orchestrate the initiation of X-inactivation, influencing the choice of which X remains active by preventing expression of the antisense Xist RNA, which is required to silence the inactive-X. Here we profile X-chromosome activity in Tsix-mutant (X-Delta Tsix) mouse embryonic epiblasts, epiblast stem cells, and embryonic stem cells. Unexpectedly, we find that Xist is stably repressed on the X-Delta Tsix in both sexes in undifferentiated epiblast cells in vivo and in vitro, resulting in stochastic X-inactivation in females despite Tsix-heterozygosity. Tsix is instead required to silence Xist on the active-X as epiblast cells differentiate in both males and females. Thus, Tsix is not required at the onset of random X-inactivation; instead, it protects the active-X from ectopic silencing once X-inactivation has commenced.