Retinoic acid accelerates downregulation of the Xist repressor, Oct4, and increases the likelihood of Xist activation when Tsix is deficient.

Background: Imbalances in X-linked gene dosage between the sexes are resolved by transcriptionally silencing one of two X-chromosomes in female cells of the early mammalian embryo. X-inactivation is triggered by expression of the non-coding Xist gene. In turn, Xist is dually regulated by the antisense Tsix RNA and by the Oct4 pluripotency factor. Although there is general agreement that Tsix is an inhibitor of Xist, some laboratories have observed ectopic Xist induction in differentiating male ES cells when Tsix is mutated, whereas we have not observed significant changes in Xist. These observational differences have led to fundamentally diverse models of X-chromosome counting. Here, we investigate if different methods of cell differentiation and use of all -trans retinoic acid (RA) could be causative factors and how they might impact Xist expression.

Results: We compared suspension and cell-adhesion cultures in the presence or absence of RA and find that RA significantly impacts Xist expression in Tsix-mutant male cells. Whereas the standard embryoid body method infrequently leads to ectopic Xist expression, adding RA generates a significant number of Xist-positive male cells. However, while normal Xist clouds in wild-type female cells are robust and well-circumscribed, those found in the RA-treated mutant males are loosely dispersed. Furthermore, ectopic Xist expression does not generally lead to complete gene silencing. We attribute the effect of RA on Xist to RA's repressive influence on Oct4, a pluripotency factor recently shown to regulate Tsix and Xist. RA-treated ES cells exhibit accelerated decreases in Oct4 RNA levels and also display accelerated loss of binding to Xist intron 1. When Tsix is deficient, the faster kinetics of Oct4 loss tip the equilibrium towards Xist expression. However, the aberrant Xist clusters are unlikely to explain elevated cell death, as X-linked silencing does not necessarily correlate with the qualitatively aberrant Xist clusters.

Conclusions: We conclude that RA treatment leads to premature downregulation of Oct4 and partial derepression of Xist irrespective of X-chromosome counting. RA-induced Xist clusters in male cells do not result in global or stable silencing, and excess cell death is not observed. These data and RA's known pleiotropic effects on ES transcription networks suggest that RA differentation bypasses normal X-inactivation controls and should be used judiciously. We propose that the likelihood of Xist expression is determined by a balance of multiple Xist activators and repressors, and that levels of Oct4 and Tsix are crucial toward achieving this balance.