A transient heterochromatic state in Xist preempts X inactivation choice without RNA stabilization

X chromosome inactivation (XCI) depends on a non-coding sense-antisense transcript pair, Xist and Tsix. At the onset of XCI, Xist RNA accumulates on one of two Xs, coating and silencing the chromosome in cis. The molecular basis for monoallelic Xistupregulation is not known, though evidence predominantly supports a posttranscriptional mechanism through RNA stabilization. Here, we test whether Tsix RNA destabilizes Xist RNA. Unexpectedly, we find that Xistupregulation is not based on transcript stabilization at all but is instead controlled by transcription in a sex-specif ic manner. Tsix directly regulates its transcription. On the future inactive X, Tsix downregulation induces a transient heterochromatic state in Xist, followed paradoxically by high-level Xist expression. A Tsix-deficient X chromosome adopts the heterochromatic state in pre-XCI cells. This state persists through XCI establishment and reverts to a euchromatic state during XCI maintenance. We have therefore identified chromatin marks that preempt and predict asymmetric Xist expression.