Xist RNA binds select autosomal genes and depends on Repeat B to regulate their expression

<i>Xist,</i> a pivotal player in X chromosome inactivation (XCI), has long been perceived as a cis-acting long noncoding RNA that binds exclusively to the inactive X chromosome (Xi). However, <i>Xist</i>'s ability to diffuse under select circumstances has also been documented, leading us to suspect that <i>Xist</i> RNA may have targets and functions beyond the Xi. Here, using female mouse embryonic stem cells (ES) and mouse embryonic fibroblasts (MEF) as models, we demonstrate that <i>Xist</i> RNA indeed can localize beyond the Xi. However, its binding is limited to ~100 genes in cells undergoing XCI (ES cells) and in post-XCI cells (MEFs). The target genes are diverse in function but are unified by their active chromatin status. <i>Xist</i> binds discretely to promoters of target genes in neighborhoods relatively depleted for Polycomb marks, contrasting with the broad, Polycomb-enriched domains reported for human <i>XIST</i> RNA. We find that <i>Xist</i> binding is associated with down-modulation of autosomal gene expression. However, unlike on the Xi, <i>Xist</i> binding does not lead to full silencing and also does not spread beyond the target gene. Over-expressing <i>Xist</i> in transgenic ES cells similarly leads to autosomal gene suppression, while deleting <i>Xist</i>'s Repeat B motif reduces autosomal binding and perturbs autosomal down-regulation. Furthermore, treating female ES cells with the <i>Xist</i> inhibitor, X1, leads to loss of autosomal suppression. Altogether, our findings reveal that <i>Xist</i> targets ~100 genes beyond the Xi, identify Repeat B as a crucial domain for its in-trans function in mice, and indicate that autosomal targeting can be disrupted by a small molecule inhibitor.