Impact of somatic XIST deletions on ongoing XIST expression and inactive X silencing and heterochromatin

The long non-coding RNA XIST is critical during early female development for establishing X-chromosome inactivation. XIST continues to be expressed from the inactive X throughout life, and can establish silenced heterochromatin when ectopically expressed; yet silencing of most genes on the inactive X has been shown to continue in the absence of XIST in somatic cells. As changes in XIST expression are observed in cancers and aging we sought to re-examine the impact of complete or partial XIST loss on gene expression and chromatin structure in the hTERT RPE-1 cell line. Loss of the A repeat region resulted in complete loss of XIST expression. While loss of an adjacent enhancer region spanning the small F repeat region impacted expression and loss of the repeat E region impacted localization, both deletions maintained partial silencing. Upon XIST loss there was reactivation of a subset of X-linked genes, with escape genes showing an average 20% increase in expression; consistent with other studies. Comparison with other studies of XIST loss in human somatic cells revealed some common XIST-dependent escapees, while autosomal genes demonstrated no consistent XIST-dependencies. Loss of XIST in hTERT RPE-1 resulted in loss of H3K27me3 and H2AK119ub; however, inhibition of each of these pathways failed to upregulate candidate escapees on the inactive X. In contrast, reduction of H3K9me3 caused partial reactivation of USP9X but not MED14. XIST regulatory elements lie within the gene, with ongoing expression dependent on the A repeat region, as well as enhancer function from the adjacent F-containing region. Unlike the E region, the F region is not involved in XIST localization. Our work, in agreement with previous studies, reveals that in somatic cells XIST expression and localization suppresses the expression of escape genes, likely through loss of XIST-dependent chromatin modifications. No single chromatin modification was responsible for the XIST-dependency, varying across both genes and studied cell lines, thus implicating combinatorial action of silencing pathways on the inactive X. While disruption of autosomal gene expression is observed, there are no consistent autosomal XIST-dependencies to implicate a direct action of XIST on autosomes across cell types.