Escape from X inactivation is directly modulated by Xist non-coding RNA [cell line RNA-seq]

X-chromosome inactivation silences one copy of the X-chromosome in female cells, but a number of genes escapes this inactivation. We asked whether increased levels of Xist RNA, the molecular actor driving X-inactivation, impacts expression levels of escapees. To this end, we performed transcriptomic profiling of X-linked gene expression in clonal neural progenitor cell lines with transgenes that allow for the overexpression of Xist on the inactive X chromosome. We perform Xist overexpression experiments for up to three weeks and also test the reversibility of Xist overexpression using washout experiments. Finally, we demonstrate the role of SPEN in Xist-mediated silencing of escape using an inducible degron. Overall design: Clonal neural progenitor cell lines were generated by differentiating first generation hybrid embryonic stem cells (from a C57BL6/J x CAST/EiJ cross). All cell lines carry an inactive B6 X chromosome and a doxycycline-inducible transgene on the Xi. Doxycyclin treatment for 3, 7, 14 and 21 days was performed in the E6 cell line and allele-specific expression was measured using RNA-Sequencing. Furthermore, multiple timepoints after washing out doxycycline were analyzed to assess reversibility. To test the effect of SPEN in Xist-induced silencing, the CL30/CL31 cell lines were used in which SPEN is fused to an auxin-inducible degradation domain. Combined treatment with auxin and doxycycline was used to assess silencing in the absence of SPEN.