Escape from X inactivation is directly modulated by Xist non-coding RNA [CUT&Run]

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 CUT&RUN profiling for two Xi-enriched histone marks, H2AK119Ub and H3K27me3 in a clonal neural progenitor cell line 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. 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 7 and 21 days was performed in the E6 cell line and allele-specific histone mark occupancy was measured using CUT&RUN. Furthermore, samples after washing out of doxycycline were analyzed to assess reversibility.