Escape from X inactivation is directly modulated by Xist non-coding RNA [Capture Hi-C]

X-chromosome inactivation silences one copy of the X-chromosome in female cells, but a number of genes escapes this inactivation. Increased levels of Xist RNA, the molecular actor driving X-inactivation, results in silencing of escaping genes. To address how chromosome topology responds to changes in transcriptional activity of escapees, we performed allele-specific Capture Hi-C analysis of clusters of escaping genes on the inactive X chromosome in NPCs. We performed the experiments after 7 and 21 days of Xist induction and upon washout of doxycycline and in presence or absence of Xist's partner SPEN. We further tested changes in the 3D topology of regions surrounding known constitutive escapees in ESCs upon increased Xist levels. Overall design: TX1072.G4 first generation hybrid mouse ESCs (from a C57BL6/J x CAST/EiJ cross) were cultured and clonal neural progenitor cell lines were generated from them by in vitro differentiation. All NPC cell lines carry an inactive B6 X chromosome and a doxycycline-inducible transgene on the Xi. Allele-specific Capture Hi-C was performed to characterized the 3D spatial organization of the two X chromosomes in ESC at two regions containing known constitutive escapees (Kdm5c and Kdm6a) and in two NPC lines, E6 and CL30.7, of two clusters of genes that escape X-inactivation (Kdm5c and Hcfc1-Mecp2).