Extended Data Fig. 5: Low Xist expression levels limit its ability to spread to autosomes.

a) Top: schematic depicting expected ratios of SHARP to Xist based on increasing concentration of Xist RNA; bottom: diagrams illustrating non-stoichiometric and stoichiometric gene silencing by Xist. b) Randomized Xist localization in simulated single cells (permutations) compared to experimental data. Top: Xist localization after 48 hours of RA-differentiation in female mESCs from bulk RAP-DNA experiments16; middle: gene density across X, only genes that undergo XCI are plotted; bottom: randomized localization of 200 Xist molecules in 1000 random “cells”; Xist represented by grey squares. c) Simulation visualizing localization of Xist molecules over genes that undergo transcriptional silencing during XCI across 20 cells (zoom-in from Extended Data Fig. 5b). Percent reflects proportion of Xist molecules overlapping the gene in all 1000 permutations. d) Expression levels of Xist in female mESCs treated with increasing dox concentrations as measured by RT-qPCR. e) Comparison of Xist occupancy (black lines; 3x dox RAP data) and DNA contact frequency with Xist locus (red lines; SPRITE data61) across 1 Mb DNA regions of chr8. Xist enrichment and 3D distance are normalized to their median coverage across chromosome 8 to place them on the same relative scale. f) Scatterplot representing the frequency of 3D contacts between each 1 Mb autosomal bin with the Xist locus (SPRITE data61; y axis) and DNA sites enriched by Xist when expressed at high concentration (3x dox; RAP-DNA data; X-axis). g) Relative Xist expression upon RA-induced differentiation (left) and dox-induction (right) of SHARP-AID mESCs in the absence or presence of auxin as measured by RT-qPCR (primer pair Xist 2). Dots represent individual replicates; n = 6 for differentiation conditions; n = 3 for dox induction conditions; data represented as mean ± s.d. h) Relative Xist expression in RA differentiated wildtype cells that do not contain dox-inducible Xist (left) and TX cells with dox-inducible Xist (right) as measured by RT-qPCR (primer pairs Xist 1, 2). i) Left: images representing Xist territories in wildtype cells without dox-inducible Xist and TX cells with dox-inducible Xist; total cell numbers (right) from two independent experiments. Right: quantification of percent of nucleus occupied by Xist in the same cell lines as measured by RNA-FISH (Xist) and DAPI staining (nucleus); scale bars 10 μm. Dots represent individual replicates; red bars represent median.