(A) iDRiP schematic.
(B) RT-qPCR demonstrated the specificity of Xist pulldown by iDRiP. Xist and control luciferase probes were used for pulldown from UV-crosslinked female and control male fibroblasts. Efficiency of Xist pulldown was calculated by comparing to a standard curve generated using 10-fold dilutions of input. Mean ± standard error (SE) of three independent experiments shown. P values determined by the Student t-test.
(C) Select high-confidence candidates from three biological replicates are grouped into functional classes. Additional candidates shown in .
(D) UV-RIP-qPCR validation of candidate interactors. Enrichment calculated as % input, as in (B). Mean ± SE of three independent experiments shown. P values determined by the Student t-test.
(E) RNA immunoFISH to examine localization of candidate interactors (green) in relation to Xist RNA (red). Immortalized MEF cells are tetraploid and harbor two Xi.

(A) RNA immunoFISH of Xist (red) and H3K27me3 (green) after shRNA KD of interactors in fibroblasts (tetraploid; 2 Xist clouds). KD efficiencies (fraction remaining): SMC1a-0.48, SMC3-0.39, RAD21-0.15, AURKB-0.27, TOP2b-0.20, TOP2a-0.42, TOP1-0.34, CTCF-0.62, SMARCA4-0.52, SMARCA5-0.18, SMARCC1-0.25, SMARCC2-0.32, SMARCB1-0.52 and SUN2-0.72. Some factors are essential; therefore, high percentage KD may be inviable. All images presented at the same photographic exposure and contrast.
(B) Quantitation of RNA immunoFISH results. n, sample size. % aberrant Xist/H3K27me3 associations shown.
(C) RT-qPCR of Xist levels in KD fibroblasts, normalized to shControls. Mean± SD of two independent experiments shown.

(A) Relative GFP levels by RT-qPCR analysis in female fibroblasts stably knocked down for indicated interactors ± 0.3 μM 5'-azacytidine (aza) ± 0.3 μM etoposide (eto). Xa-GFP, control male fibroblasts with X-linked GFP. Mean ± SE of two independent experiments shown. P, determined by Student t-test.
(B) Allele-specific RNA-seq analysis: Number of upregulated Xi genes for each indicated triple-drug treatment (aza+eto+shRNA). Blue, genes specifically reactivated on Xi (fold-change, FC>2); red, genes also unregulated on Xa (FC>1.3).
(C) RNA-seq heat map indicating that a large number of genes on the Xi were reactivated. X-linked genes reactivated in at least one of the triple-drug treatment (aza+eto+shRNA) were shown in the heat map. Color key, Log2 fold-change (FC). Cluster analysis performed based on similarity of KD profiles (across) and on the sensitivity and selectivity of various genes to reactivation (down).
(D) Chromosomal locations of Xi reactivated genes (colored ticks) for various aza+eto+shRNA combinations.
(E) Read coverage of 4 reactivated Xi genes after triple-drug treatment. Xi, mus reads (scale: 0–2). Comp, total reads (scale: 0–6). Red tags appear only in exons with SNPs.

(A) Allele-specific ChIP-seq results: Violin plots of allelic skew for CTCF, RAD21, SMC1a in wild-type (WT) and Xi^ΔXist/Xa^WT (ΔXist) fibroblasts. Fraction of mus reads [mus/(mus+cas)] is plotted for every peak with ≥10 allelic reads. P values determined by the Kolmogorov-Smirnov (KS) test.
(B) Differences between SMC1a or RAD21 peaks on the Xi^WT versus Xa^WT. Black diagonal, 1:1 ratio. Plotted are read counts for all SMC1a or RAD21 peaks. Allele-specific skewing is defined as ≥3-fold skew towards either Xa (cas, blue dots) or Xi (mus, red dots). Biallelic peaks, grey dots.
(C) Table of total, Xa-specific, and Xi-specific cohesin binding sites in WT versus ΔXist (Xi^ΔXist/Xa^WT) cells. Significant SMC1a and RAD21 allelic peaks with ≥5 reads were analyzed. Allele-specific skewing is defined as ≥3-fold skew towards Xa or Xi. Sites were considered “restored” if Xi^ΔXist's read counts were ≥50% of Xa's. X-total, all X-linked binding sites. Allelic peaks, sites with allelic information. Xa-total, all Xa sites. Xi-total, all sites. Xa-spec, Xa-specific. Xi-spec, Xi-specific. Xi-invariant, Xi-specific in both WT and Xi^ΔXist/Xa^WT cells. Note: There is a net gain of 96 sites on the Xi in the mutant, a number different from the number of restored sites (106). This difference is due to defining restored peaks separately from calling ChIP peaks (macs2). Allele-specific skewing is defined as ≥3-fold skew towards either Xa or Xi.
(D) Partial restoration of SMC1a or RAD21 peaks on the Xi^ΔXist to an Xa pattern. Plotted are peaks with read counts with ≥3-fold skew to Xa^WT (“Xa-specific”). x-axis, normalized Xa^WT read counts. y-axis, normalized Xi^ΔXist read counts. Black diagonal, 1:1 Xi^ΔXist/Xa^WT ratio; red diagonal, 1:2 ratio.
(E) Xi-specific SMC1a or RAD21 peaks remained on Xi^ΔXist. Plotted are read counts for SMC1a or RAD21 peaks with ≥3-fold skew to Xi^WT (“Xi-specific”).
(F) Comparison of fold-changes for CTCF, RAD21, and SMC1 binding in X^ΔXist cells relative to WT cells. Shown are fold-changes for Xi versus Xa. The Xi showed significant gains in RAD21 and SMC1a binding, but not in CTCF binding. Method: X^WT and X^ΔXist ChIP samples were normalized by scaling to equal read counts. Fold-changes for Xi were computed by dividing the normalized mus read count in X^ΔXist by the mus read count X^WT; fold-changes for Xa were computed by dividing the normalized cas read count in X^ΔXist by the cas read count X^WT. To eliminate noise, peaks with <10 allelic reads were eliminated from analysis. P values determined by a paired Wilcoxon signed rank test.
(G) The representative examples of cohesion restoration on Xi^ΔXist. Arrowheads, restored peaks.
(H) Allelic-specific cohesin binding profiles of Xa, Xi^WT, and Xi^ΔXist. Shown below restored sites are regions of Xi-reactivation following shSMC1a and shRAD21 triple-drug treatments, as defined in .

(A) Chr13 and ChrX contact maps showing triangular domains representative of TADs. Purple shades correspond to varying interaction frequencies (dark, greater interactions). TADs called from our composite (non-allelic) HiC data at 40-kb resolution (blue bars) are highly similar to those (gray bars) called previously ().
(B) Allele-specific HiC-seq analysis: Contact maps for three different ChrX regions at 100-kb resolution comparing Xi^ΔXist (red) to Xi^WT (orange), and Xi^ΔXist (red) versus Xa (blue) of the mutant cell line. Our Xa TAD calls are shown with RefSeq genes.
(C) Fraction of interaction frequency per TAD on the Xi (mus) chromosome. The positions of our Xa TAD borders were rounded to the nearest 100 kb and submatrices were generated from all pixels between the two endpoints of the TAD border for each TAD. We calculated the average interaction score for each TAD by summing the interaction scores for all pixels in the submatrix defined by a TAD and dividing by the total number of pixels in the TAD. We then averaged the normalized interaction scores across all bins in a TAD in the Xi (mus) and Xa (cas) contact maps, and computed the fraction of averaged interaction scores from mus chromosomes. ChrX and a representative autosome, Chr5, are shown for the WT cell line and the Xist^ΔXist/+ cell line. P value determined by paired Wilcoxon signed rank test.
(D) Violin plots showing that TADs overlapping restored peaks have larger increases in interaction scores relative to all other TADs. We calculated the fold-change in average interaction scores on the Xi for all X-linked TADs and intersected the TADs with SMC1a sites (Xi^ΔXist/ Xi^WT). 32 TADs occurred at restored cohesin sites; 80 TADs did not overlap restored cohesin sites. Violin plot shows distributions of fold-change average interaction scores between Xi^WT and Xi^ΔXist. p-value deteremined by Wilcoxon ranked sum test.
(E) Restored TADs overlap regions with restored cohesins on across Xi^ΔXist. Several datasets were used to call restored TADs, each producing similar results. Restored TADs were called in two separate replicates (Rep1, Rep2) where the average interaction score was signficantly higher on Xi^ΔXist than on Xi^WT. We also called restored TADs based on merged Rep1+Rep2 datasets. Finally, a consensus between Rep1 and Rep2 was derived. Method: We calculated the fold-change in mus or cas for all TADs on ChrX and on a control, Chr5; then defined a threshold for significant changes based on either the autosomes or the Xa. We treated Chr5 as a null distribution (few changes expected on autosomes) and found the fraction of TADs that crossed the threshold for several thesholds. These fractions corresponded to a false discovery rate (FDR) for each given threshold. An FDR of 0.05 was used.

Xist RNA (red) suppresses the Xi by either recruiting repressive factors (e.g., PRC1, PRC2) or expelling architectural factors (e.g., cohesins).