Outline of the allele-specific 4C approach to interrogate X-chromosome folding. (A) Schematic outline of the allele-specific 4C approach. Restriction fragment length polymorphisms (RFLPs) were identified and used to direct the 4C analysis to either the 129SVJ or the CAST allele. (B) 4C PCR products using allele-specific primers (Jarid1C, 129SVJ-specific) and primers not designed around a RFLP (Hbb-b1) separated on an agarose gel. (C) Four genes with different characteristics, representing different genomic environments, were chosen as viewpoints for the 4C analysis. An alternative allele-specific 4C approach and details concerning the use of NGS to analyze the 4C data can be found in Supplemental Figure S1.

Long-range interactions engaged by the different viewpoints separate active from inactive chromatin on the active X chromosome. (A) Spider plots combined with domainograms depict long-range interactions identified with the four different viewpoints on the active X chromosome. Significant interactions are depicted in the spider plot by the different colored lines, each color representing a different viewpoint. Small black bars below the spider plot represent genes located on the X chromosome. Significance of interaction is indicated by the range in color used in the domainogram below each spider plot; black is low significance (P = 1) and yellow represents high significance (P = 10⁻¹⁰) of interaction. (B) Interactions with other chromosomes are depicted in Circos plots; each line represents a trans interaction. Chromosomes are plotted around the circle. Colors indicate the chromosomes that were contacted. (C) Cis interactions from A merged into one figure for comparison. Interactions are represented by the colored bars (MeCP2, blue; Jarid1C, red; Slitrk4, gold; and Pcdh11x, green). The colored asterisk indicates the position of each viewpoint on the X chromosome. (D) Spearman's correlation calculated comparing 4C profiles of Pctk1^Xa, Slitrk4^Xa, Pcdh11x^Xa, and Jarid1C^Xa to MeCP2^Xa. Further characterization of the identified interacting regions can be found in Supplemental Figure S3.

Allele-specific 4C analysis reveals dramatic changes in chromosome conformation on the inactive X chromosome. (A) Spider plots combined with domainograms depict long-range interactions identified on the inactive X chromosome. (B) Trans interactions of the different viewpoints are depicted in Circos plots; each line represents a trans interaction. Detailed analysis of the distribution of captured sequences on the X_i can be found in Supplemental Figure S4.

Escaping genes cluster in nuclear space. Jarid1C^Xi-interacting regions contained other escapees. (A) Genes known to escape XCI—(from left to right) Utx, Eif2s3x, Xist, and MidI—are indicated by arrowheads below the chromosome. The location and size of these regions are represented by the scaled black bars. The gel pictures show the result of the allele-specific cDNA analysis performed on the indicated genes (gray) located within the Jarid1C^Xi-interacting regions. Other genes present in the interrogated region are drawn in black. The asterisk indicates the PCR product of transcripts originating from the X_i. Map3k7ip3 and Drp2 locate outside Jarid1C^Xi-interacting regions and were found silenced on X_i. (B) Spider plot depicting long-range interactions of Pctk1^Xi (top) and Xist^Xi (bottom). (C) Bars representing long-range cis interactions on the X_i from Xist, Jarid1C, and Pctk1 are plotted for comparison. Asterisks indicate the position of the interrogated genes on the X chromosome. 3D FISH analysis confirming the interactions identified by 4C can be found in Supplemental Figure S5A–C.

The inactive X chromosome partially refolds to the active X chromosome upon conditional deletion of Xist. (A) Schematic representation of the Xist knockout strategy. (B) Allele-specific quantitative PCR (qPCR) result, measuring recombination efficiency. (C) The quantification of the Xist RNA FISH applied on Xist^KO and Xist^CON NPCs. A representative example of the FISH experiment is shown in Supplemental Figure S7A. (D,E) Quantification of Ezh2 and H3K27me3 clouds, respectively, identified by immunofluorescence applied on Xist^KO NPCs. (F) Bisulfite sequencing result of X-linked gene promoters in both the control NPCs and Xist^KO NPCs. Open circles represent nonmethylated CpGs, while methylated CpGs are represented by the filled dots. (G) Allele-specific expression analysis of four X-linked genes in control NPCs and Xist^KO NPCs. Allelic transcript contribution is visualized by separating digested RT–PCR products using a RFLP-recognizing restriction enzyme on an agarose gel (see also Supplemental Fig. S7). (H) Spider plots depicting long-range interactions of Pctk1^Xi and MeCP2^Xi identified in the control NPCs (top) and Xist^KO NPCs (bottom). (I) Spearman's rank correlation calculated comparing the 4C profiles identified in the control NPCs and Xist^KO NPCs with the corresponding X_a profile of the indicated viewpoints (see also Supplemental Fig. S7).