X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region1,2, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts3. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed4,5,6. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite7,8,9,10. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions, remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC–seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes in different neural progenitor clones are associated with the presence of different TAD-like structures after XCI. These findings suggest a key role for transcription and CTCF in the formation of TADs in the context of the Xi chromosome in neural progenitors.
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We thank members of the Heard, Dekker, and Chang laboratories for their help and critical insights; PICT-IBiSA@BDD (UMR3215/U934) Imaging facility of the Institut Curie. L.G. would like to thank L. Mirny for discussing gyration tensor analysis. Supported by grants from the National Institutes of Health (P50-HG007735) and Scleroderma Research Foundation (to H.Y.C.), from the National Human Genome Research Institute (R01 HG003143) and the National Institutes of Health Common Fund, National Institute of Diabetes and Digestive and Kidney Diseases (U54 DK107980) to J.D., the Human Frontier Science Program to N.K., an EMBO Fellowship to L.G., an ERC Advanced Investigator award (ERC-2014-AdG no. 671027), EU FP7 grants SYBOSS (EU 7th Framework G.A. no. 242129) and MODHEP (EU 7th Framework G.A. no. 259743), La Ligue, Fondation de France, Labex DEEP (ANR-11-LBX-0044) part of the IDEX Idex PSL (ANR-10-IDEX-0001-02 PSL) and ABS4NGS (ANR-11-BINF-0001) to E.H. J.D. is an investigator of the Howard Hughes Medical Institute.
Extended data figures
This table contains all relevant RNA-Seq data for all locations (genes) along the X chromosome. The table columns are as follows: 1, xloc; 2, chr; 3, start; 4, end; 5, gene; 6, B129T3__129S1__category; 7, B129T3__129S1__pval; 8, B129T3__129S1__reads; 9, B129T3__129S1__rpkm; 10, B129T3__129S1__status; 11, B129T3__CAST__category; 12, B129T3__CAST__pval; 13, B129T3__CAST__reads; 14, B129T3__CAST__rpkm; 15, B129T3__CAST__status; 16, GEI.72b__129S1__category; 17, GEI.72b__129S1__pval; 18, GEI.72b__129S1__reads; 19, GEI.72b__129S1__rpkm; 20, GEI.72b__129S1__status; 21, GEI.72b__CAST__category; 22, GEI.72b__CAST__pval; 23, GEI.72b__CAST__reads; 24, GEI.72b__CAST__rpkm; 25, GEI.72b__CAST__status; 26, GUR.2d__129S1__category; 27, GUR.2d__129S1__pval; 28, GUR.2d__129S1__reads; 29, GUR.2d__129S1__rpkm; 30, GUR.2d__129S1__status; 31, GUR.2d__CAST__category; 32, GUR.2d__CAST__pval; 33, GUR.2d__CAST__reads; 34, GUR.2d__CAST__rpkm; 35, GUR.2d__CAST__status. xloc is a numerical ID for each gene location. chr is the chromosome. start is the start position of the gene. end is the end position of the gene. (for positions, start > end, not re-oriented by strand) gene is the gene name. The remaining columns are broken down into groups of 5 per sample, per allele. NNNN is the sample name. ESC = GUR.2d; WT NPC = GEI.72b; ΔFT NPC = B129T3 (D9B2). XXXX is the allele, 129S1 for the 129 allele, CAST for the Cast allele. The five columns are: NNNN__XXXX_category, category assignment of expression (bi,mono,biased,na, see ref 20) NNNN__XXXX__pval, p-value of the allelic assignment. NNNN__XXXX__reads, number of allelic reads. NNNN__XXXX__rpkm, RPKM value for the allelic gene. NNNN__XXXX__status, expression status of the gene, expressed or silenced. We defined expressed as ≥ 3 RPKM.
About this article
Nature Communications (2018)