Letter | Published:

Structural organization of the inactive X chromosome in the mouse

Nature volume 535, pages 575579 (28 July 2016) | Download Citation


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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Primary accessions

Gene Expression Omnibus

Data deposits

Sequencing data have been deposited in the Gene Expression Omnibus (GEO) under accession numbers GSE72697 (Hi-C); GSE71156 (ATAC–seq); and GSE72697 (boundary deletion data).


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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.

Author information

Author notes

    • Luca Giorgetti
    • , Bryan R. Lajoie
    • , Ava C. Carter
    •  & Mikael Attia

    These authors contributed equally to this work.

    • Luca Giorgetti

    Present address: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.


  1. Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, 26 Rue d’Ulm, 75248 Paris Cedex 05, France

    • Luca Giorgetti
    • , Mikael Attia
    • , Chong Jian Chen
    •  & Edith Heard
  2. Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 368 Plantation Street, Worcester, Massachusetts 01605, USA

    • Bryan R. Lajoie
    • , Ye Zhan
    • , Noam Kaplan
    •  & Job Dekker
  3. Center for Personal Dynamic Regulomes and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Ava C. Carter
    • , Jin Xu
    •  & Howard Y. Chang
  4. Collège de France, 11 place Marcelin-Berthelot, Paris 75005, France

    • Edith Heard
  5. Howard Hughes Medical Institute, University of Massachusetts Medical School, 368 Plantation Street, Worcester, Massachusetts 01605, USA

    • Job Dekker


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E.H. and J.D. conceived the original strategy. For Hi-C, M.A. and L.G. prepared the ES cell and NPC samples, Y.Z. performed the Hi-C experiments; for ATAC–seq, H.Y.C., E.H. and J.D. designed the experiments; A.C.C. and J.X. prepared the samples and performed the experiments; for RNA-seq, M.A. and L.G. prepared the samples and performed the experiments and C.J.C. analysed the data. Integrated analysis of Hi-C sequencing, RNA-seq and ATAC–seq data was performed by B.R.L., J.X., L.G. and A.C.C., with assistance from C.J.C. and N.K. and input from J.D.; L.G. and E.H. designed the FISH experiments, L.G. and M.A. performed FISH experiment and L.G. analysed data. M.A., L.G. and E.H. designed the NPC and DXZ4 mutant strategy, M.A. and L.G. performed the experiments and analysed them. L.G., B.R.L., A.C.C., E.H. and J.D. wrote the manuscript with input from H.Y.C.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Edith Heard or Job Dekker.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table

    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.

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