The bipartite TAD organization of the X-inactivation center ensures opposing developmental regulation of Tsix and Xist


The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other’s TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.

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Fig. 1: Genomic inversion of the Xist/Tsix loci switches their promoters into each other’s original TADs.
Fig. 2: Genomic inversion of the Xist/Tsix loci along with Xite and Jpx leads to topological changes within the Xic.
Fig. 3: Xite structural element is important for TAD boundary position and insulation.
Fig. 4: Xist/Tsix inversions lead to ectopic Xist expression, Xist RNA coating and X-linked gene silencing in male mESCs.
Fig. 5: Xist/Tsix inversions affect the initiation of XCI and the expression dynamics of Xist and Tsix during differentiation of female mESCs.

Data availability

Data have been deposited in the NCBI GEO under the accession number GSE111205. Reagents, cell lines and other data supporting the findings of this study are available from the corresponding author upon request.

Code availability

Our custom pipeline for 5C data processing, 5C-Pro, is available at Custom codes used in this study will be provided upon request.


  1. 1.

    de Laat, W. & Duboule, D. Topology of mammalian developmental enhancers and their regulatory landscapes. Nature 502, 499–506 (2013).

  2. 2.

    Dixon, J. R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).

  3. 3.

    Nora, E. P. et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485, 381–385 (2012).

  4. 4.

    Phillips-Cremins, J. E. et al. Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153, 1281–1295 (2013).

  5. 5.

    Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014).

  6. 6.

    Zhan, Y. et al. Reciprocal insulation analysis of Hi-C data shows that TADs represent a functionally but not structurally privileged scale in the hierarchical folding of chromosomes. Genome Res. 27, 479–490 (2017).

  7. 7.

    Vietri Rudan, M. et al. Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture. Cell Rep. 10, 1297–1309 (2015).

  8. 8.

    Le Dily, F. et al. Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation. Genes Dev. 28, 2151–2162 (2014).

  9. 9.

    Shen, Y. et al. A map of the cis-regulatory sequences in the mouse genome. Nature 488, 116–120 (2012).

  10. 10.

    Symmons, O. et al. Functional and topological characteristics of mammalian regulatory domains. Genome Res. 24, 390–400 (2014).

  11. 11.

    Van Bortle, K. et al. Insulator function and topological domain border strength scale with architectural protein occupancy. Genome Biol. 15, R82 (2014).

  12. 12.

    Li, Y. et al. Characterization of constitutive CTCF/cohesin loci: a possible role in establishing topological domains in mammalian genomes. BMC Genomics 14, 553 (2013).

  13. 13.

    Sofueva, S. et al. Cohesin-mediated interactions organize chromosomal domain architecture. EMBO J. 32, 3119–3129 (2013).

  14. 14.

    Nora, E. P. et al. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell 169, 930–944 e22 (2017).

  15. 15.

    Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015).

  16. 16.

    Lupianez, D. G. et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell 161, 1012–1025 (2015).

  17. 17.

    Sanborn, A. L. et al. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes. Proc. Natl Acad. Sci. USA 112, E6456–E6465 (2015).

  18. 18.

    de Wit, E. et al. CTCF binding polarity determines chromatin looping. Mol. Cell 60, 676–684 (2015).

  19. 19.

    Narendra, V. et al. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation. Science 347, 1017–1021 (2015).

  20. 20.

    Tang, Z. et al. CTCF-mediated human 3D genome architecture reveals chromatin topology for transcription. Cell 163, 1611–1627 (2015).

  21. 21.

    Lupianez, D. G., Spielmann, M. & Mundlos, S. Breaking TADs: how alterations of chromatin domains result in disease. Trends Genet. 32, 225–237 (2016).

  22. 22.

    Rastan, S. Non-random X-chromosome inactivation in mouse X-autosome translocation embryos–location of the inactivation centre. J. Embryol. Exp. Morphol. 78, 1–22 (1983).

  23. 23.

    Rastan, S. & Robertson, E. J. X-chromosome deletions in embryo-derived (EK) cell lines associated with lack of X-chromosome inactivation. J. Embryol. Exp. Morphol. 90, 379–388 (1985).

  24. 24.

    Heard, E., Avner, P. & Rothstein, R. Creation of a deletion series of mouse YACs covering a 500 kb region around Xist. Nucleic Acids Res. 22, 1830–1837 (1994).

  25. 25.

    Lee, J. T., Strauss, W. M., Dausman, J. A. & Jaenisch, R. A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell 86, 83–94 (1996).

  26. 26.

    Galupa, R. & Heard, E. X-chromosome inactivation: new insights into cis and trans regulation. Curr. Opin. Genet. Dev. 31, 57–66 (2015).

  27. 27.

    Nesterova, T. B. et al. Pluripotency factor binding and Tsix expression act synergistically to repress Xist in undifferentiated embryonic stem cells. Epigenetics Chromatin 4, 17 (2011).

  28. 28.

    Navarro, P. et al. Molecular coupling of Xist regulation and pluripotency. Science 321, 1693–1695 (2008).

  29. 29.

    Sousa, E. J. et al. Exit from naive pluripotency induces a transient X chromosome Inactivation-like state in males. Cell Stem Cell 22, 919–928 e6 (2018).

  30. 30.

    Lee, J. T. Regulation of X-chromosome counting by Tsix and Xite sequences. Science 309, 768–771 (2005).

  31. 31.

    Debrand, E., Chureau, C., Arnaud, D., Avner, P. & Heard, E. Functional analysis of the DXPas34 locus, a 3′ regulator of Xist expression. Mol. Cell Biol. 19, 8513–8525 (1999).

  32. 32.

    Lee, J. T., Davidow, L. S. & Warshawsky, D. Tsix, a gene antisense to Xist at the X-inactivation centre. Nat. Genet. 21, 400–404 (1999).

  33. 33.

    Mise, N., Goto, Y., Nakajima, N. & Takagi, N. Molecular cloning of antisense transcripts of the mouse Xist gene. Biochem. Biophys. Res. Commun. 258, 537–541 (1999).

  34. 34.

    Tian, D., Sun, S. & Lee, J. T. The long noncoding RNA, Jpx, is a molecular switch for X chromosome inactivation. Cell 143, 390–403 (2010).

  35. 35.

    Barakat, T. S. et al. RNF12 activates Xist and is essential for X chromosome inactivation. PLoS Genet. 7, e1002001 (2011).

  36. 36.

    Furlan, G. et al. The Ftx noncoding locus controls X chromosome inactivation independently of its RNA products. Mol. Cell 70, 462–472 e8 (2018).

  37. 37.

    Augui, S., Nora, E. P. & Heard, E. Regulation of X-chromosome inactivation by the X-inactivation centre. Nat. Rev. Genet. 12, 429–442 (2011).

  38. 38.

    Pollex, T. & Heard, E. Recent advances in X-chromosome inactivation research. Curr. Opin. Cell Biol. 24, 825–832 (2012).

  39. 39.

    van Bemmel, J. G., Mira-Bontenbal, H. & Gribnau, J. Cis- and trans-regulation in X inactivation. Chromosoma 125, 41–50 (2016).

  40. 40.

    Hughes, J. R. et al. Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment. Nat. Genet. 46, 205–212 (2014).

  41. 41.

    Davies, J. O. et al. Multiplexed analysis of chromosome conformation at vastly improved sensitivity. Nat. Methods 13, 74–80 (2016).

  42. 42.

    Dostie, J. et al. Chromosome conformation capture carbon copy (5C): a massively parallel solution for mapping interactions between genomic elements. Genome Res. 16, 1299–1309 (2006).

  43. 43.

    Merkenschlager, M. & Nora, E. P. CTCF and cohesin in genome folding and transcriptional gene regulation. Annu. Rev. Genom. Hum. Genet. 17, 17–43 (2016).

  44. 44.

    Ogawa, Y. & Lee, J. T. Xite, X-inactivation intergenic transcription elements that regulate the probability of choice. Mol. Cell 11, 731–743 (2003).

  45. 45.

    Stavropoulos, N., Rowntree, R. K. & Lee, J. T. Identification of developmentally specific enhancers for Tsix in the regulation of X chromosome inactivation. Mol. Cell Biol. 25, 2757–2769 (2005).

  46. 46.

    Barakat, T. S. et al. The trans-activator RNF12 and cis-acting elements effectuate X chromosome inactivation independent of X-pairing. Mol. Cell 53, 965–978 (2014).

  47. 47.

    Sun, S. et al. Jpx RNA activates Xist by evicting CTCF. Cell 153, 1537–1551 (2013).

  48. 48.

    Carmona, S., Lin, B., Chou, T., Arroyo, K. & Sun, S. LncRNA Jpx induces Xist expression in mice using both trans and cis mechanisms. PLoS Genet. 14, e1007378 (2018).

  49. 49.

    Giorgetti, L. et al. Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription. Cell 157, 950–963 (2014).

  50. 50.

    Spencer, R. J. et al. A boundary element between Tsix and Xist binds the chromatin insulator Ctcf and contributes to initiation of X-chromosome inactivation. Genetics 189, 441–454 (2011).

  51. 51.

    Jegu, T., Aeby, E. & Lee, J. T. The X chromosome in space. Nat. Rev. Genet. 18, 377–389 (2017).

  52. 52.

    Engreitz, J. M. et al. The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, 1237973 (2013).

  53. 53.

    Brockdorff, N. & Turner, B. M. Dosage compensation in mammals. Cold Spring Harb. Perspect. Biol. 7, a019406 (2015).

  54. 54.

    Brons, I. G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007).

  55. 55.

    Guo, G. et al. Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063–1069 (2009).

  56. 56.

    Franke, M. et al. Formation of new chromatin domains determines pathogenicity of genomic duplications. Nature 538, 265–269 (2016).

  57. 57.

    Rodriguez-Carballo, E. et al. The HoxD cluster is a dynamic and resilient TAD boundary controlling the segregation of antagonistic regulatory landscapes. Genes Dev. 31, 2264–2281 (2017).

  58. 58.

    Johnston, C. M., Newall, A. E., Brockdorff, N. & Nesterova, T. B. Enox, a novel gene that maps 10 kb upstream of Xist and partially escapes X inactivation. Genomics 80, 236–244 (2002).

  59. 59.

    Chureau, C. et al. Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region. Hum. Mol. Genet. 20, 705–718 (2011).

  60. 60.

    Hofmann, A. & Heermann, D. W. The role of loops on the order of eukaryotes and prokaryotes. FEBS Lett. 589, 2958–2965 (2015).

  61. 61.

    Kent, W. J. et al. The human genome browser at UCSC. Genome Res 12, 996–1006 (2002).

  62. 62.

    Pillet, N., Bonny, C. & Schorderet, D. F. Characterization of the promoter region of the mouse Xist gene. Proc. Natl Acad. Sci. USA 92, 12515–12519 (1995).

  63. 63.

    Gontan, C. et al. RNF12 initiates X-chromosome inactivation by targeting REX1 for degradation. Nature 485, 386–390 (2012).

  64. 64.

    Doetschman, T. et al. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330, 576–578 (1987).

  65. 65.

    Norris, D. P. et al. Evidence that random and imprinted Xist expression is controlled by preemptive methylation. Cell 77, 41–51 (1994).

  66. 66.

    Masui, O. et al. Live-cell chromosome dynamics and outcome of X chromosome pairing events during ES cell differentiation. Cell 145, 447–458 (2011).

  67. 67.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  68. 68.

    Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

  69. 69.

    Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171–192 (2012).

  70. 70.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

  71. 71.

    Servant, N. et al. HiTC: exploration of high-throughput ‘C’ experiments. Bioinformatics 28, 2843–2844 (2012).

  72. 72.

    Hnisz, D. et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science 351, 1454–1458 (2016).

  73. 73.

    Smith, E. M., Lajoie, B. R., Jain, G. & Dekker, J. Invariant TAD boundaries constrain cell-type-specific looping interactions between promoters and distal elements around the CFTR Locus. Am. J. Hum. Genet. 98, 185–201 (2016).

  74. 74.

    Crane, E. et al. Condensin-driven remodelling of X chromosome topology during dosage compensation. Nature 523, 240–244 (2015).

  75. 75.

    Servant, N. et al. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. Genome Biol. 16, 259 (2015).

  76. 76.

    Geiss, G. K. et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat. Biotechnol. 26, 317–325 (2008).

  77. 77.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

  78. 78.

    Mudge, J. M. & Harrow, J. Creating reference gene annotation for the mouse C57BL6/J genome assembly. Mamm. Genome 26, 366–378 (2015).

  79. 79.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  80. 80.

    McCarthy, D. J., Chen, Y. & Smyth, G. K. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 40, 4288–4297 (2012).

  81. 81.

    Borensztein, M. et al. Xist-dependent imprinted X inactivation and the early developmental consequences of its failure. Nat. Struct. Mol. Biol. 24, 226–233 (2017).

  82. 82.

    Chaumeil, J., Augui, S., Chow, J. C. & Heard, E. Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation. Methods Mol. Biol. 463, 297–308 (2008).

  83. 83.

    Engreitz, J. M. et al. RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent pre-mRNAs and chromatin sites. Cell 159, 188–199 (2014).

  84. 84.

    Chen, C. K. et al. Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing. Science 354, 468–472 (2016).

  85. 85.

    Zeng, P. Y., Vakoc, C. R., Chen, Z. C., Blobel, G. A. & Berger, S. L. In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation. Biotechniques 41, 696–698 (2006).

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We would like to thank the Heard laboratory for their technical input and critical discussions; D. Noordermeer for critical discussion and advice on Capture-C data analysis and interpretation; A. Chow from the Guttman laboratory for RAP-DNA cell culture; members of the Bourc’his laboratory; C. Reyes and A. Rapinat from the Nanostring platform and J. M. Telenius, M. Oudelaar and D. Downes from the Hughes and Higgs laboratories. This work was supported by an ERC Advanced Investigator award (ERC-2014-AdG no. 671027), Labelisation La Ligue, FRM (grant no. DEI20151234398), ANR DoseX 2017, Labex DEEP (no. ANR-11-LBX-0044), part of the IDEX Idex PSL (no. ANR-10-IDEX-0001-02 PSL) and ABS4NGS (no. ANR-11-BINF-0001) to E.H.; NWO-ALW Rubicon (no. 825.13.002) and Veni (no. 863.15.016) fellowships to J.G.v.B.; Région Ile-de-France (DIM Biothérapies) and Fondation pour la Recherche Médicale (no. FDT20160435295) fellowships to R.G.; Sir Henry Wellcome Postdoctoral Fellowship (no. 201369/Z/16/Z) to J.J.Z.; MRC Clinician Scientist Fellowship (no. MR/R008108) to J.D.; Wellcome Trust Strategic Award (no. 106130/Z/14/Z) to J.R.H.; New York Stem Cell Foundation and California Institute of Technology funds to M.G. (M.G. is a New York Stem Cell Foundation—Robertson Investigator); Novartis Foundation and European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 759366 ‘BioMeTre’) to L.G.; LabEx and EquipEx (nos. ANR-10-IDEX-0001-02 PSL, ANR-11-LBX-0044 and ‘INCa-DGOS-4654’ SIRIC11-002) to the Nanostring platform of Institut Curie; Equipex (no. ANR-10-EQPX-03), France Génomique Consortium from the Agence Nationale de la Recherche (‘Investissements d’Avenir’ program; no. ANR-10-INBS-09-08) and Canceropole Ile-de-France and by the SiRIC-Curie program—SiRIC grant (no. INCa-DGOS-4654) to the ICGex Next Generation Sequencing platform of the Institut Curie.

Author information

J.G.v.B., J.G. and E.H. conceived the study, with support from R.G., L.G. and E.P.N. J.G.v.B., N.S. (lead), R.G., A.J.S., Y.Z., E.d.W. and L.G. (equal) conducted the formal analysis. J.G.v.B and R.G. led the investigation. C.G., A.J.S., C.P., E.P.N., J.J.Z. and S.L. supported the investigation. J.D., Y.Z., L.G., J.D., J.R.H. and D.R.H. provided resources. J.G.v.B. and E.H. wrote and prepared the original draft, with support from E.P.N., R.G. and C.G and input from all authors. R.G. and E.H. led the revision and editing of the article, with support from J.G.v.B. J.G.v.B. and R.G. provided data visualization. J.G.v.B., R.G. and E.H. supervised the study, with support from J.D., D.G., S.B., M.G., J.R.H., D.R.H. and J.G. The funding was acquired by E.H., J.G.v.B. and J.G.

Correspondence to Joke G. van Bemmel or Edith Heard.

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