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Spatial partitioning of the regulatory landscape of the X-inactivation centre

Naturevolume 485, pages381–385 (2012)Cite this article

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Abstract

In eukaryotes transcriptional regulation often involves multiple long-range elements and is influenced by the genomic environment1. A prime example of this concerns the mouse X-inactivation centre (Xic), which orchestrates the initiation of X-chromosome inactivation (XCI) by controlling the expression of the non-protein-codingXist transcript. The extent ofXic sequences required for the proper regulation ofXist remains unknown. Here we use chromosome conformation capture carbon-copy (5C)2 and super-resolution microscopy to analyse the spatial organization of a 4.5-megabases (Mb) region includingXist. We discover a series of discrete 200-kilobase to 1 Mb topologically associating domains (TADs), present both before and after cell differentiation and on the active and inactive X. TADs align with, but do not rely on, several domain-wide features of the epigenome, such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters. Disruption of a TAD boundary causes ectopic chromosomal contacts and long-range transcriptional misregulation. TheXist/Tsix sense/antisense unit illustrates how TADs enable the spatial segregation of oppositely regulated chromosomal neighbourhoods, with the respective promoters ofXist andTsix lying in adjacent TADs, each containing their known positive regulators. We identify a novel distal regulatory region ofTsix within its TAD, which produces a long intervening RNA,Linx. In addition to uncovering a new principle ofcis-regulatory architecture of mammalian chromosomes, our study sets the stage for the full genetic dissection of the X-inactivation centre.

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Figure 1:Chromosome partitioning into topologically associating domains (TADs).
Figure 2:Determinants of topologically associating domains.
Figure 3:Dynamics of topologically associating domains during cell differentiation.
Figure 4:Transcriptional co-regulation within topologically associating domains.
Figure 5:5C maps reveal new regulatory regions in theXic.

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Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

High-throughput data are deposited in Gene ExpressionOmnibus under accession numberGSE35721 for all 5C experiments andGSE34243 for expression microarrays.

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Acknowledgements

We thank T. Pollex and T. Forné for experimental help; the imaging facility PICTIBiSA@BDD for technical assistance, D. Gentien and C. Hego for microarray hybridizations. We thank K. Bernhard, F. Stewart and A. Smith for protocols and material for 2i culture and EpiSC differentiation. We are grateful to members of the E.H. laboratory for critical input. This work was funded by grants from the Ministère de la Recherche et de l’Enseignement Supérieur and the ARC (to E.P.N.); a HFSP Long term fellowship (LT000597/2010-L) (to E.G.S.). EU EpiGeneSys FP7 Network of Excellence no. 257082, the Fondation pour la Recherche Medicale, ANR, ERC Advanced Investigator award no. 250367 and EU FP7 SYBOSS grant no. 242129 (to E.H.). N.B. was supported by BMBF (FORSYS) and EMBO (fellowship ASTF 307-2011). J.D., B.R.L. and N.L.v.B. were supported by NIH (R01 HG003143) and a W. M. Keck Foundation Distinguished Young Scholar Award.

Author information

Author notes

  1. Bryan R. Lajoie, Edda G. Schulz and Luca Giorgetti: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Curie, 26 rue d'Ulm, Paris F-75248, France ,

    Elphège P. Nora, Edda G. Schulz, Luca Giorgetti, Ikuhiro Okamoto, Nicolas Servant, Tristan Piolot, Emmanuel Barillot & Edith Heard

  2. CNRS UMR3215, Paris F-75248, France ,

    Elphège P. Nora, Edda G. Schulz, Luca Giorgetti, Ikuhiro Okamoto, Tristan Piolot & Edith Heard

  3. INSERM U934, Paris F-75248, France ,

    Elphège P. Nora, Edda G. Schulz, Luca Giorgetti, Ikuhiro Okamoto, Tristan Piolot & Edith Heard

  4. Department of Biochemistry and Molecular Pharmacology, Programs in Systems Biology and Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts, 01605-0103, USA

    Bryan R. Lajoie, Nynke L. van Berkum & Job Dekker

  5. INSERM U900, Paris, F-75248 France ,

    Nicolas Servant & Emmanuel Barillot

  6. Mines ParisTech, Fontainebleau, F-77300 France ,

    Nicolas Servant & Emmanuel Barillot

  7. Institute of Pathology, Charité–Universitätsmedizin, 10117 Berlin, and Institute of Theoretical Biology Humboldt Universität, 10115 Berlin, Germany ,

    Johannes Meisig & Nils Blüthgen

  8. Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, 94158-2517, California, USA

    John Sedat

  9. Department of Reproduction and Development, Erasmus MC, University Medical Center, 3000 CA Rotterdam, The Netherlands,

    Joost Gribnau

Authors
  1. Elphège P. Nora

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  2. Bryan R. Lajoie

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  3. Edda G. Schulz

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  4. Luca Giorgetti

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  9. Johannes Meisig

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  10. John Sedat

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  11. Joost Gribnau

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  14. Job Dekker

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  15. Edith Heard

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Contributions

E.P.N. performed and analysed 3C, 5C, (RT–)qPCR, immunofluorescence, RNA and DNA FISH. B.R.L. and N.L.v.B. helped in the design and/or the analysis of 3C and 5C. L.G. performed 3C, FISH and 5C analysis. E.G.S. generated the time-course transcriptomic data, which was analysed by J.M. and N.B.; I.O. performed FISH on pre-implantation embryos. J.G. donated the XTX mouse ESC line. N.S. and E.B. helped in the epigenomic and 5C analyses. J.S. and T.P. set up OMX microscopy and analysis and T.P. performed structured illumination microscopy and image analysis. The manuscript was written by E.P.N., J.D. and E.H. with contribution from E.G.S. and input from all authors.

Corresponding authors

Correspondence toJob Dekker orEdith Heard.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-15, Supplementary Methods and additional references. (PDF 10774 kb)

Supplementary Data 1

This file contains mapped 5C counts. (ZIP 8192 kb)

Supplementary Data 2

This file contains intraTAD 5C peaks. (ZIP 510 kb)

Supplementary Table 1

This file contains 5C Oligos and restriction Fragment. (XLS 562 kb)

Supplementary Table 2

This file contains cDNA microarrays. (XLS 382 kb)

Supplementary Table 3

This file contains the sequencing log. (XLS 57 kb)

Supplementary Table 4

This file contains qPCR Primers. (XLS 12 kb)

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Nora, E., Lajoie, B., Schulz, E.et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre.Nature485, 381–385 (2012). https://doi.org/10.1038/nature11049

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Editorial Summary

Genome organization revealed

The spatial organization of the genome is linked to biological function, and advances in genomic technologies are allowing the conformation of chromosomes to be assessed genome wide. Two groups present complementary papers on the subject. Bing Ren and colleagues use Hi-C, an adaption of the chromosome conformation capture (3C) technique, to investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types. Large, megabase-sized chromatin interaction domains, termed topological domains, are found to be a pervasive and conserved feature of genome organization. Edith Heard and colleagues use chromosome conformation capture carbon-copy (5C) technology and high-resolution microscopy to obtain a high-resolution map of the chromosomal interactions over a large region of the mouse X chromosome, including the X-inactivation centre. A series of discrete topologically associating domains is revealed, as is a previously unknown long intergenic RNA with a potential regulatory role.

Associated content

Topological domains in mammalian genomes identified by analysis of chromatin interactions

  • Jesse R. Dixon
  • Siddarth Selvaraj
  • Bing Ren
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