Abstract

Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions1,2. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes3,4,5. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans6, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.

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Acknowledgements

We are grateful to all members of the MPIMG transgene and mouse facility for embryonic stem cell aggregation and mouse husbandry. This work was supported by grants from the Deutsche Forschungsgemeinschaft to S.M. and F.S., the BIH to D.M.I., S.M. and A.P., and the Max Planck Foundation to S.M.

Author information

Author notes

    • Martin Franke
    •  & Daniel M. Ibrahim

    These authors contributed equally to this work.

Affiliations

  1. Max Planck Institute for Molecular Genetics, RG Development & Disease, 14195 Berlin, Germany

    • Martin Franke
    • , Daniel M. Ibrahim
    • , Guillaume Andrey
    • , Katerina Kraft
    • , Rieke Kempfer
    • , Ivana Jerković
    • , Malte Spielmann
    •  & Stefan Mundlos
  2. Institute for Medical and Human Genetics, Charité Universitätsmedizin Berlin, 13353 Berlin, Germany

    • Martin Franke
    • , Daniel M. Ibrahim
    • , Verena Heinrich
    • , Katerina Kraft
    • , Ivana Jerković
    • , Wing-Lee Chan
    • , Malte Spielmann
    •  & Stefan Mundlos
  3. Berlin Institute of Health, 10117 Berlin, Germany

    • Daniel M. Ibrahim
    • , Ana Pombo
    •  & Stefan Mundlos
  4. Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany

    • Wibke Schwarzer
    •  & Francois Spitz
  5. Max Planck Institute for Molecular Genetics, Department of Computational Molecular Biology, 14195 Berlin, Germany

    • Verena Heinrich
    • , Robert Schöpflin
    •  & Martin Vingron
  6. Max Planck Institute for Molecular Genetics, Sequencing Core Facility, 14195 Berlin, Germany

    • Bernd Timmermann
  7. Max Planck Institute for Molecular Genetics, Department Developmental Genetics, 14195 Berlin, Germany

    • Lars Wittler
  8. Institute of Human Genetics, Jena University Hospital, 07743 Jena, Germany

    • Ingo Kurth
  9. Institute of Human Genetics, Uniklinik RWTH Aachen, 52074 Aachen, Germany

    • Ingo Kurth
  10. Bambino Gesù Children's Hospital-IRCCS, 00165 Rome, Italy

    • Paola Cambiaso
  11. Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy

    • Orsetta Zuffardi
  12. Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, 5021 Bergen, Norway

    • Gunnar Houge
  13. Division of Human Genetics, National Health Laboratory Service, University of the Witwatersrand, 2000 Johannesburg, South Africa

    • Lindsay Lambie
  14. Department of Life, Health and Environmental Sciences, University of L’Aquila, 67100 L’Aquila, Italy

    • Francesco Brancati
  15. Istituto Dermopatico dell’Immacolata (IDI) IRCCS, 00167 Rome, Italy

    • Francesco Brancati
  16. Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, 13125 Berlin-Buch, Germany

    • Ana Pombo
  17. Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany

    • Stefan Mundlos

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Contributions

M.F., F.S. and S.M. conceived the study and designed the experiments. M.F. and G.A. performed 4C-seq, capture Hi-C, with analysis by V.H., D.M.I. and R.S. M.F. and W.S. performed the LacZ staining and analysis. M.F. and D.M.I. performed RNA-seq, in situ hybridizations and phenotype analysis. W.-L.C. and I.J. contributed to histological analysis. M.F., W.S., R.K., D.M.I., K.K. and L.W. generated the transgenic mouse models. I.K., P.C., O.Z., G.H., M.S., L.L. and F.B. obtained the patient samples. A.P., W.S., F.S., M.S., M.V. and B.T. contributed to scientific discussion and technical support. M.F., D.M.I. and S.M. wrote the paper with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Stefan Mundlos.

Sequencing data has been deposited in Gene Expression Omnibus (GEO) under GSE78109.

Reviewer Information Nature thanks B. Ren and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    This file contains sequences of guide RNAs für CRISPR/Cas9 experiments and primer sequences for cloning of gene targeting constructs and probes for in situ hybridization.

  2. 2.

    Supplementary Table 2

    This table contains 4C-seq primer sequences for inverse PCR and digestion strategy for 4C library preparation.

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DOI

https://doi.org/10.1038/nature19800

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