Composition and dosage of a multipartite enhancer cluster control developmental expression of Ihh (Indian hedgehog) (original) (raw)

Nature Genetics volume 49, pages 1539–1545 (2017)Cite this article

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Abstract

Copy number variations (CNVs) often include noncoding sequences and putative enhancers, but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of IHH (encoding Indian hedgehog), which cause multiple, highly localized phenotypes including craniosynostosis and synpolydactyly1,2. We show through transgenic reporter and genome-editing studies in mice that Ihh is regulated by a constellation of at least nine enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips. Consecutive deletions, resulting in growth defects of the skull and long bones, showed that these enhancers function in an additive manner. Duplications, in contrast, caused not only dose-dependent upregulation but also misexpression of Ihh, leading to abnormal phalanges, fusion of sutures and syndactyly. Thus, precise spatiotemporal control of developmental gene expression is achieved by complex multipartite enhancer ensembles. Alterations in the composition of such clusters can result in gene misexpression and disease.

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Figure 1: A cluster of enhancers interacts with the Ihh promoter during mouse development.

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Figure 2: Deletions of regulatory elements highlight additive control of Ihh expression.

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Figure 3: Duplications of enhancer elements result in Ihh over- and misexpression.

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Figure 4: 4C–seq identifies specific regulatory configurations in duplications.

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Acknowledgements

We thank the sequencing core, transgenic unit and animal facilities of the Max Planck Institute for Molecular Genetics for technical assistance. This study was supported by a grant from the Deutsche Forschungsgemeinschaft to S.M. and E.K. S.M. was supported by the European Community's Seventh Framework Programme, grant agreement 602300 (SYBIL). M.O. was supported by a Swiss National Science Foundation (SNSF) fellowship. A.V. was supported by National Institutes of Health grants R01HG003988, U54HG006997, U01DE024427 and U01DE024427.

Author information

Author notes

  1. Darío G Lupiáñez and Stefan Mundlos: These authors jointly directed this work.

Authors and Affiliations

  1. Max Planck Institute for Molecular Genetics, RG Development and Disease, Berlin, Germany
    Anja J Will, Giulia Cova, Wing-Lee Chan, Norbert Brieske, Darío G Lupiáñez & Stefan Mundlos
  2. Institute for Medical and Human Genetics, Charité–Universitätsmedizin Berlin, Berlin, Germany
    Anja J Will, Giulia Cova, Wing-Lee Chan, Darío G Lupiáñez & Stefan Mundlos
  3. MS 84-171, Lawrence Berkeley National Laboratory, Berkeley, California, USA
    Marco Osterwalder & Axel Visel
  4. Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité–Universitätsmedizin Berlin, Berlin, Germany
    Wing-Lee Chan, Darío G Lupiáñez & Stefan Mundlos
  5. Department of Developmental Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany
    Lars Wittler
  6. Department of Computational Molecular Biology, Max Planck Institute for Molecular Genetics, Berlin, Germany
    Verena Heinrich & Martin Vingron
  7. Genome Dynamics in the Immune System Laboratory, INSERM, UMR 1163, Institut Imagine, Université Paris Descartes, Sorbonne Paris Cité, Paris, France
    Jean-Pierre de Villartay
  8. Institute of Human Genetics, Biocentre, Julius Maximilians University Würzburg, Würzburg, Germany
    Eva Klopocki
  9. US Department of Energy Joint Genome Institute, Walnut Creek, California, USA
    Axel Visel
  10. School of Natural Sciences, University of California, Merced, California, USA
    Axel Visel

Authors

  1. Anja J Will
  2. Giulia Cova
  3. Marco Osterwalder
  4. Wing-Lee Chan
  5. Lars Wittler
  6. Norbert Brieske
  7. Verena Heinrich
  8. Jean-Pierre de Villartay
  9. Martin Vingron
  10. Eva Klopocki
  11. Axel Visel
  12. Darío G Lupiáñez
  13. Stefan Mundlos

Contributions

A.J.W., D.G.L. and S.M. conceived the study and designed the experiments. M.O. and A.V. performed LacZ experiments and analysis of individual enhancers, and A.J.W. performed analysis of Sleeping Beauty insertion. A.J.W. and L.W. generated transgenic mouse models. W.-L.C., J.P.d.V. and G.C. contributed to histological analysis. A.J.W., N.B. and G.C. performed qPCR, in situ hybridization and phenotype analysis. G.C., N.B. and W.-L.C. provided technical support. E.K., M.O. and A.V. contributed to scientific discussion. A.J.W. performed 4C–seq experiments, and V.H., M.V. and D.G.L. performed bioinformatic analysis. A.J.W., D.G.L. and S.M. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence toDarío G Lupiáñez or Stefan Mundlos.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Ihh interacts preferentially with its upstream neighboring gene Nhej1.

Genes are indicated by gray lines, and Ihh and Nhej1 are highlighted in blue. 4C–seq performed in E14.5 limbs using the Ihh promoter as the viewpoint is shown below. Note the increased interactions with intron 3 of the adjacent Nhej1 gene. The gray line indicates the zoomed region displayed in Figure 1. Black bars indicate the size and position of human duplications converted to mouse genome coordinates that overlap with the regulatory landscape of Ihh. Below, Capture-C data from Andrey et al. (2017) at different developmental time points. Chromatin organization is maintained during limb development.

Supplementary Figure 2 Conservation of the IHH locus between mouse and human.

The upper panel shows a representation of the mouse locus with positions indicated for the genes and regulatory elements investigated (blue and gray ovals). Below, ChIP–seq tracks for CTCF with corresponding motif orientation as well as ChIP–seq tracks for active enhancer elements (H3K4me1 and H3K27ac); all experiments were performed in developing limbs at E14.5 (ENCODE). The equivalent positions of human pathogenic duplications are shown at the bottom. The lower panel shows a representation of the human locus with the positions of genes and equivalent positions of the regulatory elements investigated in mouse (blue and gray ovals). Below, ChIP–seq tracks for CTCF with corresponding motif orientation as well as ChIP–seq tracks for active enhancer elements (H3K4me1 and H3K27ac); all are ENCODE data sets for osteoblasts. Note that the convergent orientation of CTCF at the locus is conserved between mouse and human, as well as the presence of active enhancers. The equivalent positions of human pathogenic duplications are shown at the bottom.

Supplementary Figure 3 Transgenic reporter assay (LacZ) of elements positive at E17.5.

Each element displays a lateral view of the embryo at E14.5 (scale bar, 2,000 μm), a dorsal view of the forelimbs (scale bar, 1,000 μm) and a top view of the skull at 17.5 (scale bar, 2,000 μm) together with tissue specificity scoring (bottom). All tested elements appear positive at E17.5 but not at E14.5 and are marked in Figure 1 in gray. An arrowhead indicates positive staining in the skull. The regulatory activity of the region as indicated by the inserted lacZ reporter (SB; black outline) is also displayed.

Supplementary Figure 4 _Nhej1_-knockout mice have normal skulls.

μCT analysis of adult skulls. The red square indicates enlargement of the metopic suture region, shown on the right. An enlargement of the corresponding cross-section (red arrow) of the metopic sutures is shown below. Note the normal development of sutures in _Nhej1_-knockout mice as compared to wild-type controls.

Supplementary Figure 5 Quantitative expression analysis (qPCR) of mutants at different tissues and stages.

(a) Expression analysis of Nhej1. Note that manipulations of the intronic region of the Nhej1 gene do not cause alterations in expression levels overall. (b) Expression analysis of Cnppd1 and Fam134a. Note that the increased contacts observed in 4C–seq experiments for Dup(syn) mutants (Fig. 4b, asterisk) do not cause any alteration in the expression levels of the genes. Bars represent the mean of n = 3 different individuals (circles). Two-sided Student's t test, *P < 0.05; ns, not significant.

Supplementary Figure 6 Enhancer deletions result in delayed skull ossification and reduced bone length.

Left, μCT scan of wild-type mouse forelimb and skull displaying the different regions used for measurement. Right, bone measurements for Del(4–6) and Del(7–9) mutants and wild-type age-matched controls (P70). Note the reduction in ulna and nasal suture length for Del(4–6). Del(4–6) shows a more severe effect on digit length than that observed in Del(7–9) mutants. Bars represent the mean of n = 3 different individuals (circles). Two-sided Student's t test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Supplementary Figure 7 Expression analysis of genes involved in syndactyly/interdigital cell death.

In situ hybridization analysis were performed in E14.5 forelimbs from Dup(syn)/+ mutants and corresponding wt controls. Note increased expression for Bmp4 and Nog as well as expansion of Bmp4 expression in the interdigital space (arrows). Bars represent 200μm.

Supplementary Figure 8 Pathogenic structural variants associated to the Ihh locus.

Schematic of the mouse locus with coordinates of structural variants indicated by colored bars and associated phenotypes. Positions of human duplications were transformed to the mouse genome. Enhancer elements are displayed with ovals. Duplications are depicted in green and deletions in red. All human variants are heterozygous, all mouse variants are homozygous.

Supplementary Figure 9 Limb abnormalities of Dup(syn) mice do not result from increased copies of Ihh gene.

(A) Forelimb morphology of duplications. Dup(syn)/+ mice (3 copies of Ihh gene) display 2/5 syndactyly. Skeletal stainings (right) show short and broad terminal phalanges. Dup(syn) mice were crossed to Del(2-9) or Ihh ko in order to have only 2 functional copies of Ihh, both in the duplicated allele. In both cases compound heterozygous displayed the same phenotypical effects. Bars represent 1000μm for P7 and 500μm for E17.5 autopods. (B) μCT analysis of wt mouse forelimb at P70 displaying the different regions used for measurement. None of the mutant mice displayed alterations in bone length. Bars represent mean of n ≥ 3 different individuals (circles). Two-sided Student's t test, *P< 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

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Will, A., Cova, G., Osterwalder, M. et al. Composition and dosage of a multipartite enhancer cluster control developmental expression of Ihh (Indian hedgehog).Nat Genet 49, 1539–1545 (2017). https://doi.org/10.1038/ng.3939

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