Abstract

The extracellular signal–related kinases 1 and 2 (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets1. Here, we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs. 2,3,4,5,6,7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple-suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf levels reduced to 30% of normal exhibit postnatal multiple-suture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to elements away from promoters that contain RUNX or AP-1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes.

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Acknowledgements

We thank R. Boehm, H. Care, C. Langman, J. Phipps and E. Sweeney for clinical assistance, L. Gregory, P. Piazza and staff at the High-Throughput Genomics facility at the Wellcome Trust Centre for Human Genetics for exome sequencing, C. Babbs, S. Butler, J. Frankland, C. Rode and T. Rostron for technical help, K. Kourouniotis for blastocyst injections and expert animal support, E. Giannoulatou for bioinformatics assistance, and B. Graves and P. Hollenhorst for discussions. We thank J. Heath (University of Birmingham), G. Schwabe (Charité University Hospital) and D. Rice (University of Helsinki) for the gifts of the Spp1, Runx2 and Bglap2 probes, respectively. This work was funded by the Greek Ministry of Education grants PYTHAGORAS II KA2092, PENED 03ED626, HERAKLEITOS II KA 3396 and SYNERGASIA 09SYN-11-902 to G.M., the NIHR Biomedical Research Centre, with funding from the Department of Health's NIHR Biomedical Research Centres funding scheme (S.J.L.K. and A.O.M.W.), the Oxford Craniofacial Unit Charitable Fund (V.P.S.), the Department of Health, UK, Quality, Improvement, Development and Initiative Scheme (QIDIS) (V.P.S.) and the Wellcome Trust (090532, S.J.L.K.; 093329, S.R.F.T. and A.O.M.W.). The views expressed in this publication are those of the authors and not necessarily those of the Department of Health, UK.

Author information

Author notes

    • Elena Vorgia
    • , Simon J McGowan
    • , Ioanna Peraki
    •  & Aimée L Fenwick

    These authors contributed equally to this work.

Affiliations

  1. Clinical Genetics Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

    • Stephen R F Twigg
    • , Aimée L Fenwick
    • , Vikram P Sharma
    •  & Andrew O M Wilkie
  2. Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology–Hellas, Heraklion, Greece.

    • Elena Vorgia
    • , Ioanna Peraki
    • , Maryline Allegra
    •  & George Mavrothalassitis
  3. School of Medicine, University of Crete, Heraklion, Greece.

    • Elena Vorgia
    • , Ioanna Peraki
    • , Andreas Zaragkoulias
    •  & George Mavrothalassitis
  4. Computational Biology Research Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

    • Simon J McGowan
    •  & Stephen Taylor
  5. Craniofacial Unit, Oxford University Hospitals National Health Service (NHS) Trust, Oxford, UK.

    • Vikram P Sharma
    • , David Johnson
    • , Steven A Wall
    •  & Andrew O M Wilkie
  6. National Institute for Health Research (NIHR) Biomedical Research Centre, Oxford and Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

    • Elham Sadighi Akha
    •  & Samantha J L Knight
  7. Molecular Genetics Laboratory, Oxford University Hospitals NHS Trust, Oxford, UK.

    • Helen Lord
    •  & Tracy Lester
  8. Clinical Genetics, Guy's and St. Thomas' NHS Foundation Trust, London, UK.

    • Louise Izatt
    •  & Shehla N Mohammed
  9. South East of Scotland Clinical Genetics Service, NHS Lothian, Edinburgh, UK.

    • Anne K Lampe
  10. Northern Ireland Regional Genetics Service, Belfast City Hospital, Belfast, UK.

    • Fiona J Stewart
  11. Département de Génétique, Assistance Publique–Hôpitaux de Paris (AP-HP)–Hôpital Robert Debré, Paris, France.

    • Alain Verloes
  12. North East Thames Regional Genetics Service, Great Ormond Street Hospital for Children NHS Trust, London, UK.

    • Louise C Wilson
  13. Department of Craniofacial Development and Stem Cell Biology, Dental Institute, King's College London, London, UK.

    • Chris Healy
    •  & Paul T Sharpe
  14. Molecular Medicine Unit, University College London (UCL) Institute of Child Health, London, UK.

    • Peter Hammond
  15. Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

    • Jim Hughes

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Contributions

S.R.F.T. designed and performed experiments and wrote the manuscript. M.A., I.P., A.L.F., E.V., A.Z., E.S.A., S.J.L.K., H.L. and T.L. performed experiments. S.J.M., J.H. and S.T. performed bioinformatic analyses. V.P.S. performed experiments and assessed patients. L.I., A.K.L., S.N.M., F.J.S., A.V., L.C.W., D.J. and S.A.W. identified and assessed patients. C.H. and P.T.S. performed and analyzed μCT scans. P.H. performed and analyzed three-dimensional facial imaging. G.M. conceived the project, designed experiments and wrote the manuscript. A.O.M.W. conceived the project, assessed patients, designed experiments and wrote the manuscript.

Competing interests

The Foundation for Research and Technology–Hellas has filed a patent application with the UK Intellectual Property Office for the use of animals with decreased Erf activity in drug development for ossification defects.

Corresponding authors

Correspondence to George Mavrothalassitis or Andrew O M Wilkie.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Tables 1–3, 9 and 10 and Supplementary Figures 1–10

Excel files

  1. 1.

    Supplementary Table 4

    ChIP-Seq targets identified with -FCS/+FCS >3 (non-TSS).

  2. 2.

    Supplementary Table 5

    ChIP-Seq targets identified with -FCS/+FCS >3 (TSS).

  3. 3.

    Supplementary Table 6

    Sequences near differentially bound ChIP-Seq targets (-FCS/+FCS >3; non-TSS) enriched for combinations of AP1, RUNX and ETS consensus binding sites – AP1.

  4. 4.

    Supplementary Table 7

    Sequences near differentially bound ChIP-Seq targets (-FCS/+FCS >3; non-TSS) enriched for combinations of AP1, RUNX and ETS consensus binding sites – RUNX2.

  5. 5.

    Supplementary Table 8

    Orthologous genes within 40 kb of ChIP-Seq peaks shared between datasets for Erf (-FCS/+FCS >3; non-TSS) in mouse embryonic fibroblasts (this work) and RUNX2 in human prostate carcinoma cells.38

Image files

  1. 1.

    Supplementary Movie 1

    Animated morphs of faces of 4 ERF mutation-positive individuals from Family 1.

  2. 2.

    Supplementary Movie 2

    Animated morphs of faces of 2 ERF mutation-positive individuals from Family 4.

  3. 3.

    Supplementary Movie 3

    Animated morphs of faces of 3 ERF mutation-positive individuals from Family 6.

  4. 4.

    Supplementary Movie 4

    Animated morphs of faces of 3 ERF mutation-positive individuals from Family 8.

  5. 5.

    Supplementary Movie 5

    Animated morphs of face of mutation-positive individual II-1 from Family 10.

  6. 6.

    Supplementary Movie 6

    Animated morphs of faces of 3 ERF mutation-positive individuals from Family 11.

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DOI

https://doi.org/10.1038/ng.2539

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