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.
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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.
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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.
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Supplementary Text and Figures
Supplementary Tables 1–3, 9 and 10 and Supplementary Figures 1–10 (PDF 7508 kb)
Supplementary Table 4
ChIP-Seq targets identified with -FCS/+FCS >3 (non-TSS). (XLSX 339 kb)
Supplementary Table 5
ChIP-Seq targets identified with -FCS/+FCS >3 (TSS). (XLSX 250 kb)
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. (XLSX 1153 kb)
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. (XLSX 2863 kb)
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 (XLSX 23 kb)
Supplementary Movie 1
Animated morphs of faces of 4 ERF mutation-positive individuals from Family 1. (GIF 1693 kb)
Supplementary Movie 2
Animated morphs of faces of 2 ERF mutation-positive individuals from Family 4. (GIF 805 kb)
Supplementary Movie 3
Animated morphs of faces of 3 ERF mutation-positive individuals from Family 6. (GIF 1312 kb)
Supplementary Movie 4
Animated morphs of faces of 3 ERF mutation-positive individuals from Family 8. (GIF 1233 kb)
Supplementary Movie 5
Animated morphs of face of mutation-positive individual II-1 from Family 10. (GIF 338 kb)
Supplementary Movie 6
Animated morphs of faces of 3 ERF mutation-positive individuals from Family 11. (GIF 1171 kb)
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Twigg, S., Vorgia, E., McGowan, S. et al. Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis. Nat Genet 45, 308–313 (2013). https://doi.org/10.1038/ng.2539
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DOI: https://doi.org/10.1038/ng.2539
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