Composition and dosage of a multipartite enhancer cluster control developmental expression of Indian hedgehog

Copy number variations (CNVs) often include non-coding sequence and putative enhancers but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of Indian hedgehog (IHH), causing multiple, highly localised phenotypes including craniosynostosis and synpolydactyly(1,2). We show through transgenic reporter and genome editing studies in mice that Ihh is regulated by a constellation of at least 9 enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips. Consecutive deletions show that they function in an additive manner resulting in growth defects of the skull and long bones. Duplications, in contrast, cause not only dose-dependent upregulation but also misexpression of Ihh, leading to abnormal phalanges, fusion of sutures and syndactyly. Thus, precise spatio-temporal 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.