Abstract
Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA–binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP–seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes.
Highlights
The p63 protein encoded by the TP63 gene is a transcription factor of the p53 family and functions as a master regulator of ectodermal development
Heterozygous mutations in the transcription factor p63 are found in patients with limb malformations, cleft lip/palate, and defects in skin and other epidermal appendages, through disruption of normal ectodermal development during embryogenesis
We reasoned that the identification of target genes and cis-regulatory elements controlled by p63 would provide candidate genes for defects arising from abnormally regulated ectodermal development
Summary
The p63 protein encoded by the TP63 gene is a transcription factor of the p53 family and functions as a master regulator of ectodermal development. The key function of p63 during ectodermal development is underscored by phenotypic features in p63 knockout mice [1,2] and in p63 knock-down zebrafish [3,4]. Heterozygous mutations in p63 give rise to at least seven dominantly inherited clinical conditions with three major characteristics, ectrodactyly ( known as split hand/foot malformation, SHFM), orofacial clefting and ectodermal dysplasia with defects in skin, hair, teeth, nails and exocrine glands [5,6]. The most prominent of these disorders is the Ectrodactyly Ectodermal dysplasia and Cleft lip/palate syndrome (EEC, OMIM 604292) which combines all of the three phenotypic hallmarks and is almost invariably caused by missense mutations in the DNA binding domain of p63. Ankyloblepharon Ectodermal defects Cleft lip/palate syndrome (AEC, OMIM 106260) is caused by mutations in the SAM domain of the p63 that is involved in protein interaction. The genetic basis of many of these clinically related conditions, referred to as the p63 phenotype network, is presently unknown
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