Genome-Wide Profiling of p63 DNA–Binding Sites Identifies an Element that Regulates Gene Expression during Limb Development in the 7q21 SHFM1 Locus

et al. (2010) Genome-Wide Profiling of p63 DNA-Binding Sites Identifies an Element that Regulates Gene Expression during Limb Development in the 7q21 SHFM1 Locus. PLoS Genet 6(8): e1001065. doi:10.1371/journal.pgen.1001065 Genome-Wide Profiling of p63 DNA-Binding Sites Identifies an Element that Regulates Gene Expression during Limb Development in the 7q21 SHFM1 Locus Evelyn N. Kouwenhoven 0 Simon J. van Heeringen 0 Juan J. Tena 0 Martin Oti 0 Bas E. Dutilh 0 M. Eva 0 Alonso 0 Elisa de la Calle-Mustienes 0 Leonie Smeenk 0 Tuula Rinne 0 Lilian Parsaulian 0 Emine Bolat 0 Rasa Jurgelenaite 0 Martijn A. Huynen 0 Alexander Hoischen 0 Joris A. Veltman 0 Han G. Brunner 0 Tony Roscioli 0 Emily Oates 0 Meredith Wilson 0 Miguel Manzanares 0 Jose Luis Go mez-Skarmeta 0 Hendrik G. Stunnenberg 0 Marion Lohrum 0 Hans van Bokhoven 0 Huiqing Zhou 0 Jason D. Lieb, The University of North Carolina at Chapel Hill, United States of America 0 1 Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre , Nijmegen , The Netherlands , 2 Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen , Nijmegen , The Netherlands , 3 Centro Andaluz de Biolog a del Desarrollo, Universidad Pablo de Olavide, Consejo Superior de Investigaciones Cient ficas , Sevilla, Spain, 4 Centre for Molecular and Biomolecular Informatics , Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre , Nijmegen , The Netherlands , 5 Fundacio n Centro Nacional de Investigaciones Cardiovasculares Carlos III , Madrid , Spain , 6 Department of Clinical Genetics, Children's Hospital at Westmead , Westmead , Australia , 7 Department of Cognitive Neuroscience, Donders Institute for Brain , Cognition, and Behavior , Radboud University Nijmegen Medical Centre , Nijmegen , The Netherlands 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. - Funding: This work was supported by EU: EPISTEM FP6-2004-LIFESCIHEALTH-5, Integrated Project LSH-1.2.1-3; National Foundation for Ectodermal Dysplasias USA, 2009; the Spanish Government:BFU2007-60042/BMC, Petri PET2007_0158, Proyecto de Excelencia CVI-3488, BFU2008-00838, and CSD2007-00008; and the ProCNIC Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. 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]. The developmental abnormalities in animal models are reminiscent of those in p63-associated human disorders. Heterozygous mutations in p63 give rise to at least seven dominantly inherited clinical conditions with three major characteristics, ectrodactyly (also known as split hand/foot malformation, SHFM), orofacial clefting and ectodermal dysplasia with defects in skin, hair, teeth, nails and exocrine glands [5,6]. There is a clear genotypephenotype correlation in p63-associated disorders [7]. 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. Nonetheless, mutations of p63 can explain only a minority of patients with only one of the three cardinal features, such as in patients with isolated SHFM (,10%) and in patients with isolated cleft lip/palate (,0.1%) [7]. There remains a large group of ectodermal dysplasia syndromes with phenotypes that resemble p63-associated syndromes [8]. The genetic basis of many of these clinically related conditions, referred to as the p63 phenotype network, is presently unknown. Mammalian embryonic development requires precise control of gene expression in the right place at the right time. One level of control of gene expression is through cis-regulatory elements controlled by transcription factors. Deregulation of gene expression by mutations in such cisregulatory elements has been described in developmental disorders. 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. To test our hypothesis, we carried out a genome-wide binding site analysis and identified a large number of target genes and regulatory elements regulated by p63. We further showed that one of these regulatory elements controls expression of DLX6 and possibly DLX5 in the apical ectodermal ridge in the developing limbs. Loss of this element through a microdeletion was associated with split hand foot malformation (SHFM1). The list of p63 binding sites provides a resource for the identification of mutations that cause ectodermal dysplasias and malformations in humans. There is ample evidence that diseases clustering within such phenotype networks are caused by mutations in functionally related genes that constitute a gene network [911 (...truncated)