Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects

Nadia Lo Iacono 1 2 7 Stefano Mantero 0 2 7 Anna Chiarelli 1 7 Elvin Garcia 6 7 Alea A. Mills 6 7 Maria I. Morasso 5 7 Antonio Costanzo 4 7 Giovanni Levi 3 7 Luisa Guerrini 1 7 Giorgio R. Merlo 0 2 7 0 CNR Istituto Tecnologie Biomediche , Segrate Milano , Italy 1 Department of Biomolecular Science and Biotechnology, University of Milan , Via Celoria 26, 20133 Milan , Italy 2 Dulbecco Telethon Institute, Molecular Biotechnology Center, University of Torino , Via Nizza 52, Torino, 10126 , Italy 3 Evolution des Regulations Endocriniennes CNRS, UMR5166, Museum National d'Histoire Naturelle , Paris , France 4 Department of Dermatology, University of Rome , TorVergata , Italy 5 Developmental Skin Biology Unit, NIAMS, NIH , Bethesda, MD , USA 6 Cold Spring Harbor Laboratory , Cold Spring Harbor, New York , USA 7 AS , antisense; S, sense The congenital malformation Split Hand-Foot Malformation (SHFM, or ectrodactyly) is characterized by a medial cleft of hands and feet, and missing central fingers. Five genetically distinct forms are known in humans; the most common (type-I) is linked to deletions of DSS1 and the distalless-related homeogenes DLX5 and DLX6. As Dlx5;Dlx6 double-knockout mice show a SHFM-like phenotype, the human orthologs are believed to be the disease genes. SHFM-IV and Ectrodactyly-Ectodermal dysplasia-Cleft lip (EEC) are caused by mutations in p63, an ectoderm-specific p53-related transcription factor. The similarity in the limb phenotype of different forms of SHFM may underlie the existence of a regulatory cascade involving the disease genes. Here, we show that p63 and Dlx proteins colocalize in the nuclei of the apical ectodermal ridge (AER). In homozygous p63- (null) and p63EEC (R279H) mutant limbs, the AER fails to stratify and the expression of four Dlx genes is strongly reduced; interestingly, the p63+/EEC and p63+/hindlimbs, which develop normally and have a normally stratified AER, show reduced Dlx gene expression. The p63+/EEC mutation combined with an incomplete loss of Dlx5 and Dlx6 alleles leads to severe limb phenotypes, which are not observed in mice with either mutation alone. In vitro, Np63 induces transcription from the Dlx5 and Dlx6 promoters, an activity abolished by EEC and SHFM-IV mutations, but not by Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) mutations. ChIP analysis shows that p63 is directly associated with the Dlx5 and Dlx6 promoters. Thus, our data strongly implicate p63 and the Dlx5-Dlx6 locus in a pathway relevant in the aetio-pathogenesis of SHFM. INTRODUCTION Congenital limb reduction defects occur in approximately 1:2000 live births, among which the anomalies of the central ray constitute an important subgroup (Buss, 1994). The malformation SplitHand/Foot (SHFM, MIM 183600), also known as ectrodactyly, affects the distal portion of the upper and lower limbs, and is characterized by a deep medial cleft and missing central fingers (Sifakis et al., 2001). Genetically, SHFM comprises both isolated and hereditary forms, linked to five distinct loci (types I-V). For SHFM-II (MIM 313350) and SHFM-V (MIM 606708), the disease genes have not been identified. SHFM-III (MIM 600095) is associated with genomic alterations on chromosome 10q24-q25 (deMollerat et al., 2003), which results in a complex rearrangement around the Dactylyn locus, possibly associated with gene inactivation. Dactylyn is also involved in a complex rearrangement/duplication in dactylaplasia (dac) mutant mice, which exhibit ectrodactyly-like limb defects (Chai, 1981; Johnson et al., 1995; Crackower et al., 1998). In spite of these findings, no demonstration that Dactylyn is the disease gene for SHFM-III, or in the dac mice, has been provided. *These authors contributed equally to this work Authors for correspondence (e-mails: ; ) SHFM-I, the most common form, is associated with deletions of variable extent on chromosome 7q21. The minimal common deletion includes DSS1 and the distalless-related homeogenes DLX5 and DLX6 (Simeone et al., 1994; Scherer et al., 1994a; Scherer et al., 1994b; Crackower et al., 1996). The double knock-out (DKO) of Dlx5 and Dlx6 in the mouse leads to ectrodactyly (Robledo et al., 2002; Merlo et al., 2002; Merlo et al., 2003), implicating the human orthologs DLX5 and DLX6 in this pathology. Mutations in the DLX5-DLX6 locus have not been found, therefore the molecular alteration remains unknown; however, a position effect mutagenic mechanism for SHFM-I has been proposed (Scherer et al., 2003). Dlx genes code for six distalless-related homeodomain transcription factors (Dlx1-Dlx6) that play key roles in the development and morphogenesis of the head and limb skeleton (Merlo et al., 2000; Merlo et al., 2003; Panganiban and Rubenstein, 2002). Expression of Dlx5 and Dlx6 has been detected in the apical ectodermal ridge (AER) of the embryonic limb buds, in the pharyngeal arches, in the osteoblasts of developing bones and in interneurons of the basal forebrain (Simeone et al., 1994; Acampora et al., 1999; Levi et al., 2003). In spite of known functions of distalless for the development of insect appendages, little is known about the molecular regulation of Dlx genes in mammalian limbs. Defining the upstream regulation of the Dlx5-Dlx6 locus during limb development might help to clarify the molecular basis of SHFM. SHFM-IV (MIM 605289) is caused by mutations in p63, a gene coding for a transcription factor homologous to p53 and p73 (Ianakiev et al., 2000; vanBokhoven and Brunner, 2002; BerdonZapata et al., 2004). In 50 unrelated SHFM patients, five mutations in p63 were found, suggesting that these may account for about 10% of sporadic SHFM (vanBokhoven et al., 2001; vanBokhoven et al., 2002). Mutations of p63 are also responsible for other autosomal, dominantly inherited human syndromes, including EctrodactylyEctodermal dysplasia-Cleft lip (EEC), Limb-Mammary Syndrome (LMS) and Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) (Celli et al., 1999; vanBokhoven et al., 2001; Rinne et al., 2006; Rinne et al., 2007). p63-null mice show severe defects affecting their skin, limbs, craniofacial skeleton, and they lack teeth, hair and mammary glands. In p63/ newborn animals, the hindlimbs (HLs) are absent, whereas the forelimbs (FLs) are severely truncated in the distal segments (Mills et al., 1999; Yang et al., 1999). p63 is transcribed as two classes (TA and N) of mRNAs. The use of alternative promoters drives the transcription of either TAp63 proteins, comprising a p53-related N-terminal transactivation (TA) domain, a DNA-binding (DB) and an oligomerization (OD) domain, or Np63 proteins, lacking the TA domain. Additional TA domains have been identified that account for the transcriptional activities of the N isoforms (Dohn et al., 2001; Ghioni et al., 2002; Laurikkala et al., 2006). Three alternative splicing routes at the 3 -end generate TAp63 and Np63 proteins with different C-termini, denoted , and (vanBokhoven and Brunner, 2 (...truncated)