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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02703

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Homeobox gene Dlx3 is regulated by p63 during ectoderm development: relevance in the pathogenesis of ectodermal dysplasias

Nadezda Radoja¹, Luisa Guerrini², Nadia Lo Iacono^2,3, Giorgio R. Merlo³, Antonio Costanzo⁴, Wendy C. Weinberg⁵, Girolama La Mantia⁶, Viola Calabrò^6,* and Maria I. Morasso^1,*,

¹ Developmental Skin Biology Unit, NIAMS, NIH, Bethesda, MD 20892, USA.
² Department of Biomolecular and Biotechnological Sciences, University of Milan, Via Celoria 26. 20133, Milan, Italy.
³ Dulbecco Telethon Institute c/o Istituto Tecnologie Biomediche CNR, 20100 Milan, Italy.
⁴ Department of Dermatology, University of Rome `Tor Vergata', Viale Oxford 81. 00133 Rome, Italy.
⁵ Division of Monoclonal Antibodies, CDER/FDA, Bethesda, MD 20892, USA.
⁶ Department of Structural and Functional Biology, University of Naples, Via Cinzia 26. 80126 Naples, Italy.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Transcriptional regulation of the Dlx3 promoter by p63. (A) Fold-induction increase, compared with wild type, of Dlx3-117/+60 cotransfected with vectors expressing the different p63 isoforms. (B) Sequence of the mouse Dlx3-promoter region containing the two overlapping p63-binding sites (p63 site1 and p63 site2; underlined) and CCAAT box (italics). The mutated Dlx3 sequences are shown in gray. (C-D) Dlx3-117/+60, Dlx3p63M1 and Dlx3p63M2 constructs were cotransfected with vectors expressing the TAp63 (C) and Np63 (D) isoforms, with activity shown relative to the basic activity of Dlx3-117/+60. Transient transfection experiments were performed using mouse keratinocytes (C-D) and Saos-2 cells (E). Basal activity of the reporter was set to 1. Each histogram bar represents the mean of three independent transfection duplicates. Standard deviations are indicated. Dlx3 p63M1, mutated at p63-binding-site 1; Dlx3p63M2, mutated at p63-binding-site 2.

View larger version (17K): [in this window] [in a new window]   Fig. 2. p63 and Dlx3 expression in primary mouse keratinocytes cultured in vitro. Dlx3 (A) and TAp63 (B) mRNAs were induced, and Np63 (C) mRNA was downregulated after 12- and 24-hours of high-[Ca+2] treatment. (D-F) p63 binds to the Dlx3 promoter region in vitro and in vivo. (D) EMSA assay performed with a DNA fragment that included the Dlx3 p63-binding site 1 and 2 using nuclear extract from primary keratinocytes (NE), and in the presence of 100 M excess of specific (SC) and nonspecific (NC) competitors. (E-F) ChIP analysis on mouse keratinocytes with either control IgG or p63 antibody (p63 Ab) on the region of the Dlx3 promoter containing the p63-binding sites by regular (E) and real-time (F) PCR. (G) ChIP analysis on TAp63-transfected H1299 cells with no antibody (no Ab) or p63 antibody on DLX3 and JAG2 promoters. ACHR was used as a control.

View larger version (51K): [in this window] [in a new window]   Fig. 3. p63 and Dlx3 colocalize in the embryonic ectoderm, and Dlx3 expression is downregulated in p63-KO embryos. (A) Histochemistry with anti-distal-less (pan-anti-Dlx; red) and p63 (green) antibodies on the dorsal forelimb ectoderm of E10.5 wild-type embryos (merge of Dlx and p63 expression is yellow). DAPI staining is also shown (blue). Arrows indicate the dlx-p63 double-positive nucleus. (B) Relative mRNA abundance, determined by real-time PCR, for Dlx3, TAp63 and Np63 in the anterior limb (AL) and posterior limb (PL) of E10.5, E11.5 and E12.5 wild-type embryos. The relative abundance is expressed as fold-induction relative to the value at E10.5 (set at 1). (C) Histochemistry with anti-p63 and anti-Dlx on E10.5 wild-type and p63-KO limb-bud ectoderm. (D) Whole-mount in situ hybridization on wild-type and p63-KO E10.5 embryos with a Dlx3 antisense probe. Arrows indicate limb buds. M, mesoderm; E, ectoderm. Scale bars: 10 µM in A and 40 µM in C. wt, wild type.

View larger version (47K): [in this window] [in a new window]   Fig. 4. Differential ability of p63-mutant proteins to transactivate Dlx3. (A) Schematic representation of p63 transcripts and mutations. (B-C) Transcriptional regulation of the Dlx3 promoter by TAp63 (B) and Np63 (C) wild-type and mutant proteins. H1299 cells were cotransfected with the Dlx3 reporter plasmid and expression vectors for TAp63- and Np63-mutant isoforms. The basal activity of the reporter was set to 1. Each histogram bar represents the mean of three independent transfections. Standard deviations are indicated. (D) Proteins were corroborated by western blot analysis with anti-p63 4A4 antibody (Santa Cruz). (E) The level of endogenous Dlx3 mRNA upon transfection with p63 mutants in Saos-2 cells. Top panel, lanes: 1, mock; 2, TAp63; 3, TAp63ß; 4, TAp63; 5, TAp63F518V-AEC; 6, TAp63FS-EEC; 7, TAp63E639X-SHFM; and 8, TAp63AA-LMS. Bottom panel, lanes: 1, mock; 2, Np63; 3, Np63ß; 4, Np63; 5, Np63F518V-AEC; 6, Np63FS-EEC; 7, Np63E639X-SHFM; and 8, Np63AA-LMS. GAPDH was used for normalization. (F) The level of endogenous Dlx3 mRNA upon transfection with TAp63 and TAp63 in HaCaT cells. Lanes: 1, mock; 2, TAp63 0.5 µg; 3, TAp63 2 µg; 4, TAp63 4 µg; 5, TAp63 0.5 µg; 6, TAp63 2 µg; 7, TAp63 4 µg. Cyclophillin was used for normalization.

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