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First published online 16 January 2008
doi: 10.1242/dev.006718

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P-cadherin is a p63 target gene with a crucial role in the developing human limb bud and hair follicle

¹ Department of Dermatology, Columbia University, New York, NY 10032, USA.
² Departments of Biochemistry and Molecular Biophysics, and Ophthalmology, Columbia University, New York, NY 10032, USA.
³ Department of Genetics and Development, Columbia University, New York, NY 10032, USA.

View larger version (69K): [in this window] [in a new window]   Fig. 1. Identification of mutations in the CDH3 gene of five consanguineous Pakistani families. (A) Pedigrees and clinical appearance of affected individuals of five Pakistani families (Families 1-5) with either HJMD or EEM syndrome. In Families 1-3, arrows indicate affected individuals whose clinical photographs are shown. (B) A novel mutation 490insA (arrow) in Family 1. (C) A novel mutation Ivs10-1G>T (arrows) in Families 2 and 3. (D) A novel mutation E118G in Family 4 (top) and amino-acid sequence alignment of human classical cadherins and P-cadherin of other species (bottom). The glutamic acid residue at position 118 is colored light blue. (E) The recurrent mutation N322I (arrows) in Family 5.

View larger version (70K): [in this window] [in a new window]   Fig. 2. Expression of P-cadherin in the developing mouse hair follicle, eye and limb bud. (A) P-cadherin expression is upregulated in the HF placode at E15.5. (B) P-cadherin is expressed in the pigmented epithelium of the retina (white arrow) at E17.5. (C,D) Transverse sections of forelimb bud at E9.5 (C) and E10.5 (D) show prominent expression of P-cadherin along the AER (white arrows). Note that, in the transverse section, the tip of forelimb bud corresponds to the AER. For a better view, counterstaining with DAPI is shown in red instead of blue (B-D). (E,F) Longitudinal section of forelimb bud at E15.5 shows that P-cadherin is widely expressed throughout the epithelial cells of the forelimb bud. F is a higher magnification view of E. (G,H) Whole-mount immunohistochemistry also localizes the P-cadherin in the AER of the forelimb bud at E9.5 (white arrows). Scale bars: 100 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 3. P-cadherin overlaps with p63 in the hair follicle placode and the AER in mouse. (A,B) Double-immunostaining shows overlapping expression of P-cadherin (red) and p63 (green) at the HF placode at E15.5. (C,D) Longitudinal section of forelimb bud at E9.5 (C) and transverse section of forelimb bud at E10.5 (D) demonstrate that p63 is predominantly expressed at nuclei of the AER. (E-H) Double-immunostaining using a transverse section of forelimb bud at E10.5 shows that both P-cadherin (green) and p63 (red) are expressed throughout the AER. H is the merged image of F and G. Scale bars: 100 µm in A,C,D,E; 20 µm in B,F-H.

View larger version (72K): [in this window] [in a new window]   Fig. 4. Expression of classical cadherins in the forelimb bud of mouse. (A-C) Double immunostaining of a longitudinal section of the forelimb bud at E9.5 shows that the expression of E-cadherin (green, A) and P-cadherin (red, B) overlaps along the AER (yellow in merged image, C). (D,E) Transverse section of the forelimb bud at E10.5 shows the strong expression of E-cadherin throughout the AER. (F-H) N-cadherin is strongly expressed at mesenchyme just beneath the AER (F), whereas E-cadherin is prominently expressed in the AER (G). The merged image of F and G clearly demonstrates their distinct expression patterns (H). Counterstaining with DAPI is shown in blue in C and in red in D,E. Scale bars: 100 µm in A-D,F-H; 20 µm in E.

View larger version (10K): [in this window] [in a new window]   Fig. 5. CDH3 expression is upregulated by p63 in 293 cells. HEK293 cells were transfected with TAp63, TAp63, Np63, Np63 or empty vector. At 24 hours post-transfection, the CDH3 expression levels were measured by real-time PCR. All four p63 isoforms upregulated CDH3 expression. Asterisks indicate a statistically significant difference (P<0.05) compared with the empty vector.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Identification of two distinct regions in the CDH3 promoter that are responsive to p63. (A) Schematic of the CDH3 promoter. Regions 1 and 2 that show high homology with mouse sequences are boxed. CpG islands are indicated by shaded boxes. Five templates that were cloned into the pGL3 basic vector are shown at the bottom. (B) Reporter gene assay in HeLa cells. The longest construct (-2472/+500), which contains both Regions 1 and 2, showed more than 25-fold transactivation of the reporter gene by TAp63 and Np63. Furthermore, shorter constructs that contain either Region 1 (-2472/-1833) or Region 2 (+1/+500) resulted in a much greater activation of luciferase expression by p63. (C,D) Potential p63-binding sites in Region 1 (C) and Region 2 (D) are shaded with the canonical p53-binding sequence shown either above or below. Asterisks show nucleotide residues that correspond to the `RRRCWWGYYY' sequence. The positions where primers for ChIP assay were designated are underlined. (E) Both Regions 1 and 2 showed further increase of the reporter gene activity by p63, when they were truncated to about 230 bp fragments with potential p63-binding sites. (F) Sequence of the two partially overlapping p63-binding sites in Region 1 (-2088 to -2070). Sequences of the three mutated constructs (M1-M3) are also shown. All three mutated constructs led to a marked reduction of luciferase activity by TAp63. (G) Sequence of a p63-binding site in Region 2 (+31 to +40). Sequence of the mutated construct is also shown. TAp63 showed only weak transactivation of the reporter gene in the mutated construct.

View larger version (20K): [in this window] [in a new window]   Fig. 7. A dominant-negative effect of TAp63 (R280C) mutant. (A,B) HeLa cells were transfected with the indicated amount of the expression constructs, and reporter gene activity was examined in both Region 1 (-2233/-2004, A) and Region 2 (+1/+223, B). Mutant TAp63 did not activate luciferase expression in either region. In addition, it inhibited the wild-type TAp63-induced transactivation in a dose-dependent manner in both regions.

View larger version (49K): [in this window] [in a new window]   Fig. 8. Both TAp63 and Np63 isoforms directly bind to two distinct regions in the CDH3 promoter. (A) ChIP assay in HEK293 cells in which either TAp63 or TAp63 was overexpressed. (B) In vivo ChIP assay in HaCaT cells. An antibody against p63 immunoprecipitated both Region 1 and Region 2 of the CDH3 promoter (black arrows) similar to the positive control p21 (gray arrow). The CDH3 coding region was not immunoprecipitated (black arrowhead).

View larger version (21K): [in this window] [in a new window]   Fig. 9. Schematic representation of P-cadherin protein and CDH3 mutations, and diagram of the EC1-EC2 calcium-binding site of P-cadherin. Mutations that were identified in this study are indicated in bold and underlined. Note that the mutation E118G corresponds to E11 in the mature protein. In the diagram, the side chain of E11 is colored in red. EC1-EC5, extracellular cadherin repeat domains; IC, intracellular domain; TM, transmembrane domain.

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