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.