Fig. 3. Regulation of the Dlx5 and Dlx6 promoters by p63. (A) Left histograms: co-transfection of mDlx5luc and mDlx6luc reporters (300 ng) with 50 (white bars), 100 (grey bars) or 300 ng (black bars) of TAp63 and Np63 expression plasmids. Data are reported as fold of activation relative to basal expression (reporter vector alone, set=1). Right histograms: mutated Np63 proteins EEC R279H and C306R, and SHFM-IV K193E and K194E, show reduced activity on the Dlx5 and Dlx6 promoters. The AEC L518F mutation did not affect p63 activity. Standard deviation is indicated. (B) Expression of DLX1, DLX2, DLX5 and DLX6 analysed by RT-PCR in H1299 cells induced to express Np63. White bars, no induction; black bars, induced cells (20 µM Dox). p63 induction was confirmed by western blot. (C) p63 is bound in vivo to the Dlx5 and DLX6 promoters. Specific enrichment was detected in two regions of the Dlx5 promoter, R1 (-1200/-800) and R2 (-500/-100), and in one region of the DLX6 promoter, R2 (-500/-100). Input is shown on the left. Amplification of the IKa and of the Np63 promoters is shown as a positive control. (D,E) Deletions of the mDlx5luc (D) and mDlx6luc (E) promoters. The position of the predicted p53 sites and the X5-R1, X5-R2, X6-R1 and X6-R2 regions are indicated. Luciferase activity is shown on the right.