Figure 1.

Gli1 and Gli2 repress the activity of the human ANO1 promoter. A) Schematic of the human ANO1 promoter. Previously characterized core promoter elements are indicated in black. In colors are marked the 3 putative Gli binding sites with their sequences. B) Expression of the human ANO1 promoter-luciferase chimeric constructs in the HEK293 cell line. Promoter activity was calculated by fold increase over the luciferase activity of the ANO1 promoter-luciferase chimeric construct coexpressed with the empty expression vector used for Glis. The activity of the human ANO1 promoter was significantly repressed in cells cotransfected with Gli1 and Gli2 but not Gli3 expression vectors. C) Activity of the wild-type (WT) and mutated human ANO1 promoter-luciferase chimeric constructs in the HEK293 cell line in basal conditions. Promoter activity was calculated by fold increase over the luciferase activity of the luciferase empty vector. D, E) Effect of inactivation of Gli binding sites by site-directed mutagenesis on Gli1 (D) and Gli2 (E) repression of the human ANO1 promoter. The colors indicated the mutagenesis done on the more downstream (red), the intermediate (orange) or the more upstream (red) binding sites, as indicated in the promoter schematic in A. Promoter activity was calculated by fold increase over the luciferase activity of the ANO1 promoter-luciferase chimeric construct coexpressed with the empty expression vector used for Glis (n = 6). *P < 0.05, Kruskal-Wallis with Dunn’s multiple comparisons test.