FIGURE 2.

The β-catenin/TCF4 binding region of YAP enhances SV40-driven luciferase gene expression. A, diagram of the pGL3-promoter plasmids used in luciferase assays with the SV40 minimal promoter and firefly luciferase gene shown as white rectangles and the 624 bp YAP enhancer in gray. A black vertical line represents the position of the consensus TCF motif within the YAP enhancer. The TCF sequence was changed by site-directed mutagenesis to generate the pGL3-YAP mutant plasmid (Mut.). B, HCT116 cells were transfected with vector control, pGL3-YAP wild-type (WT) or pGL3-YAP mutant (Mut.) firefly luciferase plasmids and a plasmid expressing Renilla luciferase. Twenty-four hours later, luciferase levels were measured and firefly luciferase levels were normalized to Renilla luciferase levels to control for differences in transfection efficiency between samples. C, cells transfected with the indicated reporters were untreated (black bars) or treated (gray bars) with the GSK3β inhibitor BIO for 12 h prior to measuring luciferase levels. D, cells were transfected with vector control or the pGL3-YAP (WT) luciferase reporter and, where indicated, the cells were co-transfected with a dominant negative TCF4 plasmid. Luciferase levels were measured as in B. E, nuclear proteins prepared from HCT116 cells were incubated with a 28 bp biotinylated and double-stranded DNA probe that contained either the wild-type or mutant TCF consensus sequence. Where indicated, unlabeled YAP duplexes were included in the binding reactions. Western blot analysis was used to detect bound β-catenin or TCF4. In B, C, and D, error is standard deviation.