Fig. 2.

β-catenin/TCF4 activates ZEB1 transcription. (A) SW480 and HCT116 cells were transfected with 0.5 μg of human ZEB1 promoter plus 1 μg of the empty expression vector (“−”) or equal molar amounts of expression vectors for TCF4 or TCF4_d1-30 (“+”). Transcriptional assays were performed as described in SI Discussion. The relative luciferase activity (RLU) in the two cell lines with the empty vector was set to a common value for comparison. As for the TOPFLASH reporter (Fig. S2C), basal activity of the ZEB1 promoter is lower in HTC116 cells. (B) SW480 cells were transfected with ZEB1 promoter and combinations of 1 μg of empty expression vector or equal molar amounts of TCF4, β-catenin and/or β-catenin^SA. (C) Same as in B, but in HCT116 cells. (D) TCF4 and β-catenin bind to the ZEB1 promoter. Real-time PCR quantification (qRT-PCR) of fragments of the ZEB1 and GAPDH promoters immunoprecipitated in ChIP assays from SW480 cells with Abs against TCF4, β-catenin, their respective control IgG, and with no Ab. Amplified ZEB1 promoter regions contain TCF binding sites at -578 and -161. Values represent relative binding in relation to input. (E) Endogenous β-catenin and TCF4 regulate ZEB1 transcription. As in A, SW480 cells were transfected with the ZEB1 promoter along with 100 nM siRNAs against either β-catenin (siβcat), TCF4 (siTCF4), or a control siRNA (siCtl). (F) As in B, SW480 cells were transfected with the ZEB1 promoter and different combinations of 1 μg of empty vector or equal molar amounts of TCF4, β-catenin, and TLE1.