FIGURE 3.

Functional study of SMC2 promoter activity. A, schematic representation of human SMC2 promoter. Predicted TCF response elements are also indicated; arrows indicate target sequence for ChIP PCR amplification. B, Ls174T/dnTCF4 (left) and Ls174T/pTER-β-catenin (right) cell lines were transfected with SMC2 promoter-luciferase reporter construct together with control Renilla luciferase reporter pRL-TK for normalization (RLU, relative luciferase units). Where indicated, cells were doxycline (Doxy)-treated to induce the TCF4 dominant-negative form (left) or a siRNA targeting β-catenin (right). TOP-flash vector was used as positive control for WNT signaling activity/repression. A representative result out of at least three different experiments run in triplicates is shown. C, DLD-1 or HCT116 cell lines were co-transfected with SMC2 promoter luciferase construct and pcDNA (empty vector), β-catenin, or VP16-TCF4 expression vectors. D, PCR analyses of DNA pulled down by isotypic antibody (negative control) or anti-TCF-4 monoclonal antibody in ChIP assay. c-myc promoter sequence containing TBE1 element and APC promoter region 1B sequences were amplified as positive and negative controls, respectively. Error bars indicate S.D. (Student's t test; **, p < 0.01).