FIGURE 3.

TDG interacts with TCF4 and CBP. A, TDG deletion constructs. B, the N terminus of TDG binds TCF4. HA-tagged TCF4 was cotransfected with FLAG-tagged TDG, TDG1–119, or TDG119–410 into HEK293T cells. Full-length and deleted TDG proteins were immunoprecipitated (IP) with an anti-FLAG Ab, and TCF4 was detected by Western blot analysis with an anti-HA Ab. Protein levels of TDG and TCF4 in the cell lysates were analyzed by Western blot analysis. C, the N terminus of TDG interacts with the HAT domain of CBP. HEK293T cells were transfected with HA-tagged HAT together with FLAG-tagged TDG1–119 or TDG119–410. The TDG fragments were immunoprecipitated with an anti-FLAG Ab, and the HAT fragment was analyzed by Western blot analysis with an anti-HA Ab (top blot). The expression levels of these proteins in the cell lysates were analyzed by Western blot analysis with anti-HA and anti-TDG Abs (center and bottom blots). D, TDG and CBP active TOPFlash reporter. Wnt3A was cotransfected with TOPFlash and Renilla plasmids into HEK293T cells with or without TDG and CBP. CBP enhanced TDG function in Wnt signaling. CBP inhibitor E1A abolished TDG-induced Wnt signaling. *, p < 0.05. E, TDG is required for Wnt target gene expression in colon cancer cells. HT29 colon cancer cells were infected with lentiviruses expressing a control shRNA (ctrlshRNA) or TDG shRNA. The mRNA levels of TDG and Wnt target genes, survivin, Axin2, and c-myc were analyzed by RT-PCR. β-actin was analyzed as a loading control. F, ChIP assay. HT 29 cells were treated with control shRNA or TDG shRNA. ChIP assays were performed using anti-TDG, anti-AcH3, and control Abs. The promoter sequence of the Wnt target gene, c-myc, was analyzed by PCR.