FIGURE 4.

Complex of ZIC2·TCF4 bound on DNA. A, EMSA was done by incubating a cell extract of 293T cells transfected with different expression plasmids as indicated with biotin-labeled probes containing wild-type or mutant TCF4-binding sequence. The lower panels represent Western blots of the corresponding samples as indicated. B, EMSA using 293T cells cotransfected with full-length TCF4 and its deletions together with ZIC2-expressing plasmid. Where indicated, the samples were preincubated with antibody against TCF4. C, DNA affinity precipitation of 293T cells transfected with the indicated expression vectors was performed as described under “Experimental Procedures.” The lysates were incubated with wild-type or mutant biotinylated probes corresponding to the TCF4-binding sequence, and the precipitated DNA·protein complexes were analyzed by Western blotting using the indicated antibodies. A probe with a mutant TCF-binding sequence was used as a control. D, 293T cells were transfected with ZIC2 and TCF4, and ChIP was performed with the indicated antibodies, followed by quantitative PCR using primer pairs spanning the human AXIN2 TCF4-binding site or an unrelated AXIN2 sequence as negative control. The ApoE promoter served as a positive control for ZIC2 binding. Results are presented as percentage of immunoprecipitated DNA over the input. E, luciferase reporter activity of the Wnt reporter (TOPflash) in 293T cells transfected with the expression vectors as indicated along with the β-catenin (DN90) expression vector.