FIGURE 3.

Identification of region(s) within human TAp63 promoter required for TLP-dependent transcriptional activation. A, luciferase reporter assays. HeLa cells were transiently co-transfected with the constant amount of the indicated luciferase reporter constructs bearing various length of human TAp63 promoter region (100 ng) and Renilla luciferase reporter plasmid (pRL-TK, 10 ng) together with the empty plasmid (pClneo) or with the expression plasmid for FLAG-TLP. Forty-eight hours after transfection, cells were lysed and their luciferase activities were measured as described in the legend for Fig. 2. Filled and open boxes indicate the putative TLP-responsive regions within TAp63 promoter. B, schematic drawing of TAp63 promoter region. Filled and open boxes indicate the putative TLP-responsive regions within TAp63 promoter. The positions of primer sets (#1, #2, #3, and #4) relative to the transcriptional initiation site (+1) used for ChIP assay are indicated. C, ChIP assay. HeLa cells were transiently transfected with the empty plasmid or with the expression plasmid for FLAG-TLP. Expression of FLAG-TLP was confirmed by semi-quantitative RT-PCR (right panels). Forty-eight hours after transfection, cells were cross-linked with formaldehyde and cross-linked chromatin was sonicated followed by immunoprecipitation with anti-FLAG antibody. Genomic DNA was purified from the immunoprecipitates and subjected to PCR using the indicated primer sets.