FIGURE 3.

The PHGDH promoter contains one p53 binding site. A, schematic of the promoter region in human PHGDH gene. The p53 binding site upstream of the first exon is indicated and compared with the consensus p53 responsive element (p53 RE). Pu, purine; Py, pyramidine; N, any nucleotide; BS, binding site; TSS, transcription start site. B, ChIP semiquantitative analysis of p53 enrichment at the promoter regions of PHGDH and TIGAR in H1299 cells transfected with empty or p53 vector for 24 h. The cross-linked protein-DNA complex was immunoprecipitated using full-length anti-p53 antibody (α-p53). GAPDH was used as a negative control. C, A375 cells were treated with vehicle, Nutlin-3 (10 μm), or doxorubicin (0.2 μg/ml) for 24 h, and ChIP assays for determining the enrichment of p53 on PHGDH and TIGAR promoters were carried out using anti-p53 antibody or control rabbit IgG. GAPDH was used as a negative control (Ctrl). D, EMSA showing the binding of p53 on ³²P-labeled oligonucleotides containing the p53-binding site in the human PHGDH promoter region. The DNA binding activity of purified p53 protein was enhanced with the C-terminal p53 antibody pAb421 (α-p53). The binding specificity was verified using an excess of non-radiolabeled wild-type probes (competition).