FIG. 3.
The Snk/Plk2 promoter contains three p53 binding sites and is regulated by p53. (a) Schematic representation of the human Snk/Plk2 promoter region. Three p53 DNA binding sites are located at bp −2433 (BS1), bp −2208 (BS2), and bp −1478 (BS3) upstream from the start of transcription (indicated by arrow). An alignment of the p53 consensus binding element with the p53 binding sites present in the Snk/Plk2 promoter is shown. (b) pGL3-Snk/Plk2 promoter-Δ1, pGL3-Snk/Plk2 promoter-Δ1,2, or pGL3. Snk/Plk2 promoters were cotransfected (2.88 μg) with pCEP4 or pCEP4-p53 (0.72 μg) and pCMV-β-Gal (0.4 μg) into Saos2 cells. Luciferase activity was determined 24 h later. Empty vector (pGL3-Basic) and the p210WAF1 promoter (pWWP-Luc) were used as a negative and a positive control, respectively. Induction is indicated as levels for reporters with pCEP4-p53 divided by levels for reporters with pCEP4 (empty vector). (c) Adriamycin treatment enhances p53 binding to the Snk/Plk2 promoter in vivo. Untreated or adriamycin-treated U20S cells were harvested for chromatin immunoprecipitation as described in Materials and Methods. The left panel shows DNA that coprecipitated with the immunocomplexes and was amplified by PCR. The right panel shows amplification of genomic DNA using BS1 and BS2 primers.