Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein

In the present study we present evidence for the critical role of Spl in th e mechanism of transactivation of the human cell cycle inhibitor p21(WAF1/C ip1) (p21) gene promoter by the tumor suppressor p53 protein. We found that the distal p53-binding site of the p21 promoter acts as an enhancer on the homologous or heterologous promoters in hepatoma HepG2 cells. In transfect ion experiments, p53 transactivated the p21 promoter in HaCaT cells that ex press Spl but have a mutated p53 form. In contrast, p53 could not transacti vate the p21 promoter in the Drosophila embryo-derived Schneider's SL2 cell s that lack endogenous Spl or related factors. Cotransfection of SL2 cells with p53 and Spl resulted in a synergistic transactivation of the p21 promo ter. Synergistic transactivation was greatly decreased in SL2 cells and HaC aT cells by mutations in either the p53-binding site or in the -82/-77 Spl- binding site indicating functional cooperation between Spl and p53 in the t ransactivation of the p21 promoter. Synergistic transactivation was also de creased by mutations in the transactivation domain of p53. Physical interac tions between Spl and p53 proteins were established by glutathione S-transf erase pull-down and coimmunoprecipitation assays. By using deletion mutants we found that the DNA binding domain of Spl is required for its physical i nteraction with p53. In conclusion, Spl must play a critical role in regula ting important biological processes controlled by p53 via p21 gene activati on such as DNA repair, cell growth, differentiation, and apoptosis.