Identification of the Tax-responsive elements in the p21 promoter. A, The DNA sequence of the region covering 130 nucleotides upstream and 8 nucleotides downstream of the p21 mRNA start site (is shown (Top). The p21-Luc reporter plasmid contains a 2342 bp DNA fragment that spans 2326 bp upstream and 16 bp downstream of the p21 transcriptional start site fused to the firefly luciferase reporter gene. The p21 promoter truncation p21Δ1, p21Δ2, and p21Δ3 have their 5' termini ending at 298, 100, and 60 upstream of the p21 transcriptional start site (see METHODS). HeLa/18 × 21-EGFP cells were transfected in triplicate with the respective luciferase reporter constructs with or without a Tax expression plasmid, BC12-Tax. Cell lysates were prepared 48 h after transfection for luciferase assays as previously described . All firefly luciferase reporter assays were normalized against HSV thymidine kinase (TK) promoter-driven renilla luciferase reporter activities. Standard deviations of the reporter activities and the degree of trans-activation by Tax (fold activation) are shown. B, TATA-box proximal Sp1-binding sites in the p21 promoter mediate Tax trans-activation. Luciferase reporter assays were as in (A). The constructs p21P93-Smut-1, 2, and 3 contain base substitutions in the Sp1 binding sites localized in -93 to -84, -83 to -74, and -73 to -64 regions of the p21 promoter respectively.