FIGURE 7.

Mutational analysis of the hTERT sequences associated with GRHL2. A, hTERT promoter sequences from −53 to −13 were mutated in three consecutive nucleotide sequences, yielding seven different mutant sequences as indicated in red. Underlined sequences are the Sp1-binding sites reported elsewhere (34). B, PMP was performed with the seven mutant and the wild-type hTERT promoter sequences. Western blotting revealed GRHL2 and Sp1 binding with the DNA. C, SCC4 cells were transfected with pGL3-Luc, pGL3-TRTP, or the pGL3-TRTP containing the mutant sequences (either mutant 5 or mutant 6), and pGL3-TRTPΔ(−49 to +5) containing the deletion of the sequences from −49 to +5. Firefly and Renilla luciferase activities were determined at 48 h post-transfection and plotted as normalized values. Error bars represent standard errors.