Positive effect of +-MITF and negative effect ofmi-MITF on transactivation of the mouse perforin gene.

(A) The perforin gene promoter sequences from −822,−609,−491, and −141 to +173 were inserted into the upstream of the luciferase gene of pSPLuc. These reporter constructs and empty pSPLuc were transfected into CTLL-2 cells with the pEF-BOS containing no insert (pEF-BOS) or +-MITF or mi-MITF cDNA by electroporation. The bars represent the mean ± SE of the luciferase activities obtained by 3 independent experiments: (▪) indicates +-MITF; (■),mi-MITF; (░), pEF-BOS. (B) The effect of mutations in the CANNTG and NF-P motifs on the luciferase activity. In the −609 promoter construct, the CAACTG (E1, nt −567 to −562; upstream oval) and CAGCTG (E2, nt −525 to −520; downstream oval) motifs, and NF-P motif (nt −505 to −493; black square) were mutated in various combinations. X indicates mutated motifs: CACATG to CTACAG; CAGCTG to CTGCAG; ACAGGAAGT to ACATTCCTG. Only the mutation in the NF-P motif abrogated the transactivation effect of +-MITF. *P < .05 by t test when compared with the values when pEF-BOS containing no insert was cotransfected.