FIG. 1.

Repression of TRP-1 in melanocytes is not mediated by MSF. (A) Schematic showing the elements required for regulation of the TRP-1 promoter. The M box is recognized by the transcription factor microphthalmia, while the MSEu and MSEi are targets for MSF and for negative regulation. (B) The LS-MSEi mutation relieves repression of TRP-1 in melanocytes. The melan-c melanocyte cell line was transfected with the indicated WT or LS-MSEi mutant TRP-1–CAT reporters, and CAT activity was determined. (C) Repression mediated by the MSEu in melanocytes. Melan-c cells were transfected with the USF-TK-CAT reporter or the indicated derivatives containing WT or mutant MSEu elements, and CAT activity was determined 48 h posttransfection. (D) MSF binds the WT and mutants LS-MSEi elements. A radioactive oligonucleotide probe containing the MSEu was used in a band shift assay with B16 melanoma cell nuclear extract, and MSF binding was competed with 10, 50, and 250 ng of unlabeled oligonucleotides containing the MSE, MSEu, or LS-MSEi. Only the bound DNA is shown. The sequence of each oligonucleotide probe and competitor is shown in Fig. 6.