Involvement of mi-transcription factor in expression of alpha-melanocyte-stimulating hormone receptor in cultured mast cells of mice

The microphthalmia (mi) locus encodes a member of the basic-helix-loop-heli x-leucine zipper (bHLH-Zip) protein family of transcription factors (MITF). We have reported that expression of several genes was impaired in cultured mast cells (CMCs) of mi/mi mice due to a defective transactivation ability of mutant MITF (mi-MITF). We also found that mi/mi CMCs did not express a receptor (MC1R) for alpha-melanocyte-stimulating hormone. The overexpressio n of the wild-type (+/+) MITF but not mi-MITF normalized the expression of the MC1R in mi/mi CMCs, indicating the involvement of +-MITF in the MC1R ge ne expression. Next, we analyzed the promoter region of the MC1R gene by th e transient cotransfection assay. The luciferase construct under the contro l of the MC1R promoter and the cDNA-encoding +-MITF or mi-MITF were cotrans fected into NIH/3T3 fibroblasts, The cotransfection of +-MITF but not mi-MI TF increased the luciferase activity. There were five CANNTG moths recogniz ed by bHLA-Zip-type transcription factors in the cloned promoter region. We found +-MITF bound two of five CANNTG motifs, and both moths were essentia l for the transactivation of the MC1R gene by +-MITF. These results indicat ed that +-MITF directly transactivated the MC1R gene through these two moti fs.