THE SEMIDOMINANT MI(B) MUTATION IDENTIFIES A ROLE FOR THE HLH DOMAIN IN DNA-BINDING IN ADDITION TO ITS ROLE IN PROTEIN DIMERIZATION

The mouse microphthalmia (mi) locus encodes a basic helix-loop-helix-l eucine zipper (bHLH-Zip) transcription factor called MITF (microphthal mia transcription factor). Mutations at mi affect the development of s everal different cell types, including melanocytes, mast cells, osteoc lasts and pigmented epithelial cells of the eye. Here we describe the phenotypic and molecular characterization of the semidominant Micropht halmia(brownish) (Mi(b)) mutation. We show that this mutation primaril y affects melanocytes and produces retinal degeneration. The mutation is a G to A transition leading to a Gly244Glu substitution in helix 2 of the HLH dimerization domain. This location is surprising since othe r semidominant mi mutations characterized to date have been shown to a ffect DNA binding or transcriptional activation domains of MITF and ac t as dominant negatives, while mutations that affect MITF dimerization are inherited recessively. Gel retardation assays showed that while t he mutant MITF(Mi-b) protein retains its dimerization potential, it is defective in its ability to bind DNA. Computer modeling suggested tha t the Gly244Glu mutation might disrupt DNA binding by interfering with productive docking of the protein dimer onto DNA. The Mi(b) mutation therefore appears to dissociate a DNA recognition function of the HLH domain from its role in protein dimerization.