THE MICROPHTHALMIA GENE-PRODUCT INTERACTS WITH THE RETINOBLASTOMA PROTEIN IN-VITRO AND IS A TARGET FOR DEREGULATION OF MELANOCYTE-SPECIFIC TRANSCRIPTION

Little is known of the molecular mechanisms underlying the differentia tion of the melanocyte from the melanoblast or the progression from th e melanocyte to a malignant melanoma. Since the adenovirus E1A product s have proved a useful tool for understanding control of differentiati on in other systems, we explored the possibility of using E1A as a pro be for factors controlling melanocyte-specific gene expression and dif ferentiation. The results obtained show that the adenovirus E1A 13S, b ut not the 12S, product can transform the highly pigmented and TPA-dep endent melanocyte cell line melan-a, Transformation is characterised b y a morphological change, loss of TPA-dependence, the ability to grow in soft agar and strikingly, loss of pigmentation which correlates wit h loss of expression of the melanocyte-specific TRP-1 and tyrosinase g enes, Cotransfection assays demonstrated that repression of TRP-1 by E 1A correlated with E1A binding to p105Rb and p300, with the target in the TRP-1 promoter being the M-box, an 11 bp basic-Helix-loop-Helix (b HLH) factor-binding motif conserved between melanocyte-specific promot ers, Consistent with the M-box acting as a target for E1a-mediated tra nscription repression, we also show that the basic-helix-loop-helix-le ucine zipper (bHLH-LZ) protein (Mi) encoded by the microphthalmia gene (mi), which is required for pigment cell differentiation, is a positi ve acting transcription factor which can interact with the retinoblast oma product in vitro and activate the TRP-1 promoter, Moreover, expres sion of the mi gene was reduced around 50-fold in the non-pigmented E1 a-transformed melan-a cells compared to the nontransformed melan-a cel l line, with ectopic expression of Mi able to prevent repression of th e tyrosinase and TRP-1 promoters in the presence of E1A, Mi therefore appears to play a crucial role in melanocyte-specific gene expression. The parallels between repression of myogenesis and muscle cell bHLH f actors, and Mi and melanocyte differentiation are discussed.