TARGETING THE MICROPHTHALMIA BASIC HELIX-LOOP-HELIX LEUCINE-ZIPPER TRANSCRIPTION FACTOR TO A SUBSET OF E-BOX ELEMENTS IN-VITRO AND IN-VIVO

The development of melanocytes, which are pigment-producing cells resp onsible for skin, hair, and eye color, is absolutely dependent on the action of the microphthalmia basic helix-loop-helix-leucine zipper (bH LH-LZ) transcription factor (Mi); mice lacking a functional Mi protein are entirely devoid of pigment cells. Mi has been shown to activate t ranscription of the tyrosinase, TRP-1, TRP-2, and QNR-71 genes through specific E-box elements, most notably the highly conserved hi box. We investigated the mechanism which enables Mi to be recruited specifica lly to a restricted subset of E boxes in target promoters while being prevented from binding E-box elements in other promoters. We show both in vitro and in vivo that the presence of a T residue Ranking a CATGT G E box is an essential determinant of the ability of Mi to bind DNA, and we successfully predict that the CATGTG E box from the P gene woul d not bind Mi. In contrast, no specific requirement for the sequences Ranking a CACGTG E box was observed, and no binding to an atypical E b ox in the c-Kit promoter was detected. The relevance of these observat ions to the control of melanocyte-specific gene expression was highlig hted by the fact that the E-box elements located in the tyrosinase, TR P-1, TRP-2, and QNR-71 promoters without exception possess a 5' flanki ng T residue which is entirely conserved between species as diverse as man and turtle. The ability of Mi to discriminate between different E -box motifs provides a mechanism to restrict the repertoire of genes w hich are likely to be regulated by Mi and provides insight into the ab ility of bHLH-LZ transcription factors to achieve the specificity requ ired for the precise coordination of transcription during development.