Mitf (Microphthalmia transcription factor), a basic-helix-loop-helix zipper protein, encoded at the microphthalmia (Mitf) locus, regulates the transcription of the gene encoding tyrosinase, the rate-limiting enzyme in melanin biosynthesis, by binding the DNA sequence CATGTG. This binding site is present also in the genes encoding two tyrosinase related proteins, TRP-1 and TRP-2. To gain insight into the function of Mitf in vivo, we determined whether there was a difference in the levels of these proteins in the RPE/choroid of the vitiligo (Mitfvit) mouse, in which there is a mutation of the Mitf gene. This mouse has alteration of RPE pigmentation and function that presumably leads to slow progressive loss of photoreceptor cells. The RPE/choroid was dissected from eyes of vitiligo and C57BL/6 wild-type mice at postnatal ages 2, 4, 7, 10, 14, 21 and 42 days. Extracts of pooled tissues were subjected to electrophoresis and immunoblotting. The levels of tyrosinase, TRP-1 and TRP-2 were determined densitometrically following immunodetection with rabbit antipeptide antisera. In addition, the tyrosine hydroxylase activity of tyrosinase as assayed radiometrically. Levels of TRP-1 were 3-7 fold greater in control RPE/choroid compared with mutants. This marked difference in protein level was observed at the earliest age examined (P2) and persisted throughout the first two weeks. Tyrosinase levels in mutants were similar to controls at P2 and P4, but were reduced at P10 and beyond. Tyrosinase activity was diminished also in mutants by P10. Levels of TRP-2 were similar between mutants and controls, although the typical decrease seen in controls after P14 was attenuated in the mutant mice. There is a significant reduction in the level of TRP-1 in the RPE/choroid of the Mitfvit mouse. The data suggests that transcription of the gene encoding TRP-1 is extremely dependent upon functional Mitf. It provides in vivo evidence that Mitf regulates the transcription of the gene encoding TRP-1 as well as tyrosinase.
Copyright 1998 Academic Press Limited.