Melanocyte-specific gene expression: role of repression and identification of a melanocyte-specific factor, MSF.

Abstract

For a gene to be transcribed in a tissue-specific fashion, expression must be achieved in the appropriate cell type and also be prevented in other tissues. As an approach to understanding the regulation of tissue-specific gene expression, we have analyzed the requirements for melanocyte-specific expression of the tyrosinase-related protein 1 (TRP-1) promoter. Positive regulation of TRP-1 expression is mediated by both an octamer-binding motif and an 11-bp element, termed the M box, which is conserved between the TRP-1 and other melanocytespecific promoters. We show here that, consistent with its ability to activate transcription in a non-tissue-specific fashion, the M box binds the basic-helix-loop-helix factor USF in vitro. With the use of a combination of site-directed mutagenesis and chimeric promoter constructs, additional elements involved in regulating TRP-1 expression were identified. These include the TATA region, which appears to contribute to the melanocyte specificity of the TRP-1 promoter. Mutational analysis also identified two repressor elements, one at the start site, the other located at -240, which function both in melanoma and nonmelanoma cells. In addition, a melanocyte-specific factor, MSF, binds to sites which overlap both repressor elements, with substitution mutations demonstrating that binding by MSF is not required for repression. Although a functional role for MSF has not been unequivocally determined, the location of its binding sites leads us to speculate that it may act as a melanocyte-specific antirepressor during transcription of the endogenous TRP-1 gene.

The melanocyte inducing factor MITF is stably expressed in cell lines from human clear cell sarcoma

“…pRSVCAT contains the RSV LTR linked to CAT as previously described (Gorman et al, 1982). pMITF-MCAT was obtained by insertion of CAT and MITF-M promoter sequence from the position À395 into pSuppCT (Yavuzer and Goding, 1994). pMCRE contains two ATF sites from the MITF-M promoter upstream of the RSV TATA box and transcription site linked to CAT.…”

Tyrp1 and Oculocutaneous Albinism Type 3

“…The differences in catalytic function between murine Tyrp1 and human TYPR1 is however not surprising due to differences in a) the placement of cis-regulatory regions between murine Tyrp1 and human TYRP1 (12, 21, 75), b) amino acid composition [reviewed in Boissy et al, 1998 (66)] and c) transcriptional activities, e.g. Mitf (76)(77)(78), i.e. the amino acid residues between mouse and humans are not conserved in these regions.…”

microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family.

“…We have tested the ability of mi to activate transcription of a reporter driven by the M-box pigmentation gene pro moter element (Shibahara et al 1991;Lowings et al 1992;Yavuzer and Coding 1994) because of our demon stration that Mi is capable of binding its CATGTG core sequence in vitro (Fig. ID).…”

“…A great deal has been learned about pigmentation enzymes and their regulation through study, for exam ple, of albino (tyrosinase) mutants (for review, see Halaban and Moellmann 1993). Determination of regulatory elements critical for pigmentation have revealed an 11-bp sequence known as the M box containing the core element CATGTG, which is highly conserved in the pro moters of the three major pigmentation enzyme genes tyrosinase, and tyrosinase-related proteins 1 and 2 (Shibahara et al 1991;Lowings et al 1992;Yavuzer and Coding 1994). The presence of tissue-specific transcrip tion factors capable of interacting with and transcrip tionally activating elements such as this may shed light on the regulation of pigmentation and perhaps other melanocyte-specific functions.…”