Positive and negative elements regulate a melanocyte-specific promoter.

Abstract

Melanocytes are specialized cells residing in the hair follicles, the eye, and the basal layer of the human epidermis whose primary function is the production of the pigment melanin, giving rise to skin, hair, and eye color. Melanogenesis, a process unique to melanocytes that involves the processing of tyrosine by a number of melanocyte-specific enzymes, including tyrosinase and tyrosinase-related protein 1 (TRP-1), occurs only after differentiation from the melanocyte precursor, the melanoblast. In humans, melanogenesis is inducible by UV irradiation, with melanin being transferred from the melanocyte in the epidermis to the surrounding keratinocytes as protection from UV-induced damage. Excessive exposure to UV, however, is the primary cause of malignant melanoma, an increasingly common and highly aggressive disease. As an initial approach to understanding the regulation of melanocyte differentiation and melanocyte-specific transcription, we have isolated the gene encoding TRP-1 and examined the cis-and trans-acting factors required for cell-type-specific expression. We find that the TRP-1 promoter comprises both positive and negative regulatory elements which confer efficient expression in a TRP-1-expressing, pigmented melanoma cell line but not in NIH 3T3 or JEG3 cells and that a minimal promoter extending between -44 and +107 is sufficient for cell-type-specific expression. Assays for DNA-protein interactions coupled with extensive mutagenesis identified three factors, whose binding correlated with the function of two positive and one negative regulatory element. One of these factors, termed M-box-binding factor 1, binds to an 11-bp motif, the M box, which acts as a positive regulatory element both in TRP-l-expressing and -nonexpressing cell lines, despite being entirely conserved between the melanocyte-specific tyrosinase and TRP-1 promoters. The possible mechanisms underlying melanocyte-specific gene expression are discussed.

Microphthalamia-associated transcription factor: a critical regulator of pigment cell development and survival

“…A rate-limiting step in pigment production is catalyzed by the enzyme tyrosinase, which is transcriptionally regulated via a tissue-restricted cis-acting promoter containing a canonical E-box (Ganss et al, 1994;Bertolotto et al, 1996). Identical E-boxes are also found in the promoters of the two other key pigment enzymes tyrosinase-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct), suggesting a conserved mode of transcriptional regulation (Lowings et al, 1992;Yokoyama et al, 1994;Yasumoto et al, 1997;Bertolotto et al, 1998b). In vitro experiments have shown that these conserved sequences are bound and potently transactivated by the tissue-restricted transcription factor MITF.…”

The Gene Encoding the T-box Factor Tbx2 Is a Target for the Microphthalmia-associated Transcription Factor in Melanocytes

“…In the melanocyte-specific Tyrosinase Tyrp-1 and Dct2 promoters, the specific E-box elements that are essential for their expression have been shown to bind both the bHLH-LZ transcription factors USF1 and Mitf (4,6,7,26,35,36). To determine whether USF1 could recognize the E box present in the Tbx2 promoter, we performed a DNA binding band shift assay using a radiolabeled oligonucleotide probe spanning the E box and in vitro transcribed/translated (ITT) USF1.…”

“…Mitf has been shown previously to regulate a number of genes, including Tyrosinase, Tyrp-1, and Dct involved in the manufacture of the pigment melanin, a process specific to melanocytes and the retinal pigment epithelium. The sequences recognized in all these Mitf target promoters are E boxes with the sequence CATGTG, including the highly conserved M-box motif (35).…”

In B16 melanoma cells, the inhibition of melanogenesis by TPA results from PKC activation and diminution of microphthalmia binding to the M-box of the tyrosinase promoter

“…It also plays a crucial role in the control of the tissue-speci®c expression of the melanogenic genes (Ganss et al, 1994;Lowings et al, 1992;Yavuzer et al, 1995). Further, in a previous study, we have shown that microphthalmia, through the binding to the M-box upstream from the TATA box, mediates the e ect of cAMP on the tyrosinase promoter (Bertolotto et al, 1996).…”

“…These enzymes are restricted to melanocytes suggesting the existence of a tissue-speci®c regulatory mechanism which controls the transcription of their genes. A regulatory element, thought to be involved in the cell speci®c expression of the melanogenic enzymes, is conserved in the promoter of the three enzymes (Ganss et al, 1994;Lowings et al, 1992;Yokoyama et al, 1994a). This element, called M-box, binds microphthalmia a transcription factor of the basic-helix ± loop ± helix-leucine-zipper family (b-HLH-LZ) which strongly transactivates tyrosinase and TRP1 promoter activities (Ganss et al, 1994;Lowings et al, 1992;Yavuzer et al, 1995).…”