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MELANOGENIC FACTORS: REGULATION OF GENE EXPRESSION J. Vachtenheim¹ and J. Duchon² Laboratory of Molecular Biology, Institute of Chest Diseases, Prague 8 and IInd Department of Medical Chemistry and Biochemistry, 1st Faculty of Medicine, Charles University

Melanogenesis faktory: Regulace genové exprese. Vachtenheim J., Duchori J. Sborn. Iék., Vol. 97 (1996 No. 1, p. 41-47. * This paper is dedicated to the 50th anniversary of IInd Department of Medical Chemistry and Biochemistry 1st Faculty of Medicine, Charles University
Mailing address/Adresa autora: MUDr. J. Vachtenheim, CSc., Laboratory of Molecular Biology, Institute of Chest Diseases, 18000 Prague 8 (Czech Republic).
SUMMARY: Melanogenesis is a multistep biochemical process resulting in the formation of melanin in pigment cells in the skin and the eye. Three melanogenic factors, tyrosinase, TRP 1, and TRP2 participate in the pathway. Here, the regulation of gene expression of these melanocyte-specific markers is shortly reviewed. ---------------------------------- SOUHRN: Melanogenese je biochemický proces s mnoha stupni, vedoucí k tvorbe melaninu v pig mentových bunkach v kuzi a oku. Tii melanogenní faktory, tyrosinasa, TRP1 a TRP2, se úcastni této biochemické cesty. Zde jsou kratce shrnuty poznatky o regulaci genové exprese techto pro melanocyty specifickych markeru. ------------------------------------
Melanocytes, highly differentiated cells producing the pigment melanin, are derived from the neural crest and are unique in that they possess enzymes converting tyrosine and low-molecular intermediates into the dark polymeric pigment through a specific biochemical pathway. For many years, tyrosinase has been considered to be the only enzyme responsible for the production of melanin in melanocytes. It catalyzes two initial steps in the formation of melanin, the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and the oxidation of L-DOPA to DOPAquinone. Apart from these two well-known catalytic functions, a third catalytic activity of tyrosinase has been shown (I). This activity, the oxidation of dihydroxyindol (DHI), seems to be true tyrosinase activity and could be immunoinhibited by antibodies directed against electrophoretically purified tyrosinase (2). Recently, Kobayashi et al. have shown that extract from fibroblasts transfected with the tyrosinase expression construct was able to utilize DHI as a substrate and melan-c cells (lacking functional tyrosinase) had no ability to oxidize DHI (3). These results clearly demonstrate the DHI-oxidase activity of tyrosinase. Tyrosinase is not the only enzyme that catalyzes the reactions in the melanogenic pathway. Two other regulatory factors, namely tyrosinase-related proteins, TRP1 and TRP2, have been described and Their activities determined. The action of TRP1 is more distal than TRP1- in the melanogenic pathway. TRP1 has been shown to possess dihydroxyindol carboxilic acid (DHICA) oxidase activity (3) but low tyrosine hydroxylase was also associated with this protein (see references in 3). In experiments analogous to tyrosinase expression. TRP1 expression in fibroblasts resulted in DHICA conversion in transfected cell extracts (3). TRP2 was shown to have DOPAchrome tautomerase (DT) activity isomerizing the rnelanogenic intermediate DOPAchrome to DHICA (4), a relatively stable jntermediate. Similarly the expression of TRP2 in HeLa (5) or COS 6) cells results in DOPAchrome conversion activity in cell extracts. Thus, besides tyrosinase. two other factors facilitate the formation of melanin in melanocytes: TRP2 produces DHICA and is therefore responsible for preserving carboxyl group in melanins The product of TRP2, DHICA, serves as a substrate for TRP1. Since TRP 1 was shown to be associated with the production of eumelanin rather than phaeomelanin in human melanoma cell lines (7), this factor probably has the specific role to facilitate the very distal step in the production of black type of pigment eumelanin, i.e. the oxidation of DHICA before the polymerization takes place (3).

TYROSINASE GENE FAMILY

In the past years, by using techniques of molecular biology, cDNA for tyrosinase as well as for the two proteins mentioned above: (TRP1 and TRP2) that participate in melanogenesis has been cloned. Human tyrosinase cDNA clone encodes a protein with the predicted molecular weight 62.6kD (8). The sequence shows several potential N-glycosylation sites and a signal peptide characteristic for membrane associated proteins (8,9). Another isolated tyrosinase cDNA clone has a sequence very similar to the first clone (10). When expressed in mouse fibroblasts which normally do not synthesize melanin, tyosinase was capable to induce melanin pigmentation (10). Mouse tyrosinase maps to the c albino locus on chromosome 7 (8) and its amino acid sequence is &9% homologous to the human sequence (9). The mouse and human tyrosinase genomic structures an also similar: the protein sequences are encoded in 5 exons spanning about 70 kb in the genomes 11-13). Human tyrosinase is located on 11q14-q21 (14) but a tyrosinase-related segment corresponding only to exons 4 and 5 and adjacent noncoding sequences has been found on 11p11.2 (12). The TRP 1 and TRP2 map at the b (brown) and slt (slaty) locus in the mouse, respectively (15, 16). Although initially thought to be a tyrosinase clone the mouse TRP1 has been the first gene isolated among the melanogenic proteins (17). Both TRP1 and TRP2 from the mouse are highly homologous to their human counterparts: TRP1 exerts higher homology (93 %) than TRP2 (84%) (5, 18). The degree of homology of the three individual melanogenic enzymes, tyrosinase, TRP1 and TRP2, to each other is much lower (about 40-50%) in the mouse and in human. The chromosomal loci at which TRP1 and TRP2 reside has also been determined. In humans, TRP1 maps at the short arm of chromosome 9 and TRP2 maps at 3q31-932. These regions correspond to homologous regions on mouse chromosomes 3 and 14, respectively (19, 20). In summary, the three proteins are coded at distinct genes Iocated on different chromosomes, have different enzymatic activities in the melanogenic pathway, share some sequence similarity and constitute a "tyrosinase family" of proteins.

TRANSCRIPTION REGULATION OF PIGMENT CELL-SPECIFIC GENE EXPRESSION

Tissue-specific gene expression and cell differentiation are governed by complex interaction of specific transcription factors interacting with DNA elements in gene promoters. In melanocytes, the regulatory 5'-sequences of tyrosinase, TRP1 and TRP2 genes have been isolated and analysed for the presence of positive and negative elements crucial for pigrnent cell-specific gene expression. All the three pigment cell-specific enzymes mentioned above are expressed in normal melanocytes. However, some or all of these markers are often repressed in melanoma cell lines (7, 9). For the mouse tyrosinase gene transcription, a 270-bp sequence flanking the transcriptional start site is sufficient to confer melanocyte-specific transcription of the reporter gene in a human and a mouse melanoma cell line (21). This promoter sequence was also sufficient for expression of the tyrosinase transgene both in skin melanocytes derived from the neural crest and pigmented retinal cells derived from the optic cup in mice. Tyrosinase in pigmented retinal cells was expressed from day 10,5 and in hair folicles from day 16,5 onwards in the mouse embryo (21a); thus, the mouse tyrosinase promoter was sufficient to provide cell-type and developmental regulation of tyrosinase in skin melanocytes and pigmented cells in the retina. The pattern of transgene expression mimicked the sites of expression of the endogenous tyrosinase (21, 21a). Similarly, in human tyrosinase promoter, only 115-bp segment of the upstream sequence was sufficient to confer tissue-specific transcription 122). Apart from the proximal promoter sequences required for cell-specific transcription, a short (39bp) distal enhancer element (at position about - 1800-bp upstream from the transcriptionaI start) was identified in the 5'-flanking region of the human tyrosinase gene (23). This elernent, further narrowed to only 20-bp sequence binds, however, factors from nuclear extract from both the melanoma and HeLa cells (24). The tyrosinase promoter differs from the TRP1 promoter because it does not contain typical TATA box or CCAAT box present in the tyrosinase promoter (25, 26). A 5'-flanking segment of 370-bp of the mouse TRP 1 is sufficient to direct cell-specific expression of the reporter gene in mouse melanoma cells (25). However, despite the differences in the TRP 1 and tyrosinase gene promoters, there is an important 1 bp element conserved in both promoters in both mouse and human genes (25, 27). This motif, termed M-box (ATGCATGTGCT), contains a 6bp sequence (CANNTG), a E-box known to be recognized by a family of basic-helix-loop-helix (bHLH) transcription factors comprising proteins with either specific or ubiquitous pattern of gene transactivation. The M-box was shown to be a positive functional element within the tyrosinase (28) and TRP 1 (25, 27) gene promoters. Almost identical M-box has been detected by sequencing the human TRP2 gene promoter (29) suggesting further the possible irnportance of this element for melanocyte-specific gene transcription. The functional analysis of the human TRP2 promoter sequences has been also performed (29): a 32-bp distal sequence located -447 to -415bp upstream to the transcriptional start was sufficient for pigment cell-specific expression but the simultaneous presence of another more proximal region (containing M-box) was also required (29). It should be noted that TRP2 is the first melanocyte-specific marker that is expressed, detecting the early migratory melanoblasts at day 10 in the mouse embryo (30).
By a detailed deletional and mutational analysis of the mouse TRP1 promoter, both positive and negative elements were identified (27). The stretch between -44 and +107 (containing the M-box) was able to confer cell-specific transcription of the gene. Two positive and one negative element correlated with protein binding and it was surprising that one of the positive element, the M-box, was capable to regulate positively the expression in both TRP 1-expressing and -nonexpressing cell lines (27). It was further demonstrated that M-box bound the ubiquitous bHLH transcription factor USF (31). In the TRP1 promoter, the initiator element (-1 to +5) that binds putative melanocyte specific factor, seems to be important for melanocyte-specific expression. The same factor interacts also with identical upstream sequence (31). These two elements, however, overlap two negative regulatory elements. When the initiator element was mutated, dramatic increase of the reporter expression was observed and the expression was detected also in nonpigmented cells not expressing TRP1 (31). Additional upstream octamer binding motif acts as a positive regulator of TRP 1 expression (31).
Recently, the gene that maps to the mi (microphthalmia) locus of the mouse has been identified (32, 33). Many different mutated mi alleles have been described and mice homozygous for the mutated mi gene have defects in melanin pigmentation, small eyes and are deaf (due to the lack of melanocytes in the inner ear) (34). In human, mutations at the mi locus causes Waardenburg syndrome type: 2 (35) and the human homologue of mi has also been cloned (36). Since the mi protein is a member of the bHLH-leucine-zipper protein family of transcription factors, it has become an excellent candidate for the melanocyte-specific transcripton factor. Indeed, it has been shown that it can transactivate transcription from the promoter containing the M-box (22, 24, 37). Mi protein, however, binds the CANNTG motif (37) and might probably stimulate transcription through the interaction with this motif at the initiator sequence of the human tyrosinase promoter (22). Microphthalmia protein binds to DNA as homodimer or heterodimer with factors TFEB, TFE3 or TFEC. This group of proteins constitute a novel MiT gene family (37). The mi mutations, when analysed for their DNA recognition and oligomerization abilities in vitro, can explain the effects of mutations known in mi mutant mice strains (37). Microphthalmia gene therefore seems to be crutial for the melanocyte differentiation and function. It is expressed in cells producing melanin and, surprisingly, in heart in high levels (32). Additionally, microphtalmia gene seems to be a target for the repression by adenoviral Ela oncogene which is known to repress many differentiation-specific genes. The arteficial expression of mi gene from CMV promoter prevents the repression of tyrosinase and TRP 1 genes seen in Ela-expressing cells (38). Moreover, the mi protein interacts with the retinoblastoma protein in vitro (38).
Although the precise role of microphtalmia protein and other potential transcription factors in the transcriptional regulation of the tyrosinase family of genes and the regulation of expression of the mi gene itself remains to be determined, the function of melanogenic enzymes and the regulation of expression of genes encoding these factors are now more clear than several years ago. Besides the three melanogenic factors other proteins such as pmel17/silver locus gene product (39) were suggested to be structural part of the melanosome matrix and might also prove to be important for melanogenesis in subcellular particles - melanosomes. However, little is known about the molecular mechanisms that operate in the conversion of normal to malignant melanocytes and the role of differentiation-specific genes in the process.

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