Cloning of the human DOPAchrome tautomerase/tyrosinase-related protein 2 gene and identification of two regulatory regions required for its pigment cell-specific expression.
We have cloned and sequenced the human genomic DNA segments encoding the 5'-flanking region and the first two exons of the DOPAchrome tautomerase (DT)/tyrosinase-related protein 2 (TRP-2) gene. The DT gene is a member of the tyrosinase gene family and specifically expressed in melanin-producing cells. A transcriptional initiation site of the DT gene was identified by S1 nuclease-mapping and primer-extension analyses using RNA prepared from human pigmented melanoma cells. To study the mechanism for pigment cell-specific expression of the human DT gene, we analyzed the promoter function of its 5'-flanking region by transient expression assays. The fusion genes, containing the DT gene promoter upstream from a firefly luciferase reporter gene, were introduced into human pigmented melanoma cells and HeLa cells, and the pigment cell-specific promoter activity was evaluated by comparing the luciferase activity expressed in both cell lines. A series of 5' deletion studies of the human DT gene promoter revealed that the 32-bp element, located between -447 and -415, is sufficient to confer pigment cell-specific expression of a reporter gene on a homologous promoter, but not on a heterologous simian virus 40 promoter. Internal deletion studies using a homologous or a heterologous promoter revealed that the pigment cell-specific expression of a reporter gene mediated by the 32-bp element is dependent on the presence of another region of the DT gene spanning from -268 to -56, which was termed the proximal region. However, the proximal region by itself is not sufficient to confer cell type-specific expression. These results indicate that the presence of two regulatory regions, the 32-bp element and the proximal region, is required for pigment cell-specific expression of the DT gene. Both regulatory regions contain a CANNTG motif, a well known binding site for a large family of transcription factors possessing a basic helix-loop-helix structure.
Yokoyama
K
K
Department of Applied Physiology and Molecular Biology, Tohoku University School of Medicine, Miyagi, Japan.
Yasumoto
K
K
Suzuki
H
H
Shibahara
S
S
D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D045563:Indolequinones; D007211:Indoles / Q000378:metabolism; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000235:genetics*; D008546:Melanoma, Experimental / Q000378:metabolism*; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics; D005810:Multigene Family / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011809:Quinones / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D045563:Indolequinones; D007211:Indoles; D011809:Quinones; C001123:dopachrome; D014442:Monophenol Monooxygenase; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase