Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene.

Abstract

Tyrosinase is a rate-limiting enzyme in melanin biosynthesis and is specifically expressed in differentiated melanocytes. We have identified the enhancer element in the 5'-flanking region of the human tyrosinase gene that is responsible for its pigment cell-specific transcription and have termed it tyrosinase distal element (TDE) (positions -1861 to -1842). Transient expression assays showed that TDE confers efficient expression of a firefly luciferase reporter gene linked to the tyrosinase gene promoter in MeWo pigmented melanoma cells but not in HeLa cells, which do not express tyrosinase. TDE was specifically bound by nuclear proteins of MeWo and HeLa cells, the binding properties of which were indistinguishable in gel mobility shift assays. TDE contains the CATGTG motif in its center, and mutation analysis indicates that the CA dinucleotides of this motif are crucial for protein binding and pigment cell-specific enhancer function. The CATGTG motif is consistent with the consensus sequence recognized by a large family of transcription factors with a basic helix-loop-helix structure, which prompted us to examine the possible involvement of a ubiquitous transcription factor, USF, and a novel factor, microphthalmia-associated transcription factor (MITF), recently cloned as the human homolog of the mouse microphthalmia (mi) gene product. The mi phenotype is associated with a mutant mi locus and characterized by small eyes and loss of melanin pigments. Both USF and MITF are predicted to contain a basic helix-loop-helix structure and a leucine zipper structure. We provide evidence that USF binds to TDE, whereas we were unable to detect the DNA-binding activity of MITF. Transient coexpression assays showed that MITF specifically transactivates the promoter activity of the tyrosinase gene through the CATGTG motif of TDE but not the promoter of the ubiquitously expressed heme oxygenase gene, while USF is able to activate both promoters. These results indicate that MITF is a cell-type-specific factor that is capable of activating transcription of the tyrosinase gene.

FermentedViola mandshuricaInhibits Melanogenesis in B16 Melanoma Cells

“…CEFV was found to inhibit mRNA and protein levels of MITF in a dose-dependent manner, MITF is known as a major transcription factor in the regulation of tyrosinase expression, and thus plays a critical role in melanin biosynthesis. [8][9][10][11] Our results are consistent with the findings of a recent report 37) that xanthohumol (XH) significantly decreased mRNA and protein levels of MITF, suggesting that XH inhibits MITF transcription.…”

“…6,7) In addition, it has been reported that MITF strongly stimulates tyrosinase promoter activities, indicating that it is an important transcriptional regulator of melanogenesis. 8) Cyclic AMP-elevating agents such as -melanocyte stimulating hormone ( -MSH), isobutymethylxanthine (IBMX) and forskolin also stimulate melanin synthesis. Moreover, it is well known that -MSH, as a signal transducer, potently induces MITF expression and increases melanin synthesis.…”

Transcriptional regulation of osteopontin gene in vivo by PEBP2αA/CBFA1 and ETS1 in the skeletal tissues

“…Soluble extracts were then assayed for luciferase activity with a luminometer LB96P (Berthold GmbH, Wildbad, Germany) and for bgalactosidase activity. The luciferase activity was normalized by the galactosidase activity and total protein concentration according to the method described by Yasumoto et al (1994). The normalized value was divided by that obtained with co-transfection of the reporter and pEF-BOS, and is expressed as the relative luciferase activity.…”

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

“…In vitro experiments have shown that these conserved sequences are bound and potently transactivated by the tissue-restricted transcription factor MITF. Although the full functionality of the conserved E-box site remains to be ascertained, MITF shows high preferential binding to the canonical E-box CACGTG as well as the nonpalindromical CACATG (Bentley et al 1994;Hemesath et al, 1994;Yasumoto et al, 1994). MITF binds DNA as a homo-or heterodimer with TFE3, TFEB, or TFEC, which together constitute the MiT-family of bHLHzip-proteins (Hemesath et al, 1994).…”