Pigment Cell-specific Expression Research Articles

Genetics of pigment cells: lessons from the tyrosinase gene family.

In mammals, the melanin pigment is produced in two cell types of distinct developmental origins. The melanocytes of the skin originate form the neural crest whereas the retinal pigment epithelium (RPE) of the eye originates from the optic cup. The genetic programs governing these two cell types are thus quite different but have evolved to allow the expression of pigment cell-specific genes such as the three members of the tyrosinase-related family. Tyrosinase, Tyrp1 and Dct promoters contain a motif termed E-box which is bound by the transcription factor Mitf. These E-boxes are also found in the promoters of the corresponding fish genes, thus highlighting the pivotal role of Mitf in pigment cell-specific gene regulation. Mitf, which displays cell type-specific isoforms, transactivates the promoters of the tyrosinase gene family in both pigment cell lineages. However, specific DNA motifs have been found in these promoters, and they correspond to binding sites for RPE-specific factors such as Otx2 or for melanocyte-specific factors such as Sox10 or Pax3. The regulation of pigment cell-specific expression is also controlled by genetic elements located outside of the promoter, such as the tyrosinase distal regulatory element located at -15 kb which acts as a melanocyte-specific enhancer but also protects from spreading of condensed chromatin. Thus, by using the tyrosinase gene family as a model, it is possible to define the transcription factor networks that govern pigment production in either melanocytes or RPE.

Pigment cell-specific expression of the tyrosinase gene in ascidians has a different regulatory mechanism from vertebrates

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5′ upstream regions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5′ upstream region of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the −152bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to −1.8kb. Surprisingly, in the −152bp minimal promoter or in other regions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed.

TheFugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.

Genomic organization and FISH mapping of human Pmel 17, the putative silver locus.

The Pmel 17 gene is expressed preferentially in pigment cells. It has been mapped to human chromosome 12 pter-q21 and mouse chromosome 10, near the silver locus. The Pmel 17 gene contains an insertional mutation at its carboxyl terminus in the silver mouse, suggesting that the silver locus might correspond to the gene. In the current studies, we have isolated and characterized human Pmel 17 genomic clones and employed FISH mapping for a precise localization of this gene in the human chromosome. The FISH mapping placed the Pmel 17 gene at human chromosome 12 q12-q13. The human gene consists of nine exons and eight introns, and the entire coding region of the gene spans approximately 7.9 kb of the human chromosome 12. The putative functional domains, such as the signal sequence, histidine-rich, 26-amino acid repeats, cysteinerich, transmembrane and cytoplasmic domains, were encoded by separate exons. Cistranscription elements such as a TATA, a CAT and other potential elements for pigment cell-specific gene expression were found within 1100 base pairs of the 5' flanking region.

Transcriptional activation of the melanocyte-specific genes by the human homolog of the mouse Microphthalmia protein.

Mi protein encoded at the mouse microphthalmia (mi) locus is a transcription factor with a basic helix-loop-helix/leucine zipper structure. To assess the function of the human homolog of Mi protein, termed microphthalmia-associated transcription factor (MITF), we analyzed the effects of MITF on the promoter function of the mouse tyrosinase and tyrosinase-related protein 1 (TRP-1) genes. These two gene promoters are able to direct transcription preferentially in melanin-producing cells, and an enhancer element M box of 11 bp, containing a CATGTG motif, is conserved in both promoters. By transient expression assays, we have localized the cis-acting element of the tyrosinase gene responsible for pigment cell-specific expression to the proximal 82-bp region, which contains a CATGTG motif (positions -12 to -7) but lacks the M box (positions -107 to -97). We also provide evidence that the 82-bp region and the M box are involved in the transactivation of the tyrosinase promoter by MITF and that the M box is bound by MITF in vitro. Furthermore, MITF activated the TRP-1 gene promoter possibly through the M box (positions -44 to -34). These results suggest that MITF is a common factor regulating transcription of the pigment cell-specific genes.

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.

Functional Analysis of the Tyrosinase Gene and Brown-Locus Protein Gene Promoters

Tyrosinase is a rate-limiting enzyme of melanin biosynthesis and the brown (b)-locus protein is responsible for the formation of black melanin rather than brown. To identify the cis-acting element(s) required for pigment cell-specific gene transcription, we analyzed the promoter function of two pigment cell-specific genes encoding mouse tyrosinase and b-locus protein using a cell-free transcription system prepared from mouse melanoma cells. Functional and structural analysis of both gene promoters reveals that three elements are conserved in both genes at equivalent positions, suggesting that these elements may be responsible for pigment cell-specific transcription. We discuss possible mechanisms for pigment cell-specific expression of the tyrosinase and b-locus protein genes.

Molecular characterization of the mouse tyrosinase gene: pigment cell-specific expression in transgenic mice.

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest-derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well-characterized c-locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type-specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell-type-specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type-specificity and developmental regulation in melanocytes and the pigment epithelium.

A cis-acting element involved in mouse tyrosinase gene expression and partial purification of its binding protein.

The tyrosinase gene is specifically expressed in melanocytes. Understanding the molecular basis of tissue-specific expression of the tyrosinase gene will greatly explain the mechanisms controlling pigmentation. We report a nucleotide sequence, TGATGTATTC, located -236 base pairs upstream of the transcription start site, that enhances tyrosinase gene expression in mouse melanoma cells. The sequence is referred to as the tyrosinase element-1 (TE-1). TE-1 was protected from DNase I cleavage by pigment cell nuclear extracts but was not protected by non-pigment cell nuclear extract. Partial purification of TE-1 binding protein (TEBP-1) was performed from the B16 mouse melanoma cell nuclear extract using biotin-cellulose affinity chromatography. The affinity-purified fraction exhibited binding to the DNA fragment containing TE-1, and to a synthetic oligomer representing TE-1. UV-cross-linking indicated that the size of TEBP-1 is approximately 49 kD. TE-1 also directed enhanced CAT activity in the B16 melanoma cells but not in non-pigment cells. These data indicate that TE-1 may be an enhancer element that is responsible for pigment cell specific expression of the tyrosinase gene.

Identification of a cis-acting element that enhances the pigment cell-specific expression of the human tyrosinase gene.

To identify the cis-acting element that is responsible for the pigment cell-specific expression of the human tyrosinase gene, we analyzed the promoter activity of its 5'-flanking region by transient expression assays. The fusion genes were constructed by inserting the 5'-flanking region of the human tyrosinase gene upstream from the firefly luciferase gene and were introduced into human melanoma cells and HeLa cells. We thus found the element, located between 2.7 and 1.8 kilobase pairs upstream from the transcription initiation site, that enhances the transient expression of the luciferase reporter gene in melanoma cells, but not in HeLa cells, the tyrosinase gene expression of which is not detectable. Using the fusion genes containing putative enhancer elements under the control of the heterologous simian virus 40 promoter, we identified the pigment cell-specific enhancer of approximately 200 base pairs (bp) between -2.0 and -1.8 kilobase pairs and localized the core sequence to a 39-bp region. This 39-bp core element was then confirmed to direct the melanoma cell-specific expression of the reporter gene under the tyrosinase gene promoter. We thus propose that this core element is responsible for the pigment cell-specific expression of the human tyrosinase gene.

Downstream region of the human tyrosinase-related protein gene enhances its promoter activity

We have cloned and sequenced the human genomic DNA segments encoding the 5′-flanking region and the first two exons of the tyrosinase-related protein (TRP) gene, a pigment cell-specific gene. Functional analysis of its promoter suggests that the downstream region of the TRP gene, including the first intron, enhances the transient expression of the luciferase gene under control of the TRP gene promoter about 16- to 20-fold. This enhancer-like activity is detected not only in melanoma cells but also in HeLa cells whose TRP gene expression is assumed to be repressed. We suggest a possibility that the downstream region is not sufficient to confer pigment cell-specific expression, but is required for efficient transcription of the TRP gene in pigment cells.

Lens and pigment cell specific gene expression in multipotent dedifferentiated cells derived from chick embryo pigment epithelial cells [formula omitted]: [formula omitted], [formula omitted]∗ and [formula omitted]. Natl. Inst. Basic Biology, Okazaki 444 and ∗Aichi Med. Univ., Nagakute 480-11, Japan

Lens and pigment cell specific gene expression in multipotent dedifferentiated cells derived from chick embryo pigment epithelial cells [formula omitted]: [formula omitted], [formula omitted]∗ and [formula omitted]. Natl. Inst. Basic Biology, Okazaki 444 and ∗Aichi Med. Univ., Nagakute 480-11, Japan