Microphthalmia-associated Transcription Factor Gene Research Articles

Post-Transcriptional Regulation of Melanin Biosynthetic Enzymes by cAMP and Resveratrol in Human Melanocytes

Upregulation of microphthalmia-associated transcription factor (MITF) expression has been proposed to mediate melanogenesis stimulated by cAMP, whereas downregulation of MITF has been suggested to underlie the depigmentary effects of resveratrol, a promising chemotherapeutic found in red wine. We have assessed the contribution of MITF to pigmentation regulation by treating primary cultures of normal human melanocytes with the adenylate cyclase activator forskolin and/or resveratrol, then quantifying mRNA and protein levels for MITF, tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase (DCT). The inhibition of tyrosinase activity by resveratrol was not due to alterations in MITF, but instead was explained by both direct tyrosinase inhibition and a post-transcriptional effect that reduced the amount of fully processed tyrosinase. Glycosidase digestion revealed that the basis for the tyrosinase decrease was the retention of an immature form in the ER and subsequent loss of the mature, Golgi-processed enzyme. Elevation of intracellular cAMP by forskolin markedly increased protein levels for MITF, tyrosinase and DCT, however there was no concomitant increase in tyrosinase or DCT mRNA. This indicated that elevated levels of MITF were not sufficient to promote transcription of these melanogenic genes and that the increase in their protein abundance appeared to be predominantly mediated through post-transcriptional processing events.

Identification of the Consensus DNA Sequence for Nczf Binding

The Nczf gene, which is identified as a target gene of Ncx, encodes a novel Kruppel-associated box (KRAB) zinc finger protein, which functions as a sequence-specific transcriptional repressor. We generated a fusion protein of the zinc finger domain of Nczf and glutathione S-transferase to identify Nczf-binding consensus DNA sequences with random oligonucleotides of 15 and 35 bases. The consensus binding sequence of core nucleotides contains (A/T/C)CTTT(A/G)TTNT. In a gel mobility shift assay, the probe containing these sequences bound to the fusion protein. In silico analysis, these consensus sequences were found on regulatory regions of the endothelin receptor B and the microphthalmia-associated transcription factor genes, which are involved in neural crest development. These results suggest that Nczf functions as a sequence-specific transcription repressor to regulate neural crest cell development.

Lineage dependency and lineage-survival oncogenes in human cancer

Although cell-lineage and differentiation models dominate tumour classification and treatment, the recognition that cancer is also a genomic disease has prompted a reconfiguration of cancer taxonomies according to molecular criteria. Recent evidence indicates that a synthesis of lineage-based and genetic paradigms might offer new insights into crucial and therapeutically pliable tumour dependencies. For example, MITF (microphthalmia-associated transcription factor), which is a master regulator of the melanocyte lineage, might become a melanoma oncogene when deregulated in certain genetic contexts. MITF and other lineage-survival genes therefore implicate lineage dependency (or lineage addiction) as a newly recognized mechanism that is affected by tumour genetic alterations.

A Term Newborn Who Has Abnormal Facies

A term female presents with abnormal facies.The infant was delivered by the vaginal route in vertex presentation. She made the transition to the extrauterine environment without difficulty, with Apgar scores of 9 at both 1 and 5 minutes. Her birthweight was 3.3kg, which was appropriate for gestational age.The infant failed the newborn hearing screen bilaterally.*Adapted from Jones KL, ed. Smith's Recognizable Patterns of Human Malformation. 5th ed. Philadelphia, Pa: WB Saunders Co; 1997.Diagnosed clinically according to the Waardenburg Consortium.Waardenburg syndrome (WS) is an autosomal dominant disorder characterized by varying degrees of hypopigmentation, hearing loss, and defects in neural crest cell migration. The syndrome was named in 1951 by Petrus Johannes Waardenburg, a Dutch optometrist, who first defined the syndrome with six primary elements, including lateral displacement of the inner canthi with blepharophimosis, prominent broad nasal root, hypertrichosis of the medial eyebrows (synophyrys), white forelock, heterchromia iridis, and deafness. The frequency of WS is estimated to be 1 in 200,000 in the general population of the Netherlands and 1 in 20,000 in Kenya. There is no race or sex predilection, despite these reported frequencies.Multiple terms are used to describe the abnormal ocular findings in WS. Lateral displacement of the inner canthi gives rise to short palpebral fissures, resulting in telecanthus. Telecanthus is defined as an increased distance between canthi and is synonymous with hypertelorism (increased distance between two organs). Dystopia refers to the abnormal position of part of an organ and frequently is used to describe the position of the eyes in WS (ie, dystopia canthorum). Blepharophimosis is defined as a decrease in the size of the palpebral aperture without fusion of the lid margins, which results in convergent strabismus.Infants who have WS can be identified soon after birth. Four types of WS have been described, and all forms demonstrate marked variability, even within families.Congenital deafness in WS can be bilateral or unilateral and severe or moderate. Patients who have hearing loss and WS have temporal bone anomalies, with 50% having enlargement of the vestibular aqueduct visible on computed tomography.Cutaneous abnormalities include both hypopigmented lesions and hyperpigmented lesions and can resemble the lesions found in piebaldism, tuberous sclerosis, and neurofibromatosis.Ocular color abnormalities of WS present in three patterns: heterochromia iridis, bilateral isohypochromia iridis (pale blue eyes), and hypopigmentation of the fundus.Syndromes that have similar clinical features include Rozycki syndrome (deafness and viteligo), Fisch syndrome (congenital deafness and early graying of the hair), and Woolf syndrome (albinism and deafness).According to the Waardenburg Consortium, an individual can be diagnosed if two of the following major criteria or one major and two minor criteria are present:The measurements necessary to calculate the W index in mm are: inner canthal distance (a), interpupillary distance (b), and outer canthal distance (c)WS is associated with neural crest-derived melanocyte deficiency caused by mutations in transcription factors. More than 90% of individuals who meet the clinical diagnostic criteria for WS Type I have mutations in the PAX3 gene. The PAX3 gene is located on chromosome 2 and controls some aspects of the development of the face and inner ear. Approximately 15% of individuals who have Type II WS are heterozygous for mutations in the microphthalmia-associated transcription factor (MITF) gene. The MITF gene is found on chromosome 3 and is involved in the development of the ear. Type III WS is considered an extreme presentation of Type I, with some affected individuals being homozygous for the PAX3 gene mutation. Type IV WS can be caused by mutations in the genes for endothelin-3 or one of its receptors, EDNRB and EDN3 genes.

Three Cases of Waardenburg Syndrome Type 2 in a Korean Family

Waardenburg syndrome (WS) is a rare, autosomal dominant disorder characterized by sensorineural hearing loss, pigmentary disturbances of the skin, hair, and iris, and other developmental defects such as lateral displacement of both medial canthi and lacrimal puncta called dystopia canthorum. While mutations of the PAX3 (paired box) gene have been identified in about 99% of WS type 1 cases, WS type 2 is a heterogeneous group, with about 15% of cases caused by mutations in microphthalmia associated transcription factor (MITF). We have experienced three cases of typical WS type 2 in a Korean family, for whom full ocular examination and genetic studies were performed. The genetic studies revealed no mutation in either PAX3 or MITF genes. The genetic basis, as yet unknown for most cases of WS type 2, might be found with further investigation.

Microphthalmia-associated transcription factor in the Wnt signaling pathway.

Microphthalmia-associated transcription factor (MITF) contains a basic helix-loop-helix and leucine-zipper (bHLH-LZ) structure and consists of many isoforms with different N-termini. Melanocyte-specific MITF isoform (MITF-M) is of particular interest, because a heterozygous mutation in the MITF gene is associated with Waardenburg syndrome type 2 (WS2) that is characterized by deafness and hypopigmentation because of lack of melanocytes in the inner ear and skin. Expression of MITF-M is under the regulation of the melanocyte-specific promoter (M promoter) of the MITF gene, and transcription from the M promoter is induced by Wnt signals through a nuclear mediator, lymphoid-enhancing factor 1 (LEF-1). In addition, functional cooperation of MITF-M with LEF-1 could lead to transcriptional activation of the M promoter and the dopachrome tautomerase (DCT) gene, an early melanoblast marker. The bHLH-LZ region of MITF-M is responsible for the physical interaction with LEF-1, and beta-catenin is required for the collaboration between LEF-1 and MITF-M. Importantly, MITF-M could function as a non-DNA-binding co-factor for LEF-1. These results suggest that MITF-M may function as a self-regulator of its own expression to maintain a threshold level of MITF-M at a certain sensitive stage of melanocyte development, which could account for the dominant inheritance of WS2. MITF-M therefore plays dual roles in the Wnt signaling pathway; MITF-M represents a downstream target and a nuclear mediator of Wnt signals in melanocytes.

Waardenburg syndrome type 1

Waardenburg syndrome (WS) is a rare disease characterized by sensorineural deafness in association with pigmentary anomalies and defects of neural-crest-derived tissues. Depending on additional symptoms, WS is classified into four types, WS1, WS2, WS3 and WS4. WS1 and WS3 are attributed to mutations in PAX3, whereas WS2 is heterogeneous, being caused by mutations in the microphthalmia-associated transcription factor gene in some but not all affected families. WS4 is attributed to mutations in the endothelin-3 or the endothelin-B receptor genes and SOX10 gene. WS1 is an auditory-pigmentary disorder comprising sensorineural hearing loss and pigmentary disturbances of the iris, hair, and skin, along with dystopia canthorum. We report a case with a confirmed diagnosis of WS1 and review the relevant literature for this rare disorder.

Modulation of Microphthalmia-associated Transcription Factor Gene Expression Alters Skin Pigmentation

The microphthalmia-associated transcription factor is implicated in melanocyte development and in the regulation of melanogenesis. Microphthalmia-associated transcription factor is thought to bind to the M-box promoter elements of tyrosinase, tyrosinase-related protein-1 and dopachrome tautomerase/tyrosinase-related protein-2 and transactivate these genes, resulting in increased pigmentation. Using a luciferase reporter construct driven by the microphthalmia-associated transcription factor promoter, we identified agents that modulate microphthalmia-associated transcription factor promoter activity. Changes in endogenous microphthalmia-associated transcription factor expression levels upon treatment with these agents were confirmed using northern and western blots, and their pigmentary modulating activities were demonstrated. Ultraviolet B irradiation and traditional Chinese medicine-1, a natural extract used in traditional Chinese medicine, upregulated microphthalmia-associated transcription factor gene expression and enhanced tyrosinase activity in vitro. Dihydrolipoic acid, lipoic acid, and resveratrol reduced microphthalmia-associated transcription factor and tyrosinase promoter activities. These agents also inhibited the forskolin- and ultraviolet B-stimulated promoter activities of these genes and significantly reduced tyrosinase activity in melanocyte cultures, resulting in depigmentation. Overexpressed microphthalmia-associated transcription factor was capable of rescuing the repressive effects of these compounds on the cotransfected tyrosinase promoter. Dark-skinned Yucatan swine treated with these agents showed visible skin lightening, which was confirmed histologically, whereas ultraviolet B-induced tanning of light-skinned swine was inhibited using these agents. Our findings suggest that modulation of microphthalmia-associated transcription factor expression can alter skin pigmentation and further confirm the central role of microphthalmia-associated transcription factor in melanogenesis.

Differentiation of Murine Melanocyte Precursors Induced by 1,25-Dihydroxyvitamin D3 Is Associated with the Stimulation of Endothelin B Receptor Expression

The effects of 1,25-dihydroxyvitamin D3 on the differentiation of immature melanocyte precursors were studied. The NCC-/melb4 cell line is an immature melanocyte cell line established from mouse neural crest cells. 1,25-Dihydroxyvitamin D3 inhibited the growth of NCC-/melb4 cells at concentrations higher than 10(-8) m. That growth inhibition was accompanied by the induction of tyrosinase and a change in L-3,4-dihydroxyphenylalanine reactivity from negative to positive. Electron microscopy demonstrated that melanosomes were in more advanced stages after 1,25-dihydroxyvitamin D3 treatment. In primary cultures of murine neural crest cells, L-3,4-dihydroxyphenylalanine-positive cells were increased after 1,25-dihydroxyvitamin D3 treatment. These findings indicate that 1,25-dihydroxyvitamin D3 stimulates the differentiation of immature melanocyte precursors. Moreover, immunostaining and reverse transcription-polymerase chain reaction analysis revealed that endothelin B receptor expression was induced in NCC-/melb4 cells following treatment with 1,25-dihydroxyvitamin D3. The induction of endothelin B receptor by 1,25-dihydroxyvitamin D3 was also demonstrated in neural crest cell primary cultures, but not in mature melanocytes. The expression of microphthalmia-associated transcription factor was induced in NCC-/melb4 cells treated with 1,25-dihydroxyvitamin D3 and endothelin 3, but not by 1,25-dihydroxyvitamin D3 alone, suggesting that endothelin 3 may stimulate the expression of the microphthalmia-associated transcription factor gene after binding to the endothelin B receptor induced by 1,25-dihydroxyvitamin D3. These findings suggest a regulatory role for vitamin D3 in melanocyte development and melanogenesis, and may also explain the working mechanism of vitamin D3 in the treatment of vitiligo.

Identification of a distal enhancer for the melanocyte-specific promoter of the MITF gene.

Waardenburg syndrome (WS) is characterized by deafness and hypopigmentation because of the lack of melanocytes in the inner ear and skin. WS type 2 is associated with mutations in the gene encoding microphthalmia-associated transcription factor (MITF) that is required for melanocyte differentiation. MITF consists of multiple isoforms with different N-termini, one of which is exclusively expressed in melanocytes, named MITF-M. Its N-terminus is encoded by exon 1M that is under the regulation of the melanocyte-specific (M) promoter. Here we identify a distal regulatory region of 298 bp, located 14.5 kb upstream from exon 1M, which enhances the M promoter activity in cultured melanoma cells. This enhancer activity depends on the proximal M promoter region (-120 to -46). The MITF-M distal enhancer (MDE), thus identified, contains the binding sites for SOX10, a transcription factor responsible for another type of WS, known as Waardenburg-Hirschsprung syndrome. Characterization of MDE has suggested SOX10 as one of factors that are involved in the function of MDE. A putative MDE counterpart is located 12 kb upstream from mouse exon 1M and its role is discussed in relevance to the pathogenesis of red-eyed white Mitf mi-rw mice that exhibit small red eyes and white coat. Moreover, by in situ hybridization analysis, we suggest that Sox10 and Mitf-M (mRNA) are expressed in melanoblasts migrating toward the otic vesicle (prospective inner ear) of mouse embryos but are separately expressed in different cell types of the newborn cochlea. Thus, SOX10 regulates transcription from the M promoter in a developmental stage-specific manner.

Microphthalmia-associated transcription factor (MITF): multiplicity in structure, function, and regulation.

Microphthalmia-associated transcription factor (MITF) regulates the differentiation and development of melanocytes and retinal pigment epithelium and is also responsible for pigment cell-specific transcription of the melanogenesis enzyme genes. Heterozygous mutations in the MITF gene cause auditory-pigmentary syndromes. MITF consists of at least five isoforms, MITF-A, MITF-B, MITF-C, MITF-H, and MITF-M, differing at their N-termini and expression patterns. Here we show a remarkable similarity between the N-terminal domain of MITF-A and cytoplasmic retinoic acid-binding proteins. To date, four isoform-specific first exons have been identified in the MITF gene: exons 1A, 1H, 1B, and 1M in the 5' to 3' direction, each of which encodes the unique N-terminus of a given isoform. The 5'-flanking regions of these isoform-specific exons are termed A, H, B, and M promoters, respectively. Among these promoters, the M promoter has received particular attention, because it is functional only in melanocyte-lineage cells and is upregulated by Wnt signaling via the functional LEF-1-binding site. Moreover, the M promoter is upregulated by other transcription factors, PAX3, SOX10, and CREB. The activity and degradation of MITF-M are regulated by extracellular signals via protein phosphorylation, such as c-Kit signaling. Together, multiple signals appear to converge on the M promoter as well as on MITF proteins, leading to the proper regulation of MITF-M in melanocytes and other MITF isoforms in many cell types.

The Transcription Factor Onecut-2 Controls the Microphthalmia-Associated Transcription Factor Gene

Microphthalmia-associated transcription factor (MITF) is essential for melanocyte differentiation. MITF mutations are associated with some cases of Waardenburg syndrome (WS) type 2. WS is a dominantly inherited disease characterized by auditory-pigmentary defects that result from the absence of melanocytes. The lack of mutation in MITF coding sequences in some WS2 patients suggests that unidentified factors controlling MITF expression might be involved. We show here that the cut-homeodomain transcription factor Onecut-2 (OC-2) is expressed in melanocytes and binds to the MITF gene promoter. Overexpression of OC-2 in transfected cells stimulates MITF promoter activity. Mutations that prevent OC-2 binding decrease MITF promoter activity by 75%. Based on these results, we searched in 56 WS2 patients for mutations in the OC2 gene or in OC-2 binding sites in the MITF promoter, but none was found. These results show that OC-2 stimulates MITF expression and that OC2 is a candidate gene, but not a common cause, of WS.

Regulation of tyrosinase-related protein-2 (TYRP2) in human melanocytes: relationship to growth and morphology.

Treatment of human melanoma cells with the differentiation-inducing agent hexamethylene bisacetamide (HMBA) results in reciprocal changes in expression of melanocyte-specific genes tyrosinase-related proteins-1 and -2 (TYRP1 and TYRP2). In this study, we investigated the effects of HMBA on cultured neonatal human cutaneous melanocytes. Flow cytometric analysis showed that HMBA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent growth of melanocytes by reducing the population of cells entering the DNA synthesis phase of cell cycle. Melanocyte growth inhibition was accompanied by an increase in the number of cells exhibiting polydendritic morphology. This morphologic change was less pronounced when HMBA was added to melanocytes in the absence of TPA. Northern blot analyses of total cellular RNA showed that expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), TYRP1, Silver (SILV/Pmel17) gene was down-regulated by HMBA, while TYRP2 mRNA was up-regulated (> 10-fold). When the inducer was added to cells in the absence of TPA, there was > 50-fold increase in TYRP2 mRNA with a moderate increase in MITF, tyrosinase and SILV gene mRNAs and complete repression of TYRP1 gene. Studies using inhibitors for protein kinases involved in cell signaling pathways suggested that stress-activated kinase p38 and mitogen-activated protein kinase kinase MEK are involved in TPA-independent regulation of TYRP2 expression in melanocytes. These data show that treatment of proliferating melanocytes with the differentiation inducer HMBA results in a distinct change in morphology and up-regulation of TYRP2, while quiescent melanocytes respond by a dramatic increase in expression of TYRP2 without change in morphology. These results suggest an inverse relationship of TYRP2 gene regulatory mechanisms to melanocyte growth regulatory pathways.

Regulation of the Microphthalmia-associated Transcription Factor Gene by the Waardenburg Syndrome Type 4 Gene,SOX10

The absence of melanocytes from the cochlea and epidermis is responsible of deafness and hypopigmentation, two symptoms shared by the four Waardenburg syndrome (WS) subtypes. Microphthalmia-associated transcription factor (MITF) controls melanocyte survival and differentiation. Mutations, which impair MITF function or expression, result in an abnormal melanocyte development leading to the WS2. WS1 and WS3 are caused by mutation in the gene encoding the transcription factor Pax3, which regulates MITF expression. Recently, mutations in SOX10, a gene encoding a SRY-related transcription factor, have been reported in patients with WS4. However, the molecular basis of the defective melanocyte development in these patients remained to be elucidated. In the present report, we demonstrate that Sox10 is a strong activator of the MITF promoter, and we identify a Sox10 binding site between -264 and -266 of the MITF promoter. Finally, we show that three SOX10 mutations found in WS4 abolish the transcriptional activity of the resulting Sox10 proteins toward the MITF promoter. Taken together, our observations bring new and meaningful information concerning the molecular process that leads to a defective melanocyte development in WS4 patients with SOX10 mutations.

Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome.

Waardenburg syndrome (WS) is an autosomal dominant disorder with an incidence of 1 in 40 000 that manifests with sensorineural deafness and pigmentation defects. It is classified into four types depending on the presence or absence of additional symptoms. WS1 and WS3 are due to mutations in the PAX3 gene whereas some WS2 cases are associated with mutations in the microphthalmia-associated transcription factor (MITF) gene. The WS4 phenotype can result from mutations in the endothelin-B receptor gene (EDNRB), in the gene for its ligand, endothelin-3 (EDN3), or in the SOX10 gene. PAX3 has been shown to regulate MITF gene expression. The recent implication of SOX10 in WS4 prompted us to test whether this transcription factor, known to cooperate in vitro with PAX3, is also able to regulate expression from the MITF promoter. Here we show that SOX10, in synergy with PAX3, strongly activates MITF expression in transfection assays. Analyses revealed that PAX3 and SOX10 interact directly by binding to a proximal region of the MITF promoter containing binding sites for both factors. Moreover, SOX10 or PAX3 mutant proteins fail to transactivate this promoter, providing further evidence that the two genes act in concert to directly regulate expression of MITF. In situ hybridization experiments carried out in the dominant megacolon (DOM:) mouse, confirmed that SOX10 dysfunction impairs MITF: expression as well as melanocytic development and survival. These experiments, which demonstrate an interaction between three of the genes that are altered in WS, could explain the auditory-pigmentary symptoms of this disease.

MITF: a stream flowing for pigment cells.

Microphthalmia-associated transcription factor (MITF) is a transcription factor with a basic-helix-loop-helix-leucine zipper (bHLHZip) structure. Mutations of the MITF gene cause a variety of phenotypes, most notably in pigmented cells, in several species. In humans, haploinsufficiency of MITF causes Waardenburg syndrome type 2, while a dominant-negative mutation causes Tietz syndrome. Four isoforms have been cloned so far: MITF-M is the most abundant and is expressed in neural-crest-derived melanocytes; MITF-A is expressed in various cultured cells including retinal pigment epithelium (RPE) and enriched in RPE of embryonal and developing eyes; MITF-H are expressed in many types of cultured cells and in the heart tissue; MITF-C is expressed in many types of cultured cells, but not in melanocytes. Many growth factor signaling pathways have been implicated for regulation of MITF at both protein and promoter levels. Most notably, Steel factor/c-Kit signaling pathway was linked to phosphorylation of MITF at Ser73 and Ser409 through activation of MAP kinase and RSK-1, respectively. Phosphorylation of MITF is also conducted at Ser298 through GSK3beta, although the signaling pathway for this event still remains to be elucidated. IGF-1 and HGF/SF pathways may merge with the c-Kit signaling pathway. WNT and MSH signaling pathways regulate MITF positively at the promoter level. Endothelins may regulate MITF at the protein and promoter levels. MITF is involved in the differentiation, growth and survival of pigment cells, employing a number of signaling pathways.

Molecular bases of congenital hypopigmentary disorders in humans and oculocutaneous albinism 1 in Japan.

The molecular bases of various types of congenital hypopigmentary disorders have been clarified in the past 10 years. Homozygous gene mutations of enzymes functional in melanogenesis such as tyrosinase, P protein and DHICA oxidase, result in oculocutaneous albinism (OCA) 1, OCA 2, and OCA 3, respectively. The genes responsible for Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS) have also recently been isolated and cloned. The transcription factor paired box 3 (PAX3) works at the promoter region of the microphthalmia-associated transcription factor (MITF) gene, and the MITF transcription factor orders the expression of c-kit, which encodes the receptor for stem-cell factor, which in turn stimulates melanoblast migration from the neural tube to the skin in the embryo. Heterozygous mutations of PAX3, MITF, or c-kit genes induce Waardenburg syndrome (WS) 1/3, WS 2 or Piebaldism, respectively. A defect of endothelin-3 or the endothelin-B receptor produces WS 4. In our examination of 26 OCA 1 patients in Japan, all were found to have homozygous or heterozygous tyrosinase gene mutations at codons 77 or 310. Therefore, mutations at codons 77 and 310 are the major ones in Japanese patients with OCA 1. An autosomal dominant pigmentary disease of dyschromatosis symmetrica hereditaria (DSH) is well known in Japan, and is characterized by a mixture of hypo- and hyper-pigmented macules of various sizes on the backs of the hands and feet. The disease gene and its chromosomal localization have not been identified yet. Our trial of linkage analysis and positional cloning to determine the disease gene is presented.

Structural organization of the human microphthalmia-associated transcription factor gene containing four alternative promoters

Microphthalmia-associated transcription factor (MITF) affects the development of many types of cells, including melanocytes and retinal pigment epithelium (RPE). MITF consists of at least three isoforms, MITF-A, MITF-H and MITF-M, differing at their amino-termini and expression patterns. Here, we characterize the structural organization of the human MITF gene. The gene contains at least four isoform-specific first exons, exons 1A, 1H, 1B and 1M in the 5′ to 3′ direction, each of which encodes the unique amino-terminus of a given isoform, including newly identified MITF-B. The 5′-flanking regions of these isoform-specific exons are termed promoters A, H, B and M, respectively, which showed different promoter activities, as judged by transient transfection assay. Promoter A directs the expression of a reporter gene in RPE, cervical cancer and melanoma cells, whereas promoter M is functional only in melanoma cells. Promoter H showed the significant activity in RPE and cervical cancer cells but not in melanoma cells. In contrast, the 1.7 kb 5′-flanking region of exon 1B showed no noticeable promoter activity in these cell lines. Therefore, alternative promoters provide the MITF gene with the diversity in transcriptional regulation and the capability of generating structurally different protein isoforms.

Point mutation in the MITF gene causing Waardenburg syndrome type II in a three-generation Indian family

Waardenburg syndrome (WS) is an autosomal-dominant neural crest cell disorder phenotypically characterized by hearing impairment and disturbance of pigmentation. A presence of dystopia canthorum is indicative of WS type 1, caused by loss of function mutation in the PAX3 gene. In contrast, type 2 WS (WS2) is characterized by normally placed medial canthi and is genetically heterogeneous; mutations in MITF (microphthalmia associated transcription factor) associated with WS2 have been identified in some but not all affected families. Here, we report on a three-generation Indian family with a point mutation in the MITF gene causing WS2. This mutation, initially reported in a Northern European family, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking the HLH-Zip or Zip structure necessary for normal interaction with its target DNA motif. Comparison of the phenotype between the two families demonstrates a significant difference in pigmentary disturbance of the eye. This family, with the first documented case of two unrelated WS2 families harboring identical mutations, provides additional evidence for the importance of genetic background on the clinical phenotype.

The rat microphthalmia-associated transcription factor gene (Mitf) maps at 4q34-q41 and is mutated in the mib rats.

The rat gene encoding the microphthalmia-associated transcription factor (Mitf) was assigned to rat Chromosome (Chr) 4q34-q41, as well as the Gata2 and Mem1 genes. Rat Chr 4 is homologous to mouse Chr 6 and human Chr 3, which carry the Mitf (MITF) gene in these species (MMU 6, 40.0 cM, and HSA 3p14.1-p12.3). mib/mib rats, which are characterized by depigmentation, microphtalmy, osteopetrosis, and neurological disorders were shown to bear a deletion covering several kilobases of genomic DNA in the Mitf gene and to lack Mitf mRNA. The Mitf mutation in the mib/mib rats is thus very likely to be a Mitf null mutation, causing a phenotype similar to the one observed in the miVGA-9 mice, but including osteopetrosis as an additional feature.