Previous work has demonstrated that two key melanocyte-specific elements termed the MSEu and MSEi play critical roles in the expression of the melanocyte-specific tyrosinase-related protein 1 (TRP-1) promoter. Both the MSEu and MSEi, located at position ؊237 and at the initiator, respectively, bind a melanocyte-specific factor termed MSF but are also recognized by a previously uncharacterized repressor, since mutations affecting either of these elements result in strong up-regulation of TRP-1 promoter activity in melanoma cells. Here we demonstrate that repression mediated by the MSEu and MSEi also operates in melanocytes. We also report that both the MSEu and MSEi are recognized by the brachyury-related transcription factor Tbx2, a member of the recently described T-box family, and that Tbx2 is expressed in melanocyte and melanoblast cell lines but not in melanoblast precursor cells. Although Tbx2 and MSF each recognize the TRP-1 MSEu and MSEi motifs, it is binding by Tbx-2, not binding by MSF, that correlates with repression. Several lines of evidence tend to point to the brachyury-related transcription factor Tbx2 as being the repressor of TRP-1 expression: both the MSEu and MSEi bind Tbx2, and mutations in either element that result in derepression of the TRP-1 promoter diminish binding by Tbx2; the TRP-1 promoter, but not the tyrosinase, microphthalmia, or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) promoter, is repressed by Tbx2 in cotransfection assays; a high-affinity consensus brachyury/Tbx2-binding site is able to constitutively repress expression of the heterologous IE110 promoter; and a low-affinity brachyury/Tbx2 binding site is able to mediate Tbx2-dependent repression of the G3PDH promoter. Although we cannot rule out the presence of an additional, as yet unidentified factor playing a role in the negative regulation of TRP-1 in vivo, the evidence presented here suggests that Tbx2 most likely is the previously unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box family member.In attempting to understand how the precise temporal and spatial pattern of gene expression necessary for the development of an organism is achieved, consideration should be given not only to the question of why a specific gene is expressed in a particular cell type at any given time, but also to why it is not expressed elsewhere or at other times. Analysis of tissue-specific promoters in many cell types has revealed that they often contain binding sites for widely expressed transcription factors which may act together with factors with a more restricted tissue distribution. However, while these tissue-specific factors may be present in only a very limited number of cell types, they frequently fall into transcription factor families, members of which have identical or highly similar DNA-binding properties. For example, the CANNTG E-box motif is recognized by members of the basic helix-loop-helix (bHLH) class of transcription factors, and w...

Constitutive activation of the Wnt/␤-catenin signaling pathway is a notable feature of a large minority of cases of malignant melanoma, an aggressive and increasingly common cancer. The identification of target genes downstream from this pathway is therefore crucial to our understanding of the disease. The POU domain transcription factor Brn-2 has been implicated in control of proliferation and melanoma survival, and its expression is strongly upregulated in melanoma. We show here that in vivo Brn-2 is expressed in melanocytes but not in embryonic day 11.5 melanoblasts and that its expression is directly controlled by the Wnt/␤-catenin signaling pathway in melanoma cell lines and in transgenic mice. Moreover, silent interfering RNA-mediated inhibition of Brn-2 expression in melanoma cells overexpressing ␤-catenin results in significantly decreased proliferation. These results, together with the observation that BRAF signaling also induces Brn-2 expression, reveal that Brn-2 is a focus for the convergence of two key melanoma-associated signaling pathways that are linked to cell proliferation.Melanocytes originate in the neural crest as undifferentiated nonpigmented melanoblasts that migrate to the epidermis and hair follicles, where they differentiate and are responsible for skin and hair color. As melanocytes are not essential for survival and as mutations affecting the survival or differentiation of the melanocyte lineage are reflected in an obvious pigmentation phenotype, the melanocyte system represents an excellent model for understanding how signal transduction pathways coordinate the program of gene regulation underlying the genesis of a specific cell type. Importantly, constitutive activation of signaling pathways normally operating during melanocyte development is linked to the transformation of a melanocyte to a malignant melanoma (5, 16), a highly aggressive cancer, the incidence of which is increasing at an alarming rate (33).The Wnt signaling pathway (for reviews of Wnt signaling, see references 3 and 12) is critically required for development of the melanocyte lineage; in both zebrafish and mice, overexpression of components of the Wnt signaling pathway result in an increase in the number of melanocytes at the expense of neurons and glia (9, 11), and disruption of the Wnt-1 and Wnt-3a genes leads to complete loss of melanoblasts (24). Wnt proteins interact with frizzled receptors and lead to the inhibition of serine-threonine kinase glycogen synthase kinase 3␤. Phosphorylation of ␤-catenin by glycogen synthase kinase 3␤ is associated with the destabilization of ␤-catenin. Thus, increased Wnt signaling leads to stabilization of ␤-catenin and its translocation from the cytoplasm to the nucleus, where it can activate transcription via association with the Lef1 and Tcf transcription factors (1,22,29). A key role for Wnt signaling in melanocyte development is the activation of the promoter for the gene encoding the microphthalmia-associated transcription factor Mitf (10, 43). Mitf (19,23) is essential fo...

Malignant melanoma, an aggressive and increasingly common cancer, is characterized by a strikingly high rate (70%) of mutations in BRAF, a key component of the mitogen-activated protein (MAP) kinase signaling pathway. How signaling events downstream from BRAF affect the underlying program of gene expression is poorly understood. We show that the Brn-2 POU domain transcription factor is highly expressed in melanoma cell lines but not in melanocytes or melanoblasts and that overexpression of Brn-2 in melanocytes results in increased proliferation. Expression of Brn-2 is strongly upregulated by Ras and MAP kinase signaling. Importantly, the Brn-2 promoter is stimulated by kinase-activating BRAF mutants and endogenous Brn-2 expression is inhibited by RNA interference-mediated downregulation of BRAF. Moreover, silent interfering RNA-mediated depletion of Brn-2 in melanoma cells expressing activated BRAF leads to decreased proliferation. The results suggest that the high levels of Brn-2 expression observed in melanomas link BRAF signaling to increased proliferation.The mitogen-activated protein (MAP) kinase signaling pathway, comprising the RAS-RAF-MEK-ERK cascade, plays a key role in the cellular response to extracellular signals, and deregulation of the pathway, for example, as a result of activating mutations in Ras, is a feature of many cancers. Three Raf genes have been described (ARAF, BRAF, and CRAF) that are regulated by interaction with Ras, and RAF protein can phosphorylate and activate the MAP kinase kinase MEK. The importance of this signaling pathway in cancer has recently been highlighted by the observation that almost 70% of melanomas and primary nevi exhibit kinase-activating mutations in BRAF (11,26). Understanding how signaling by the MAP kinase pathway affects the underlying program of gene expression is therefore a particularly important issue for melanoma, and it is presumed that deregulation of MAP kinase signaling results in altered expression or activity of key transcription factors, leading to aberrant growth control. The identification of BRAF target genes currently represents a major goal in melanoma biology.The POU domain transcription factor Brn-2 (also called N-Oct3 and POU3F2) (see reference 28 for a review) has been implicated in neuronal differentiation (16) and activation of the corticotropin-releasing hormone gene (22,29). Targeted disruption of the Brn-2 gene in the mouse results in loss of specific neuronal lineages in the hypothalamus and consequent loss of the posterior pituitary gland (25, 29). Brn-2-negative mice therefore die within 10 days after birth, although the specific cause of death is not apparent. Although Brn-2 clearly plays a major role in neuronal development, evidence also implicates it in melanoma growth and survival. Thus, Brn-2 mRNA is overexpressed in melanoma compared to normal melanocytes (15,33,34), and Brn-2 expression is substantially downregulated by differentiating agents (32). Importantly, downregulation of Brn-2 with an antisense strategy resulted in ...

Previous work has established that the melanocytespecific tyrosinase-related protein-1 (TRP-1) promoter is regulated positively by the microphthalmia-associated transcription factor Mitf, acting through the conserved M box and negatively by the T-box factor Tbx2, which can bind two "melanocyte-specific elements" termed the MSEu and MSEi. Both the MSEu and MSEi, which share a 6-base pair GTGTGA consensus, are also recognized by a previously unidentified melanocytespecific factor, MSF. Here we show using a combination of DNA binding assays, proteolytic clipping, and antiPax3 antibodies that MSF is indistinguishable from Pax3, a paired homeodomain transcription factor implicated genetically in melanocyte development and the regulation of the Mitf promoter. Consistent with Pax3 being able to bind the TRP-1 promoter, Pax3 is expressed in melanocytes and melanomas, and TRP-1 promoter activity is up-regulated by Pax3. The results identify a novel role for Pax3 in the expression of TRP-1, and the potential role of Pax3 in the melanocyte lineage is discussed.The development of the melanocyte lineage presents a fascinating opportunity to analyze the complex interplay between signal transduction pathways and transcription factors, which underlies development. Because melanocytes are not essential for viability and variations in pigmentation are obvious (1), over 70 independent genetic loci have been implicated in the development or function of these melanin-producing cells. Of the 20 or so that have been cloned to date, some, such as the genes encoding tyrosinase or tyrosinase-related protein-1 (TRP-1), 1 have a clearly defined function in the genesis of pigment. On the other hand, genes such as the endothelin B (2-4) and c-Kit receptors (5), and the microphthalmia (6 -8), Sox10 (9, 10), and Pax3 (11) transcription factors have been implicated in the developmental pathway leading to the genesis of the mature pigment-producing melanocyte from a nonpigmented melanoblast precursor cell originating in the trunk neural crest.Particularly interesting are mutations affecting the Pax3-paired homeodomain transcription factor, exemplified by the splotch allele (11), which expresses a truncated Pax3 protein.Although splotch homozygotes die in utero, heterozygous splotch mice exhibit pigmentation defects resulting from the loss of a proportion of the melanoblasts migrating away from the neural crest. The loss of melanocytes in splotch mice may be explained by the fact that Pax3 has recently been shown to activate expression from the promoter for the gene encoding the microphthalmia-associated basic helix-loop-helix-leucine zipper transcription factor (Mitf) (12); mice devoid of functional Mitf lack all pigment cells, and a decrease in Mitf levels resulting from monoallelic loss of Pax3 would account for the pigmentation defect exhibited by splotch mice. The ability of Pax3 to regulate expression of Mitf is paralleled by the role of Pax3 in skeletal muscle formation where it is required for expression of the basic helix-loop-helix tran...

We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Heterokaryons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.