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• W2016114470 abstract "Commitment to the melanocyte lineage is characterized by the onset of microphthalmia-associated transcription factor (Mitf) expression. Mitf plays a fundamental role in melanocyte development, with mice lacking Mitf being entirely devoid of pigment cells. In the absence of functional Mitf protein, melanoblasts expressing Mitf mRNA disappear around 2 days after their first appearance either by apoptosis or by losing their identity and adopting an alternative cell fate. The role of Mitf must therefore be to regulate genes required for melanoblast survival, proliferation, or the maintenance of melanoblast identity. Yet to date, Mitf has been shown to regulate genes such as Tyrosinase,Tyrp-1, and Dct, which are required for pigmentation, a differentiation-specific process. Because expression of these genes cannot account for the complete absence of pigment cells in Mitf-negative mice, Mitf must regulate the expression of other as yet uncharacterized genes. Here we provide several lines of evidence to suggest that Mitf may regulate the expression of the Tbx2 transcription factor, a member of the T-box family of proteins implicated in the maintenance of cell identity. First, isolation and sequencing of the entire murine Tbx2 gene revealed that the Tbx2promoter contains a full consensus Mitf recognition element; second, Mitf could bind the promoter in vitro and activateTbx2 expression in vivo in an E box-dependent fashion; and third, Tbx2 is expressed in melanoma cell lines expressing Mitf, but not in a line in which Mitf expression was not detectable. Taken together, with the fact that Tbx2 is expressed in Mitf-positive melanoblasts and melanocytes, but not in Mitf-negative melanoblast precursor cells, the evidence suggests that the Tbx2 gene may represent one of the first known targets for Mitf that is not a gene involved directly in the manufacture of pigment. Commitment to the melanocyte lineage is characterized by the onset of microphthalmia-associated transcription factor (Mitf) expression. Mitf plays a fundamental role in melanocyte development, with mice lacking Mitf being entirely devoid of pigment cells. In the absence of functional Mitf protein, melanoblasts expressing Mitf mRNA disappear around 2 days after their first appearance either by apoptosis or by losing their identity and adopting an alternative cell fate. The role of Mitf must therefore be to regulate genes required for melanoblast survival, proliferation, or the maintenance of melanoblast identity. Yet to date, Mitf has been shown to regulate genes such as Tyrosinase,Tyrp-1, and Dct, which are required for pigmentation, a differentiation-specific process. Because expression of these genes cannot account for the complete absence of pigment cells in Mitf-negative mice, Mitf must regulate the expression of other as yet uncharacterized genes. Here we provide several lines of evidence to suggest that Mitf may regulate the expression of the Tbx2 transcription factor, a member of the T-box family of proteins implicated in the maintenance of cell identity. First, isolation and sequencing of the entire murine Tbx2 gene revealed that the Tbx2promoter contains a full consensus Mitf recognition element; second, Mitf could bind the promoter in vitro and activateTbx2 expression in vivo in an E box-dependent fashion; and third, Tbx2 is expressed in melanoma cell lines expressing Mitf, but not in a line in which Mitf expression was not detectable. Taken together, with the fact that Tbx2 is expressed in Mitf-positive melanoblasts and melanocytes, but not in Mitf-negative melanoblast precursor cells, the evidence suggests that the Tbx2 gene may represent one of the first known targets for Mitf that is not a gene involved directly in the manufacture of pigment. basic helix-loop-helix-leucine zipper base pair(s) fetal calf serum polymerase chain reaction reverse transcription bacterial artificial chromosome in vitrotranscribed/translated microphthalmia-associated transcription factor upstream stimulatory factor 1 upstream melanocyte-specific element initiator melanocyte-specific element phosphate-buffered saline Tyrosinase-related protein-1 dopachrome tautomerase Understanding how specific cell lineages are established and maintained lies at the heart of developmental biology. The melanocyte lineage arises in the neural crest as nonpigmented precursor cells, termed melanoblasts, which then migrate to their final destinations in the epidermis and hair follicles, where they differentiate into mature pigment-producing melanocytes. Little is known of the precise program of events leading from a multipotent neural crest cell to a melanoblast, but the switch from a melanoblast precursor cell to a melanoblast is characterized by the onset of expression of the basic-helix-loop-helix-leucine zipper (bHLH-LZ)1microphthalmia-associated transcription factor, Mitf (1.Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (942) Google Scholar, 2.Moore K.J. Trends Genet. 1995; 11: 442-448Abstract Full Text PDF PubMed Scopus (196) Google Scholar). Mitf plays a critical but poorly understood role in melanocyte development. Mice lacking Mitf generate neural crest-derived melanoblasts, as measured by the expression of Mitf mRNA, but these cells are no longer detectable around 2 days after their first appearance (3.Opdecamp K. Nakayama A. Nguyen M.T. Hodgkinson C.A. Pavan W.J. Arnheiter H. Development. 1997; 124: 2377-2386Crossref PubMed Google Scholar), resulting in a mouse devoid of all pigment cells. It is likely that the loss of melanoblasts in Mitf-negative mice reflects either the failure of the melanoblasts to survive or an inability to maintain their identity. Although Mitf has been shown to regulate the expression of genes such as Tyrosinase and Tyrp-1 (4.Yasumoto K. Yokoyama K. Shibata K. Tomita Y. Shibahara S. Mol. Cell. Biol. 1994; 14: 8058-8070Crossref PubMed Scopus (359) Google Scholar, 5.Yavuzer U. Keenan E. Lowings P. Vachtenhein J. Currie G. Goding C.R. Oncogene. 1995; 10: 123-134PubMed Google Scholar, 6.Bentley N.J. Eisen T. Goding C.R. Mol. Cell. Biol. 1994; 14: 7996-8006Crossref PubMed Scopus (417) Google Scholar, 7.Hemesath T.J. Steingrimsson E. McGill G. Hansen M.J. Vaught J. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Fisher D.E. Genes Dev. 1994; 8: 2770-2780Crossref PubMed Scopus (545) Google Scholar, 8.Ganss R. Schutz G. Beermann F. J. Biol. Chem. 1994; 269: 29808-29816Abstract Full Text PDF PubMed Google Scholar), which are involved in the manufacture of the pigment melanin, expression of the pigmentation genes is not required for the genesis of the melanocyte lineage. This implies that Mitf must regulate other genes required for the survival, proliferation of melanoblasts, or the maintenance of their cell identity. However, apart from the pigmentation genes, no targets for Mitf in the melanocyte lineage have been identified to date. Over the past few years considerable excitement has been generated by the discovery, by Bollag et al., (9.Bollag R.J. Siegried Z. Cebra-Thomas J.A. Garvay N. Davison E.M. Silver L.M. Nat. Genet. 1994; 7: 383-389Crossref PubMed Scopus (195) Google Scholar) of a novel family of transcription factors defined by the conservation of the T box, which corresponds approximately to the DNA binding domain (10.Kispert A. Herrmann B.G. EMBO J. 1993; 12: 3211-3220Crossref PubMed Scopus (280) Google Scholar, 11.Müller C.W. Herrmann B.G. Nature. 1997; 389: 884-889Crossref PubMed Scopus (265) Google Scholar). The T-box family plays a critical role in embryonic development (for reviews see Refs. 12.Smith J. Curr. Opin. Genet. Dev. 1997; 7: 474-480Crossref PubMed Scopus (113) Google Scholar, 13.Papaioannou V.E. Silver L.M. BioEssays. 1998; 20: 9-19Crossref PubMed Scopus (269) Google Scholar, 14.Papaioannou V.E. Trends Genet. 1997; 13: 212-213Abstract Full Text PDF PubMed Scopus (42) Google Scholar) and is highly evolutionarily conserved, with family members being found in a wide range of organisms, including human, mouse, chicken, Xenopus, zebra fish, andDrosophila, with at least 21 T-box proteins being identified by sequence homology on completion of the sequence of theCaenorhabditis elegans genome (15.Chervitz S.A. Aravind L. Sherlock G. Ball C.A. Koonin E.V. Dwight S.S. Harris M.A. Dolinski K. Mohr S. Smith T. Weng S. Cherry J.M. Botstein D. Science. 1998; 282: 2022-2028Crossref PubMed Scopus (361) Google Scholar). Consistent with the high degree of conservation within the T-box family, mutations within the family have a dramatic affect on development. For example, the prototype for the family, the T gene (16.Herrmann B.G. Labeit S. Poustka A. King T.R. Lehrach H. Nature. 1990; 343: 617-622Crossref PubMed Scopus (668) Google Scholar), encodes the Brachyury transcription factor (17.Kispert A. Koschorz B. Herrmann B.G. EMBO J. 1995; 14: 4763-4772Crossref PubMed Scopus (254) Google Scholar), which is essential for mesoderm induction, whereas mutations in the human Tbx3 andTbx5 genes result in the autosomal dominant ulnar-mammary (18.Bamshad M. Lin R.C. Law D.J. Watkins W.C. Krakowiak P.A. Moore M.E. Franceschini P. Lala R. Holmes L.B. Gebuhr T.C. Bruneau B.G. Schinzel A. Seidman J.G. Seidman C.E. Jorde L.B. Nat. Genet. 1997; 16: 311-315Crossref PubMed Scopus (443) Google Scholar) and Holt-Oram (19.Li Q.Y. Newbury-Ecob R.A. Terrett J.A. Wilson D.I. Curtis A.R. Yi C.H. Gebuhr T. Bullen P.J. Robson S.C. Strachan T. Bonnet D. Lyonnet S. Young I.D. Raeburn J.A. Buckler A.J. Law D.J. Brook J.D. Nat. Genet. 1997; 15: 21-29Crossref PubMed Scopus (750) Google Scholar) syndromes, respectively. Both syndromes are characterized by developmental defects; in ulnar-mammary syndrome the limb and apocrine glands are affected, whereas Holt-Oram syndrome is characterized by abnormalities in the cardiac septum and forelimbs. In addition to these naturally occurring mutations, targeted disruption of the mouse Tbx6 gene results in an embryo in which the somites are transformed into neural tubes (20.Chapman D.L. Papaioannou V.E. Nature. 1998; 391: 695-697Crossref PubMed Scopus (338) Google Scholar), whereas theTbx4 and Tbx5 genes regulate limb identity (21.Takeuchi J.K. Koshiba-Takeuchi K. Matsumoto K. Vogel-Höpker A. Naitoh-Matsuo M. Ogura K. Takahashi N. Yasuda K. Ogura T. Nature. 1999; 398: 810-814Crossref PubMed Scopus (208) Google Scholar,22.Rodriguez-Estaban C. Tsukui T. Yonei S. Magallon J. Tamura K. Balmonte J.C.I. Nature. 1999; 398: 814-818Crossref PubMed Scopus (240) Google Scholar). Together the evidence suggests that T-box family members may play a crucial role in the maintenance of cell identity and that at least one family member may be expressed in all cell types. We have shown previously that, in the melanocyte lineage, a single T-box factor gene Tbx2, is expressed in melanoblasts and melanocytes but not in neural crest-derived melanoblast precursor cells (23.Carreira S. Dexter T.J. Yavuzer U. Easty D.J. Goding C.R. Mol. Cell. Biol. 1998; 18: 5099-5108Crossref PubMed Scopus (152) Google Scholar), that is, Tbx2 expression appears to occur after commitment to the melanocyte lineage. Given the critical role of the T-box family in the maintenance of cell identity in development, understanding the controls operating on Tbx2 expression should provide an important insight into the regulatory mechanisms operating during the genesis of the melanocyte lineage. To this end, we have isolated and sequenced the entire murine Tbx2 gene, characterized the intron/exon boundaries, and mapped the transcription start site. We demonstrate that the Tbx2 promoter functions in a cell type-specific fashion and can be regulated by Mitf through a specific Mitf-consensus binding site. Thus Tbx2 may represent one of the first known targets for Mitf in the melanocyte lineage other than those genes directly involved in the manufacture of pigment. Two primers corresponding to the 3′-end of the mouse Tbx2 cDNA clone were synthesized: 5′-CTCAGCCAAAGAGGCG-3′ and 5′-CCCATTCTGTGCTGTACACG-3′. These were used to amplify by polymerase chain reaction (PCR) a 192-base pair (bp) Tbx2 cDNA fragment. This fragment was used as a probe to screen a mouse ES genomic library. A genomic clone of approximately 100 kilobases in a P1 vector was obtained, digested by BamHI or XhoI andSalI, and subcloned into either the BamHI orSalI sites of pBluescript-SK+. The locations of the intron/exon boundaries were determined by sequencing either genomic PCR products or direct sequencing of the subclones, and the transcription start site was located by primer extension analysis. Primer extension was performed with a primer (5′-TCATCGGGACATCCGGCCCAGGCTCCAGG-3′) derived from the previously published Tbx2 cDNA sequence (9.Bollag R.J. Siegried Z. Cebra-Thomas J.A. Garvay N. Davison E.M. Silver L.M. Nat. Genet. 1994; 7: 383-389Crossref PubMed Scopus (195) Google Scholar). Isolation of RNA and blotting procedures were described previously (24.Easty D.J. Guthrie B.A. Maung K. Farr C.J. Lindberg R.A. Toso R.J. Herlyn M. Bennett D.C. Cancer Res. 1995; 55: 2528-2532PubMed Google Scholar). For the reverse transcription (RT)-PCR, total RNA isolated from various cell lines was subjected to RT with avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals) followed by a first strand cDNA synthesis (First Strand cDNA synthesis kit; Amersham Pharmacia Biotech). PCR was performed as described previously (23.Carreira S. Dexter T.J. Yavuzer U. Easty D.J. Goding C.R. Mol. Cell. Biol. 1998; 18: 5099-5108Crossref PubMed Scopus (152) Google Scholar), and the primers used were as follows: 5′-CAGACAGACAGTGCGTC-3′ and 5′-ACTGGGCTCACGGCTATTTC-3′ for Tbx2 and 5′-CCAACTGCTTAGCCCCCCTGGCCAAG-3′ and 5′-CTCCTTGGAGGCCATGTAGGCCATG-3′ for the glyceraldehyde-3-phospho-dehydrogenase(G3PDH) gene yielding PCR products of 599 and 550, bp respectively. In performing the RT-PCR we took aliquots of the reactions for analysis every four cycles to determine the kinetics of the PCR reaction, and the samples for analysis were taken from the “log phase” of the PCR reaction after the same number of cycles. The band shift assays for USF1were performed in a final volume of 20 μl containing 20 mmHepes (pH 7.9), 10% glycerol, and 112 mm KCl; nuclear extracts were prepared as described previously (25.Yavuzer U. Goding C.R. Mol. Cell. Biol. 1994; 14: 3494-3503Crossref PubMed Scopus (60) Google Scholar). The conditions for binding of Mitf were as described (26.Bertolotto C. Busca R. Abbe P. Bille K. Aberdam E. Ortonne J.-P. Ballotti R. Mol. Cell. Biol. 1998; 18: 694-702Crossref PubMed Scopus (286) Google Scholar) except that the DNA-protein complexes were resolved by electrophoresis in a 6% polyacrylamide gel (37.5:1 acrylamide:bis) containing 10% glycerol. In vitrotranscribed/translated (ITT) protein was made by using a TNT T7 Quick Coupled transcription kit as instructed by the manufacturer (Promega). Nuclear extracts or ITT USF1 (27.Aksan I. Goding C.R. Mol. Cell. Biol. 1998; 18: 6930-6938Crossref PubMed Scopus (176) Google Scholar) were preincubated at 0 °C with 1 μg of poly(dIdC-dIdC) for 20 min before adding 10, 50, or 250 ng of cold competitor or USF1 polyclonal antibody (Santa Cruz). After a further incubation of 20 min, the radiolabeled probe was added and the assays were carried out as described previously (23.Carreira S. Dexter T.J. Yavuzer U. Easty D.J. Goding C.R. Mol. Cell. Biol. 1998; 18: 5099-5108Crossref PubMed Scopus (152) Google Scholar). The monoclonal anti-SV5 epitope antibody was purchased from Serotec, and the polyclonal anti-Mitf antibodies were raised in rabbits against either a C-terminal peptide, or against the N-terminal 70 amino acids fused to glutathione S-transferase. The sequence of double stranded oligonucleotides used as probes and competitors, where the lowercase letters indicate mutated bases and the underlined sequences indicate the E-box elements, are as follow:Tbx2 E box, 5′-ctagaGAACAGGGCAGGACACATGTGAGATAGTCACAt-3′ and 5′-ctagaTGTGACTATCTCACATGTGTCCTGCCCTGTTCt-3′; Tbx2 E box mut, 5′-ctagaGAACAGGGCAGGACACcTtTGAGATAGTCACAt-3′ and 5′-ctagaTGTGACTATCTCAaAgGTGTCCTGCCCTGTTCt-3′. The Tbx2 promoter luciferase reporters were made by inserting the NcoI (−859/+121), and BamHI (−230)-NcoI (+121) fragments of the Tbx2 gene into either the NcoI or the BglII and NcoI sites, respectively, of the luciferase reporter vector pGL3-Basic (Promega). The −11/+121 promoter mutants was made by deletion of a SmaI fragment from the full-length promoter in the pGL3- Basic vector. The pCMV-USF1 vector was constructed by cloning the full-length human USF1 cDNA between the BamHI and EcoRI sites of pCMV19a. HeLa and COS cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The mouse melanocyte and melanoma cell lines were grown in RPMI 1640 with 10% fetal calf serum, and 12-O-tetradecanoylphorbol-13-acetate was added to a final concentration of 200 nm for the melan-c and melan-a cells. Transfections were performed by using Fugene (Roche Molecular Biochemicals) or the Transfast (Promega) reagents. Cells were plated at 2 × 104/ml in 24-well plates 1 day before transfection. Transfections were carried out as instructed by the manufacturers, and the vector pCH110 containing the SV40 promoter driving expression of a LacZ reporter was used as an internal control for transfection efficiency. All transfections were repeated multiple times using different preparations of DNA. For each transfection used to obtain the results presented (see Fig. 2), we compared the values obtained for the lacZ reporter in the two cell lines and, assuming that any difference (which was around 20%) represented a difference in transfection efficiency, we used these values to adjust the relative levels of luciferase activity obtained using the Tbx2promoter-reporter. By giving the luciferase activity from theTbx2-luciferase reporter a value of 100% in B16 cells, we arrived for each transfection experiment at a value for the activity of the same reporter in HeLa cells as a percentage of that observed in B16 melanoma cells. We then took an average of the HeLa cell results and compared that with the result from B16 cells presented as 100%. The lacZ reporter was also used in other transfection experiments as a control for transfection efficiency, and the values obtained using the various Tbx2-luciferase reporters were adjusted accordingly. The luciferase assays were performed as instructed by the manufacturer of the luciferase assay reagent (Promega) and were quantitated with a Bertholdt Microlumat LB 96V plate luminometer. The origin and culture of the melanocyte and melanoma cell lines used for the RT-PCR have been described previously (28.Bassi M.T. Incerti B. Easty D.J. Sviderskaya E.V. Ballabio A. Genome Res. 1996; 6: 880-885Crossref PubMed Scopus (28) Google Scholar, 29.Bennett D.C. Cooper P.J. Hart I.R. Int. J. Cancer. 1987; 39: 414-418Crossref PubMed Scopus (397) Google Scholar, 30.Fidler I.J. Gruys E. Cifone M.A. Barnes Z. Bucana C. J. Natl. Cancer Inst. 1981; 67: 947-956PubMed Google Scholar, 31.Goss P. Parsons P.G. Cancer Res. 1977; 37: 152-156PubMed Google Scholar) with the MM96 and K1735 cell lines being provided Dr. Dot Bennett. To gain an insight into the regulation of Tbx2 gene expression in the melanocyte lineage, it was necessary to isolate the sequences controlling its expression. To this end a mouse BAC library was screened for clones containing the Tbx2 gene. A single BAC clone was isolated, which hybridized to probes derived from the 5′- and 3′-ends of the mouse Tbx2 cDNA. The BAC DNA was initially digested withBamHI and subcloned into pUC19. One subclone, containing approximately 9 kilobases of DNA, hybridized to probes derived from the 5′- and 3′-ends of the Tbx2 cDNA and was therefore likely to contain the entire Tbx2 gene. Sequence analysis confirmed that this was the case, and eventually the sequence of the wholeTbx2 gene was obtained. The mouse Tbx2 gene was found to comprise seven exons, the relative locations of which are depicted in Fig.1 A. Previous work (32.Campbell C. Goodrich K. Casey G. Beatty B. Genomics. 1995; 28: 255-260Crossref PubMed Scopus (58) Google Scholar) had identified the intron/exon boundaries of the human gene, which are aligned with those deduced for the mouse gene in Fig. 1 B, and the contribution of each exon to the Tbx2 protein is depicted in Fig. 1 C. The overall structure of the murine Tbx2gene is remarkably conserved in terms of number and position of exons when compared with that of the human gene. The evolutionary implications of the conservation of gene structure within the T-box family has been discussed previously (33.Yi C.-H. Terrett J.A. Li Q.-Y. Ellington K. Packham E.A. Armstrong-Buisseret L. McClure P. Slingsby T. Brook D.J. Genomics. 1999; 55: 10-20Crossref PubMed Scopus (50) Google Scholar, 34.Wattler S. Russ A. Evans M. Nehls M. Genomics. 1998; 48: 24-33Crossref PubMed Scopus (64) Google Scholar). Although the structure of the T-box genes is of considerable interest to evolutionary biologists, our primary concern here was to isolate the Tbx2 promoter and to gain some clues as to its regulation. Sequencing revealed that the initialBamHI fragment containing the Tbx2 gene extended only some 350 bp 5′ from the ATG initiation codon, and it was not clear whether this 5′ sequence contained a significant region of theTbx2 promoter. To obtain additional Tbx2 promoter sequence, the original BAC DNA was restricted with both XhoI and Sal1 and subcloned into pUC19. The clones isolated were screened using a probe derived from the 5′ end of the Tbx2cDNA and positive clones sequenced. The results obtained enabled us to determine the sequence of an additional 829 bp of theTbx2 promoter. The full sequence of this region is shown in Fig. 2 A and contains potential binding sites for several transcription factors, including AP1, AP2, SP1, SIF-1, GATA, HIF-1, and EGR-2 (not shown). However, although such an analysis might give some indication as to the potential of the promoter to be regulated, an extensive mutational analysis would be required to determine whether any of these binding sites might be functional. Moreover, because all of these factors are known to be expressed in a wide range of tissues, they were not of immediate interest in terms of the regulation of Tbx2 expression in the melanocyte lineage. In contrast, an E-box element, CATGTG, located between 332 and 327 bp 5′ to the initiator ATG, attracted our attention. E boxes are recognized by members of the bHLH and bHLH-LZ families of transcription factors. In the melanocyte lineage, the bHLH-LZ transcription factor Mitf plays a critical role; mice devoid of a functional Mitf lack all pigment cells. Mitf has been shown previously to regulate a number of genes, including Tyrosinase, Tyrp-1, andDct involved in the manufacture of the pigment melanin, a process specific to melanocytes and the retinal pigment epithelium. The sequences recognized in all these Mitf target promoters are E boxes with the sequence CATGTG, including the highly conserved M-box motif (35.Lowings P. Yavuzer U. Goding C.R. Mol. Cell. Biol. 1992; 12: 3653-3662Crossref PubMed Scopus (128) Google Scholar). Although such E-box elements are present in many genes, we recently established that Mitf can only recognize a subset of E-box motifs in vitro and in vivo (27.Aksan I. Goding C.R. Mol. Cell. Biol. 1998; 18: 6930-6938Crossref PubMed Scopus (176) Google Scholar). Thus Mitf will recognize a CATGTG E box only if it is flanked by a specific 5′ T residue. In other words, Mitf recognizes TCATGTG, CATGTGA, or TCATGTGA. In addition, these elements may also be targeted by a second bHLH-LZ factor, USF1, which is ubiquitously expressed. As we discuss below, whether Mitf or USF1 will recognize these elements in vivowill depend on a number of factors related to the relative abundance of DNA-binding competent USF1 and Mitf in the cell at a given time. Before undertaking an analysis of the potential role of this E-box element in the Tbx2 promoter function, we first identified the transcription initiation site by primer extension analysis using a primer corresponding to sequences located between 24 and 52 bp 5′ to the ATG translation initiation codon and mRNA derived from a B16 melanoma cell line. The primer location was chosen because previously isolated Tbx2 cDNAs extended up to a maximum of 57 bp 5′ from the initiator ATG. The results (Fig. 2 B) revealed the presence of two bands corresponding to cDNAs extending some 117 bp 5′ to the initiator ATG and 60 bp 5′ to the end of the longest previously published cDNAs. Because no cDNAs extending beyond these positions were obtained using other primers located within the putative Tbx2 leader sequence (not shown), we believe that the 5′ ends of the cDNAs derived by primer extension most likely correspond to the location of the transcription initiation site. These are indicated in Fig. 2 A. Prior to examining the requirements for Tbx2 expression in melanocytes, we asked whether the gene was expressed in a number of cell lines that are regularly used for the analysis of melanocyte-specific gene expression. We had previously shown that Tbx2 was expressed in both melanoblasts and all melanocyte cell lines tested but was not expressed in a melanoblast precursor cell line (23.Carreira S. Dexter T.J. Yavuzer U. Easty D.J. Goding C.R. Mol. Cell. Biol. 1998; 18: 5099-5108Crossref PubMed Scopus (152) Google Scholar). However, we had not determined whether Tbx2 was also present in melanoma cell lines. We therefore asked whether Tbx2 was expressed in twoMitf-positive melanoma cell lines, B16 and MM96, oneMitf-negative melanoma line, K1735, and the melanocyte cell line melan-c, whereas melan-a melanocytes were used as a positive control. The results, shown in Fig. 3, reveal that Tbx2 was expressed in all cell lines that also expressed Mitf, that is, melan-a, melan-c, and the two melanoma lines B16 and MM96. However, intriguingly, given the presence of the potential Mitf binding site in the Tbx2 promoter, no expression was detected in the Mitf-negative cell line K1735. These results were confirmed independently by Northern blotting. 2D. Bennett, personal communication. To determine whether the Tbx2 promoter isolated was sufficient to direct cell type-specific expression, sequences between −859 and +121, relative to the transcription start site, were cloned upstream from a luciferase reporter (Fig.4 A), and promoter activity was determined following transfection into B16 melanoma cells. As a control, the Tbx2 promoter-luciferase reporter was also transfected into HeLa cells, which we have shown previously do not express Tbx2 (23.Carreira S. Dexter T.J. Yavuzer U. Easty D.J. Goding C.R. Mol. Cell. Biol. 1998; 18: 5099-5108Crossref PubMed Scopus (152) Google Scholar), and the relative promoter strength was determined by comparison to the activity of a cotransfected SV40-lacZ reporter. The results shown in Fig. 4 B demonstrate that theTbx2 promoter used was sufficient to direct expression specifically in the melanoma cell line; relative to the activity of the lacZ reporter, the Tbx2 promoter was at least 30-fold better expressed in B16 cells than in HeLa cells. We next determined whether the minimal promoter, which still retained the E-box motif, was functional using promoter deletion mutants (Fig.5 A). Deletion of theTbx2 promoter to −230 resulted in no more than around a 3-fold decrease in promoter activity relative to the promoter extending to −859 (Fig. 5 B). However, mutating the CATGTG E box motif to CCTTTG in the context of the −230 deletion resulted in a further 5-fold decrease in promoter activity, suggesting that this element plays a significant role in Tbx2 promoter function. The importance of this element was highlighted by the fact that the activity of the mutated promoter extending to position −230 was no more than 2-fold greater than when the promoter was almost entirely deleted to −11. The results obtained so far suggest that the Tbx2 promoter is cell type-specific and contains a functional E-box motif that contributes substantially to promoter function. In the melanocyte-specific Tyrosinase Tyrp-1 and Dct2 promoters, the specific E-box elements that are essential for their expression have been shown to bind both the bHLH-LZ transcription factors USF1 and Mitf (4.Yasumoto K. Yokoyama K. Shibata K. Tomita Y. Shibahara S. Mol. Cell. Biol. 1994; 14: 8058-8070Crossref PubMed Scopus (359) Google Scholar, 6.Bentley N.J. Eisen T. Goding C.R. Mol. Cell. Biol. 1994; 14: 7996-8006Crossref PubMed Scopus (417) Google Scholar, 7.Hemesath T.J. Steingrimsson E. McGill G. Hansen M.J. Vaught J. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Fisher D.E. Genes Dev. 1994; 8: 2770-2780Crossref PubMed Scopus (545) Google Scholar, 26.Bertolotto C. Busca R. Abbe P. Bille K. Aberdam E. Ortonne J.-P. Ballotti R. Mol. Cell. Biol. 1998; 18: 694-702Crossref PubMed Scopus (286) Google Scholar, 35.Lowings P. Yavuzer U. Goding C.R. Mol. Cell. Biol. 1992; 12: 3653-3662Crossref PubMed Scopus (128) Google Scholar, 36.Yasumoto K. Yokayama K. Takahashi K. Tomita Y. Shibahara S. J. Biol. Chem. 1997; 272: 503-509Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). To determine whether USF1 could recognize the E box present in theTbx2 promoter, we performed a DNA binding band shift assay using a radiolabeled oligonucleotide probe spanning the E box andin vitro transcribed/translated (ITT) USF1. The sequence of the probe is shown in Fig. 6 A, and the results of the DNA binding assay are shown in Fig.6 B. The ability of USF1 to recognize the Tbx2 E box was demonstrated by the presence of a band that was absent when unprogrammed reticulocyte lysa" @default.
• W2016114470 created "2016-06-24" @default.
• W2016114470 creator A5002433296 @default.
• W2016114470 creator A5023173745 @default.
• W2016114470 creator A5085732690 @default.
• W2016114470 date "2000-07-01" @default.
• W2016114470 modified "2023-10-13" @default.
• W2016114470 title "The Gene Encoding the T-box Factor Tbx2 Is a Target for the Microphthalmia-associated Transcription Factor in Melanocytes" @default.
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