TFE3, a Transcription Factor Homologous to Microphthalmia, Is a Potential Transcriptional Activator of Tyrosinase and TyrpI Genes

Carole Verastegui, Corine Bertolotto, Karine Bille, Patricia Abbe, Jean Paul Ortonne and Robert Ballotti

Institut National de la Santé et de la Recherche Médicale (INSERM) U385 Faculté de Médecine 06107 Nice Cedex 2, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Microphthalmia gene encodes a basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor involved in the development of the melanocyte lineage and plays a key role in the transcriptional regulation of the melanogenic enzymes, tyrosinase and TyrpI. Recently, we have shown that Microphthalmia mediates the melanogenic effects elicited by {alpha}MSH that up-regulates the expression of tyrosinase through the activation of the cAMP pathway. Therefore, Microphthalmia appears as a principal gene in melanocyte development and functioning. Among the transcription factors of the bHLH-Zip family, TFE3 and TFEB show a remarkably elevated homology with Microphthalmia. These observations prompted us to investigate the role of TFE3 and TFEB in the regulation of tyrosinase and TyrpI gene transcription. We show in this report that overexpression of TFE3 stimulates the tyrosinase and TyrpI promoter activities, while TFEB acts only on the TyrpI promoter. TFE3 and TFEB elicit their effects mainly through the binding to Mbox (AGTCATGTGCT) and Ebox motifs (CATGTG) of tyrosinase and TyrpI promoters. In B16 melanoma cells, the high basal expression of TFE3 is down-regulated by forskolin and by {alpha}MSH. Interestingly, endogenous TFE3 cannot bind as homodimers to the Mbox, and we did not detect TFE3/Mi heterodimers. According to these data, TFE3 is clearly endowed with the capacity to regulate tyrosinase and TyrpI gene expression. However, TFE3 binding to the melanogenic gene promoters is hindered, thereby preventing its potential melanogenic action. In specific physiological or pathological conditions, the recovery of its binding function would make TFE3 an important element in melanogenesis regulation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Melanocytes and cells from the pigmented retina epithelium possess a specific enzymatic machinery responsible for melanin synthesis. Three enzymes, tyrosinase, TyrpI (tyrosinase related protein-1), and Dct, are involved in this process, which transforms tyrosine to melanin pigments. Tyrosinase catalyzes the conversion of tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA) and of DOPA to DOPA-quinone (1, 2, 3, 4). TyrpI catalyzes the oxidation of 5,6-dihydroxyindol-2-carboxylic acid (DHICA) to indole-5,6-quinone-2-carboxylic acid (5, 6), and Dct possesses a tautomerase activity, converting the DOPA-chrome to DHICA (7, 8, 9). The expression of these enzymes is restricted to melanocytes and to the pigmented retina epithelium, suggesting the presence of tissue-specific regulatory elements in the promoters of the melanogenic enzymes. Among these regulatory elements, a 11-bp motif (AGTCATGTGCT) termed Mbox, found in both tyrosinase and TyrpI promoters, has been shown to play a crucial role in the regulation of tyrosinase and TyrpI promoter activities. This motif has been demonstrated to bind Microphthalmia (Mi), a transcription factor that strongly increases tyrosinase and TyrpI promoter activities (10, 11, 12, 13). In the Dct promoter, a motif closely related to the Mbox is also present, but its role in the regulation of the promoter activity is much less important, and consequently, the transactivating effect of Mi appears less pronounced (14).

{alpha}MSH is one of the most potent physiological stimulators of melanogenesis. {alpha}MSH binds to a seven-transmembrane receptor (MC1r) coupled to an {alpha}s G protein, leading to an activation of adenylate cyclase and an increase in the intracellular cAMP content. {alpha}MSH and cAMP elevating agents elicit their melanogenic effects by increasing tyrosinase expression; furthermore they have been shown to increase Mi expression and binding to the Mbox. Inhibition of Mi activity by the expression of a Mi-dominant negative mutant prevents cAMP-induced melanogenic effects (15), thus demonstrating the pivotal role of Mi in the acute regulation of melanogenesis by {alpha}MSH.

The consensus hexamere motif CANNTG, which matches with the core sequence of the Mbox, is recognized by transcription factors of the basic helix-loop-helix (bHLH) family (16). The basic domain and the HLH dimerization motif confer DNA binding and dimerization specificities (17, 18, 19). Among the bHLH proteins, a subfamily of transcription factors contains an additional dimerization motif consisting of leucine repeat region called leucine zipper (Zip) that has been reported to strengthen the dimerization specificity (20). The bHLH-Zip family includes Myc, Max (21), Mad (22), Mxi1 (23), USF (24), AP4 (25), FIP (26), TFE3 (27), TFEB (28), and Mi (29). Interestingly, TFE3 and TFEB share a strong homology with Mi and thereby define a new class among the bHLH-Zip transcription factors (30). These observations led us to hypothesize that TFE3 and TFEB could participate in regulation of melanogenesis through the binding to Mbox motifs and the regulation of tyrosinase and TyrpI expression.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TFE3 and TFEB have been shown to be expressed in a wide variety of tissues and cells (30, 31, 32). However, their expression in melanocytes has not been reported. Northern blot analysis of mRNA from different cell types was performed using probes specific to TFE3 or TFEB (Fig. 1AGo). Comparable levels of TFE3 and TFEB messengers were found both in fibroblasts (lanes 1 and 3) and in melanocytes (lanes 2 and 4) derived from mouse (lanes 1 and 2) or human (lanes 3 and 4). Western blot analysis (Fig. 1BGo), showed a strong TFE3 basal expression in all the cells tested, while Mi is expressed only in melanocyte cells (lanes 2 and 4).



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Figure 1. TFE3 and TFEB Are Expressed in Melanocyte Cells

A, mRNA from mouse NIH3T3 cells (lane 1), mouse B16 cells (lane 2), human fibroblasts (lane 3), and human melanocytes (lane 4) were analyzed by Northern blot using TFE3, TFEB, and GAPDH probes. B, Protein extracts from mouse NIH3T3 cells (lane 1), mouse B16 cells (lane 2), human fibroblasts (lane 3), and human melanocytes (lane 4) were analyzed by Western blot using anti-TFE3 or anti-Mi antibodies.

 
The presence of TFE3 and TFEB transcripts in melanocytes prompted us to investigate the role of these transcription factors in the regulation of the expression of the melanogenic enzymes. Hence, we studied the effect of these transcription factors on tyrosinase and TyrpI promoter activities. For this purpose, we used luciferase reporter plasmids containing either a 2.2-kb fragment of the tyrosinase promoter (pTYRO) or a 1.2-kb fragment of the TyrpI promoter (pTyrpI) that carry the elements accountable for melanocyte-specific expression of the enzymes (11, 33). B16 cells were transiently transfected with pTYRO or pTyrpI together with expression plasmids encoding Microphthalmia, TFE3, TFEB, USF, or AP4. As shown in Fig. 2AGo, pTYRO expression was strongly stimulated by Mi and to a lesser extent by TFE3, whereas TFEB or the two other bHLH-Zip transcription factors, AP4 and USF, had no effect on pTYRO activity. In cells transfected with pTyrpI, Microphthalmia, TFE3, and TFEB increased the luciferase activity 12-, 20-, and 8-fold, respectively. Neither USF nor AP4 had an effect on the luciferase activity in these cells. As control we showed that none of the bHLH-Zip transcription factors used in this study increased the luciferase activity in cells transfected with a luciferase reporter plasmid containing the SV40 promoter (pSV40). The same experiments performed in NIH3T3 (Fig. 2BGo) showed that pTYRO activity was increased by Microphthalmia and to a lesser extent by TFE3 and TFEB. Similar activation of pTyrpI was observed with Mi, TFE3, or TFEB. Thus, TFE3 transactivates tyrosinase and TyrpI promoters, while TFEB is only able to transactivate the TyrpI promoter. This suggests a potential role of these transcription factors in the regulation of melanogenesis.



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Figure 2. TFE3 and TFEB Transactivate Tyrosinase (pTYRO) and TyrpI (pTyrpI) Promoter Activities

B16 melanoma (A) or NIH3T3 (B) cells were transiently transfected with 0.15 µg of the reporter plasmid (pTYRO, pTyrpI, or pSV40) and 0.15 µg of pCDNA3 empty vector or pCDNA3 encoding TFE3, TFEB, Mi, USF, or AP4. Basal luciferase activity (in arbitrary light units x 10-3) are 32 and 1.2 for pTYRO, 43 and 1.8 for pTyrpI, and 93 and 76 for pSV40 in B16 melanoma and NIH3T3 cells, respectively. Results are expressed as fold stimulation of the luciferase activity. Data are mean ± SE of five experiments performed in triplicate.

 
TFE3 and TFEB have been described to form heterodimers with Mi (34). In B16 cells, effects of TFE3 and TFEB on pTYRO and pTyrpI could be ascribed to such heterodimers. However, TFE3 and TFEB might also function as homodimers since they transactivate tyrosinase and TyrpI promoters in NIH3T3 cells that do not express Mi. It is noteworthy that the effects of TFE3 and TFEB in NIH3T3 are greatly reduced compared with B16 cells, suggesting that most of the effects of these transcription factors in melanoma cells are mediated by Mi-containing heterodimers. However, the effect of Mi is also markedly reduced in NIH3T3, indicating that a cell-specific mechanism, independently of Mi/TFE3 or Mi/TFEB heterodimer formation, allows Mi and TFE3 to efficiently activate tyrosinase and TyrpI promoters in B16 cells.

The transactivation of tyrosinase and TyrpI promoters by Mi is mainly mediated through the binding of Mi to an Mbox motif (AGTCATGTGCT) located upstream the TATA box present in both promoters (10). In the tyrosinase promoter, an Ebox motif (Ebox-tyro, CATGTG), located at the initiation site, also plays a crucial role in mediating the effect of Mi, while in the TyrpI promoter an Ebox (Ebox-TyrpI, CAAGTG) located at -233 bp plays a much less important role in the transactivation by Mi (13). In an attempt to study whether TFE3 and TFEB were able to bind to tyrosinase or TyrpI Mbox and Ebox, we performed gel shift experiments with in vitro translated Mi, TFE3, and TFEB proteins. In vitro translation of Mi, TFE3, and TFEB (Fig. 3AGo) gave rise, respectively, to 60-, 45-, and 60-kDa proteins. In gel shift assays (Fig. 3BGo), we observed that Mi, TFE3, and TFEB formed heavily labeled complexes with the Mbox that were specifically displaced by an excess of cold probe. Using the Ebox-tyro as a probe, we also observed strong complexes with Mi and TFE3, while the complex formed with TFEB was markedly less labeled. Mi and TFE3 formed weak but specific complexes with Ebox-TyrpI. TFEB was unable to shift this probe. These results indicate that Mi and TFE3 have a high affinity for the Mbox and, to a lesser extent, for the Ebox of the tyrosinase promoter. Mi and TFE3 bind with a much lower affinity to the Ebox of the TyrpI promoter. TFEB also binds avidly to the Mbox but poorly to the Ebox-tyro and does not interact with Ebox-TyrpI. Consistently with our results, TFE3 and TFEB bind to the µE3 motif of the immunoglobulin heavy chain gene promoter that is identical to the core sequence of the Mbox (CATGTG) (28). The similar binding specificity of Mi, TFE3, and TFEB can be explained by the remarkably high homology within the bHLH regions that are involved in DNA binding specificity of the bHLH-Zip transcription factors.



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Figure 3. Binding of TFE3 and TFEB to Mbox and Ebox Motifs

A, In vitro translated Mi, TFE3, and TFEB proteins (1 µl of the Mi or TFE3 mix and 5 µl of the TFEB mix) were analyzed by SDS-PAGE 10% acrylamide and visualized by fluorography. B, Gel shift assays were performed by using the 1 µl of the Mi or TFE3 mix and 5 µl of the TFEB mix. Double-stranded synthetic Mbox from tyrosinase or TyrpI promoter, tyrosinase promoter Ebox (Ebox-TYRO), and TyrpI promoter Ebox (Ebox-TyrpI) were end labeled with {gamma}-[32P]-ATP and T4 polynucleotide kinase. For competition experiments, unlabeled homologous oligonucleotides were added in a 50-fold molar excess.

 
Next, we investigated the role of Mbox and Ebox motifs in the transactivation of tyrosinase and TyrpI promoters by TFE3 and TFEB (Fig. 4Go). To this aim, pTYRO (Fig. 4AGo) or pTyrpI (Fig. 4BGo) reporter plasmids containing deletions or mutations in the 5'-flanking region of the promoters were cotransfected with expression plasmid encoding Mi, TFE3, and TFEB. The pTYRO-0.2 construct contains a large deletion in the 5'-flanking regions upstream from the Mbox. This construct showed the same responsiveness to Mi and TFE3 compared with pTYRO containing the 2.2-kb fragment of the tyrosinase promoter. Mutations of the Mbox (pTYRO-mMbox) or of the Ebox (pTYRO-mEbox) in pTYRO led to a dramatic decrease in the effect of Mi and TFE3 on the tyrosinase promoter activity. These results indicate that Mi and TFE3 transactivate the tyrosinase promoter through the same Mbox and Ebox motifs.



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Figure 4. Role of Mbox and Ebox Motifs in the Effect of TFE3 and TFEB

A, B16 cells were cotransfected with 0.15 µg of pTYRO, pTYRO-0.2, pTYRO-mMbox, pTYRO-mEbox, and 0.15 µg of pCDNA3 empty vector or encoding Mi, TFE3, or TFEB. B, B16 cells were cotransfected with 0.15 µg of pTyrpI, pTyrpI-0.2, pTyrpI-mMBox, pTyrpI-mEBox, and 0.15 µg of pCDNA3 empty vector or encoding TFE3 or TFEB. Results are expressed as fold stimulation of the luciferase activity. Values in brackets represent the basal luciferase activity (in arbitrary light units x 10-3) of each construct. Data are mean ±SE of five experiments performed in triplicate.

 
In the case of the TyrpI promoter, the deletion of the 5'-flanking region just upstream from the Mbox (pTyrpI-0.2) led to a moderate decrease in the effect of Mi but did not affect the activation evoked by TFE3 or TFEB. Mutation of the Mbox in pTyrpI (pTyrpI-mMbox) resulted in an almost complete loss of the promoter response to Mi, TFE3, and TFEB. We observed a slight decreased response to Mi and TFE3 but not to TFEB when the Ebox located upstream the Mbox was mutated. Thus the Mbox plays a key role in the activation of TyrpI promoter by Mi, TFE3, and TFEB. An Ebox located upstream the Mbox is also involved in Mi and TFE3 effects, but its role is much less crucial.

The importance of a motif in the regulation of promoter activity appears to correlate with the binding potentiality of Mi, TFE3, or TFEB. However, it should not be concluded that the binding of the bHLH-Zip is sufficient to activate tyrosinase or TyrpI promoters. Indeed, USF, which has been shown to bind avidly to the Mbox (35), is unable to transactivate pTYRO and pTyrpI. This observation emphasizes the role of transactivation domains that would add an additional level of specificity in the gene regulation by bHLH-Zip transcription factors. The results presented above show that TFE3 could participate in the regulation of tyrosinase and TyrpI expression by interacting with the same motifs as Mi.

Second, we wished to investigate the potential role of TFE3 in the regulation of the expression of melanogenic enzymes induced by the activation of the cAMP pathway. To this aim, we wished to compare the effect of cAMP on TFE3 and Mi expression, but since TFE3 and Mi share a strong homology, we first studied the specificity of anti-TFE3 and anti-Mi antibodies in Western blot and in gel shift assays. In Western blot, the anti-Mi antibody detected a doublet of 53 and 60 kDa in B16 cells and a single band of 45 kDa in splenocytes used as a control (Fig. 5AGo). In both B16 cells and in splenocytes, TFE3 was detected at 72 kDa by the anti-TFE3 antibody that did not recognize the doublet of Mi. In gel shift assay, in vitro translated Mi (lanes 1, 3, and 5) and TFE3 (lanes 2, 4, and 6) proteins formed complexes with labeled Mbox. The anti-TFE3 displaced both Mi- and TFE3-containing complexes while the anti-Mi antibody shifted specifically Microphthalmia-containing complexes (Fig. 5BGo). Thus the anti-TFE3 is able to recognize Mi in gel shift assay but is specific of TFE3 in Western blot. No complex was observed when transcription and translation were performed using an empty plasmid as template (lane 7).



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Figure 5. Specificity of the anti-TFE3 and anti-Microphthalmia Antibodies

A, Twenty micrograms of proteins from B16 mouse melanoma cells (B16) or from mouse splenocytes (spl) were subjected to Western blot analysis using the monoclonal anti-Mi antibody (right panel) or the polyclonal anti-TFE3 antibody (left panel). Molecular masses are expressed in kilodaltons. B, Gel shift assays using in vitro translated proteins from Microphthalmia encoding plasmid (lanes 1, 3, and 5) or TFE3 encoding plasmid (lanes 2, 4, and 6) were performed using labeled tyrosinase Mbox. The assay was performed in the absence of antibody (lanes 1 and 2), in the presence of 1 µl of anti-Mi antibody (lanes 3 and 4), or in the presence of 0.1 µl of anti-TFE3 antibody (lanes 5 and 6). Lane 7, The transcription and translation reaction was performed using an empty plasmid.

 
Next, we compared the effect of forskolin on TFE3, Mi, and tyrosinase protein expression in B16 melanoma cells (Fig. 6Go, A–C). As previously shown, forskolin increased Mi protein with a maximal effect between 3 and 5 h (Fig. 6BGo). After 24 h, Mi decreased but remained above the basal level. The effect of forskolin on tyrosinase expression can be observed after 24 h (Fig. 6CGo). Interestingly, the basal level of TFE3 protein was markedly higher than Mi basal level. However, forskolin did not increase TFE3 protein, but led to an inhibition of its expression after 24 h (Fig. 6AGo). Northern blot experiments (Fig. 6DGo) consistently showed that forskolin decreased TFE3 messengers and increased those encoding Mi. These results show that Mi and TFE3 are differentially regulated by cAMP in melanoma cells. Further, {alpha}MSH, a physiological cAMP regulator, elicited identical effects as forskolin on both TFE3 and Mi expression (Fig. 6EGo). These observations suggest that TFE3 is not involved in the stimulation of tyrosinase expression elicited by cAMP and are in total agreement with previous reports (13, 32) demonstrating that the effect of cAMP on tyrosinase expression is mediated by Mi.



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Figure 6. cAMP-Elevating Agents Decrease TFE3 Expression

Proteins (20 µg) from B16 mouse melanoma cells treated for the indicated times with 20 µM forskolin were subjected to Western blot analysis using anti-TFE3 antibody (A), anti-Microphthalmia antibody (B), or anti-tyrosinase antibody (C). Membranes were probed with an anti-ERK1 antibody to control the even loading of the gel. Molecular masses are expressed in kilodaltons. D, Northern blot analysis using 5 µg of mRNA from nonstimulated cells or cells stimulated with forskolin for the indicated times. The membrane was revealed with a TFE3 probe (upper panel, left), or a Microphthalmia probe (upper panel, right). The lower part of the membrane was incubated with a GAPDH probe (lower panels). E, Proteins (20 µg) from B16 mouse melanoma cells treated for the indicated times with {alpha}MSH (0.1 µM) were subjected to Western blot analysis using anti-TFE3 or anti-Microphthalmia. Membranes were probed with an anti-ERK1 antibody to control the even loading of the gel.

 
However, it is conceivable that the high basal level of TFE3 could participate in the regulation of tyrosinase or TyrpI expression in the absence of cAMP. Further, TFE3 could also be involved in the cAMP-induced stimulation of tyrosinase and TyrpI gene expression through the formation of heterodimers with Mi. Indeed, it has been shown that TFE3 and Mi form heterodimers both in a cell-free system and more recently in intact osteoclasts (36). Interestingly, when we performed gel shift assays with nuclear extracts from B16 melanoma cells (Fig. 7Go), the labeled Mbox formed a complex that was increased by forskolin treatment. This complex was completely shifted by the monoclonal antibody to Mi, indicating that endogenous TFE3 homodimers are not able to bind the Mbox. However, we cannot rule out the possibility that TFE3 is associated with Mi in the complexes shifted by the anti-Mi antibody. To verify this hypothesis, nuclear extracts from B16 cells treated with forskolin for 3 h were immunoprecipitated with the anti-Mi antibody. The proteins in the immune complexes were analyzed by Western blot and probed either with the anti-Mi or with the anti-TFE3 (Fig. 8Go). We observed that the anti-Mi precipitated Mi, but virtually no TFE3. The same results were obtained with nuclear extracts from nonstimulated cells (not shown). Thus, in B16 cells, Mi does not associate with TFE3 to form heterodimers. In B16 cells, the binding of the endogenous TFE3 to the Mbox appears to be prevented by a mechanism that remains to be elucidated. This process explains the strong effect of Mi overexpression on tyrosinase and TyrpI gene transcription. Indeed, if TFE3 was functional, the effect of Mi would be considerably reduced because of the strong basal expression that would be induced by TFE3. It could be proposed that the binding of TFE3 to the Mbox is prevented through the interaction with other proteins containing an HLH domain but not a DNA-binding domain. When TFE3 is overexpressed, it can outnumber these inhibitory proteins, and then it binds and activates tyrosinase or TyrpI promoters.



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Figure 7. TFE3 from B16 Nuclear Extracts Does Not Bind the Tyrosinase Mbox

Nuclear extracts from B16 melanoma cells nontreated (-) or treated with forskolin for 3 h (+) were used in gel shift assay with labeled tyrosinase Mbox. The extracts were incubated without antibody (none), with a preimmune serum (control), or with the monoclonal anti-Microphthalmia antibody (Mi).

 


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Figure 8. TFE3 Does Not Form Heterodimers with Mi

Nuclear extracts from B16 melanoma cells nontreated (-) or treated with forskolin for 3 h (+) were immunoprecipitated with the anti-Microphthalmia antibody (anti-Mi). Then total nuclear extracts (no) or immunoprecipated proteins (anti-MI) were subjected to Western blot analysis with the anti- Microphthalmia (left panel) or the anti-TFE3 (right panel) antibodies.

 
Now, it remains to determine whether TFE3 is inactivated during all the melanocyte differentiation process and whether other melanogenic agents (endothelin-1, thymine dimers) are able to restore its binding to the Mbox. In such conditions, TFE3 could become an efficient regulator in melanocyte differentiation and pigment production.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RNA Isolation and RT-PCR assays
RNA isolated by a modification of the method of Chomczynski and Sacchi (37) was reverse transcribed using the Promega Corp. (Madison, WI) reverse transcription system and subjected to 35 PCR cycles (57 C, 45 sec; 72 C, 1 min. 30 sec; 94 C, 30 sec). cDNAs from B16 melanoma cells, melb-a melanoblasts, and NIH3T3 fibroblasts were amplified using specific mouse TFE3 primers (5'-AGCTGCCTAACATCAAACGC-3' and 5'-CCCTCCTCCTCCATCAGAAT-3'). cDNAs from human melanocytes and human keratinocytes were amplified with human TFE3 primers (5'-GGCAGGAAGAAAGACAATCAC-3' and 5'-TCCTCCTCCTCCATCAGAAT-3'). cDNAs from human melanocytes, human fibroblasts, Hela cells, and human keratinocytes were amplified with specific TFEB primers (5'-AGGAGCGGCAGAAGAAAGAC-3' and 5'-AGGTGATGGAATGGGGACGG-3'). Control amplifications were performed using specific primers for mouse (from CLONTECH Laboratories, Inc., Palo Alto, CA) or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and 5'-CATGTGGGCCATGAGGTCCACCAC-3'). The PCR products were analyzed on 1% agarose gels and stained with ethidium bromide before visualization under UV light.

Transfection and Luciferase Assays
B16 melanoma or NIH3T3 cells were seeded in 24-well dishes, and transient transfections were performed the following day as previously described (38) using 2 µl of lipofectamine, 0.15 µg of the reporter plasmid (pTYRO, pTYRO-0.2, pTYRO-mMbox, pTYRO-mEbox, pTyrpI, pTyrpI-0.2, pTyrpI-mMBox, pTyrpI-mEBox or pSV40), and 0.15 µg of pCDNA3 empty vector or pCDNA3 encoding TFE3, TFEB, Mi, USF, or AP4. After 48 h, cells were harvested in 50 µl of lysis buffer and assayed for luciferase activity. Promoters used for these experiments were previously described (13, 38). SV40-luc was used as an external control of the transfection. Results are expressed as fold stimulation of the luciferase activity. Data are mean ± SE of five experiments performed in triplicate.

Gel Shift Assays
Nuclear extracts from B16 mouse melanoma cells or mouse splenocytes prepared as previously described (13) were used for gel shift assay and Western blot experiments. Mi, TFE3, and TFEB were in vitro transcribed and translated in the presence of 35S-labeled methionine using TNT-coupled reticulocyte lysate from Promega Corp.. Labeled proteins were analyzed by SDS-PAGE 10% acrylamide and visualized by fluorography. Gel shift assays were performed using nuclear extracts or in vitro translated proteins. Double-stranded synthetic Mbox (5'-GAAAAAGTTAGTCATGTGCTTTGCAGAAGA-3'), tyrosinase promoter Ebox (Ebox-TYRO, 5'-GGTCTTAGCCAAAACATGTG-ATAGTCACTCCAG-3'), and TyrpI promoter Ebox (Ebox- pTyrpI, 5'-GAAAATACAAGTGTGACATTG-3') were end labeled with {gamma}-[32P]-ATP and T4 polynucleotide kinase. For competition experiments, unlabeled homologous oligonucleotides were added in a 50-fold molar excess. DNA-protein complexes were resolved by electrophoresis on a 4% polyacrylamide gel in Tris acetate EDTA (TAE) buffer.

Northern Blot Analysis
Poly-A mRNAs were purified by oligo-dT column from total RNAs isolated from control and forskolin-treated B16 melanoma cells. RNAs were denatured 5 min at 65 C in formaldehyde/formamide mixture, separated by electrophoresis in a 1% agarose/2.2 M formaldehyde gel, transferred to a nylon membrane (Hybond N, Amersham Pharmacia Biotech, Arlington Heights, IL) in 20xSSPE (pH 7.7, 3.5 M NaCl, 0.2 M sodium, 0.02 M Na2EDTA). Membranes were hybridized with Mi, TFE3, TFEB, and GAPDH probes labeled by random priming with {alpha}-[32P]-dCTP (Amersham Pharmacia Biotech). The Microphthalmia probe was the 1.3 kb HindIII/NotI fragment containing the full open reading frame of Mi. The mouse TFE3 probe was a 314-bp StuI fragment corresponding to the 3'-end of the TFE3 coding sequence. The human TFE3 probe (413–968 bp) and the mouse TFEB probe (851–1377 bp) were isolated by PCR. The human TFEB probe (528–1579 bp) was isolated after a PST1/ECORV digestion.

Western Blot Assays
Nuclear proteins were separated on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. Microphthalmia was detected with C5 monoclonal antibody at 1:10 dilution (13). TFE3 protein was detected with polyclonal antibody directed against the N-terminal part of the protein at 1:300 dilution (39). Proteins were visualized with a peroxidase-conjugated secondary antimouse antibody or antirabbit antibody at 1:3000 dilution and the enhanced chemiluminescence (ECL) system from Amersham Pharmacia Biotech.


    ACKNOWLEDGMENTS
 
We thank Philippe Pognonec (INSERM U 470, Université de Nice, Nice, France) for the plasmid containing USF coding sequence, Robert Tjian (Howard Hugues Medical Institute, University of California, Berkeley, CA) for AP4 cDNA, David. E. Fisher (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA) for plasmid containing TFEB sequence, Kathryn Calame (Colombia University College of Physicians and Surgeons, New York, NY) for plasmid containing TFE3 sequence and for anti-TFE3 antibody. We are grateful to R. Buscà for critical reading of this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Robert Ballotti, Institut National de la Santé et de la Recherche Médicale U385, Avenue de Valombrose, 06107 Nice Cedex 2, France.

This work was supported by Association pour la Recherche sur le Cancer (ARC Grant 9402), Ligue Contre le Cancer, SANOFI-Vaincre le Mélanome, Centre de Recherches et d’Investigations Epidermiques et Sensorielles (CERIES), Institut National de la Santé et de la Recherche Médicale, and Université de Nice-Sophia Antipolis.

Received for publication March 25, 1999. Revision received November 12, 1999. Accepted for publication December 6, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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