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
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ABSTRACT
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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
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
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.
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INTRODUCTION
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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).
MSH is one of the most potent physiological stimulators of
melanogenesis.
MSH binds to a seven-transmembrane receptor (MC1r)
coupled to an
s G protein, leading to an activation of adenylate
cyclase and an increase in the intracellular cAMP content.
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
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.
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RESULTS AND DISCUSSION
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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. 1A
). 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. 1B
), 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.
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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. 2A
, 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. 2B
) 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.
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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. 3A
) gave rise, respectively, to 60-, 45-,
and 60-kDa proteins. In gel shift assays (Fig. 3B
), 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
-[32P]-ATP and T4 polynucleotide kinase. For
competition experiments, unlabeled homologous oligonucleotides were
added in a 50-fold molar excess.
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Next, we investigated the role of Mbox and Ebox motifs in the
transactivation of tyrosinase and TyrpI promoters by TFE3 and TFEB
(Fig. 4
). To this aim, pTYRO (Fig. 4A
) or
pTyrpI (Fig. 4B
) 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.
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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. 5A
). 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. 5B
).
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.
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Next, we compared the effect of forskolin on TFE3, Mi, and tyrosinase
protein expression in B16 melanoma cells (Fig. 6
, AC). As previously shown, forskolin
increased Mi protein with a maximal effect between 3 and 5 h (Fig. 6B
). 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. 6C
). 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. 6A
). Northern blot experiments (Fig. 6D
) 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,
MSH, a physiological
cAMP regulator, elicited identical effects as forskolin on both TFE3
and Mi expression (Fig. 6E
). 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 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.
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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. 7
), 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. 8
).
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.
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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.
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MATERIALS AND METHODS
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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
-[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
-[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 (413968 bp) and the mouse TFEB probe
(8511377 bp) were isolated by PCR. The human TFEB probe (5281579
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
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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 dInvestigations 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.
 |
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