bcn-1 Element-dependent Activation of the Laminin γ1 Chain Gene by the Cooperative Action of Transcription Factor E3 (TFE3) and Smad Proteins*

Laminin is a major component of the extracellular matrix. The laminin γ1 chain is the least variant component of the laminin heterotrimeric assembly. The laminin γ1 chain gene (LAMC1) expression is induced by several factors, including transforming growth factor-β (TGF-β). LAMC1 promoter contains a highly conserved transcriptional element, bcn-1. We screened cDNA libraries with the yeast one-hybrid system to identify transcriptional factors that are recognized by the bcn-1 motif. Using this strategy we isolated the basic helix-loop-helix/leucine zipper (bHLHzip) E-box-binding transcription factor, TFE3. Until now, the E-box was the only element known to recruit the bHLHzip transcription factors. Although the bcn-1 element only remotely resembles the E-box sequence, we show that TFE3 binds and activates the bcn-1 element. TFE3 cooperates with Smad proteins in the activation of the LAMC1 promoter in cells, an effect that is critically dependent not only on the bcn-1 element but also on one of the Smad-binding elements (SBE). The cooperative induction of theLAMC1 promoter and the endogenous LAMC1 gene by TFE3 and Smad3 is augmented by the TGF-β signaling pathway. Thus, the bcn-1 is a novel TFE3-dependent TGF-β target element that regulates LAMC1 gene expression.

Laminin is a large glycoprotein component of the extracellular matrix localized in basement membranes (1). Laminin is a multifunctional protein that regulates cell-to-matrix interactions, including cell adhesion, spreading, migration, and differentiation. Laminin exerts its effects on cellular behavior by interacting with cellular receptors that recognize specific sites on the laminin molecule. Laminin exhibits all the attributes of an extracellular signaling molecule that regulates cellular architecture, for example that of glomeruli (2,3). The best studied laminin receptors are the members of the integrin family of proteins (1). Laminin is composed of three polypeptide chains, ␣, ␤, and ␥, held together by disulfide bonds, forming a cruciform structure (4). There are five ␣ (␣1-5), three ␤ (␤1-3), and three ␥ (␥1-3) chains that account for the twelve known laminin trimeric assemblies (Laminin 1-12) (4).
The ␥1 chain, found in ten of the twelve known trimeric laminin isoforms, is the most widely expressed laminin chain (4). Laminin ␥1 is required for basement membrane formation, and its absence causes early lethality in mouse embryos (5,6). These observations suggest that the synthesis of ␥1 chain is essential for the laminin heterotrimeric assembly. LAMC1 gene expression is responsive to extracellular signals such as TGF-␤ 1 (7), interleukin-1␤ (8), and glucose (9). Because of its vital role, transcription of the laminin ␥1 chain gene has been intensely studied (7, 10 -14). The 5Ј-flanking regions of the human (15), murine (12), and rat (13) laminin ␥1 chain gene have been cloned. Although the human and the rodent promoters share only a small degree of sequence similarity, they have several common features that may be functionally be similar. (i) There are no TATA or CAAT boxes in either promoter, motifs common in genes transcribed by RNA polymerase II (16). Although uncommon, TATA-less promoters have been described for many genes (17). Similar to other TATA-less promoters (18), the rat, mouse, and human LAMC1 promoters have multiple transcription initiation sites (13) providing greater flexibility to initiate transcription. (ii) The murine and rat LAMC1 promoters have a number of repeats of a unique consensus sequence, CCCG(T)CCCA(T)CCT. An identical motif is present in the human laminin ␥1 chain gene promoter as well (12,13,15). (iii) Rat, mouse, and human promoters contain a number of "GC"rich motifs similar to the binding site (GGGCGG) for the transcriptional factor, Sp1 (19,20). (iv) Finally, the rat, murine, and human promoters contain an identical bcn-1 transcriptional element (CCCCGCCCACCTCGCGC) (7).
Because the bcn-1 element may play an important role in the regulation of LAMC1 gene expression, in the present study we set out to identify and characterize transcription factors that act from this element. Using the yeast one-hybrid screen we identified TFE3 as one of the factors that regulates the bcn-1 element. We show that TFE3 cooperates with Smad proteins in the activation of the LAMC1 gene, an effect that is bcn-1-and SBE-dependent and is augmented by the TGF-␤ signaling pathway.

Yeast One-hybrid Screen
The yeast one-hybrid screen analysis was carried out according to the manufacturer's protocol (MATCHMAKER One-Hybrid System, CLONTECH). Briefly, three tandem copies of the 17-bp bcn-1 motif (5Ј-ccccgcccacctcgcgc-3Ј) were inserted upstream of the His3 and LacZ reporter genes by designing sense and antisense 3ϫ bcn-1 oligonucleotides with EcoRI and XbaI or SalI sites at the ends. Sense and antisense oligonucleotides were annealed and subcloned into the EcoRI/XbaIdigested, pHISi-1 or EcoRI/SalI-digested pLacZi reporter plasmids (CLONTECH). Inserts were sequenced to confirm the presence and integrity of the bcn-1 elements. Both bcn-1-reporter constructs were integrated into the genome of YM4271 strain to generate bcn-1-reporter strains. Background HIS3 expression was ablated using 10 mM 3-aminotrizole (3-AT) (Sigma). Screening bcn-1-reporter strains with a pGAD-MC cDNA library identified genes encoding bcn-1-binding proteins. Plasmids were isolated from yeast clones capable of growing in the presence of 10 mM 3-AT in (Ϫ) leucine and (Ϫ) histidine media. Plasmids from these clones were transformed into competent DH5␣ bacteria, and each cDNA insert was sequenced and compared with known sequences in the GenBank TM data base. False positives were excluded using the LacZ reporter gene assay.

In Vitro Transcription and Translation
The partial or complete cDNAs of TFE3 were used as a template for in vitro transcription by SP6 or T7 DNA-dependent RNA polymerases to generate mRNAs for in vitro translation as previously described (24). In vitro translation in a rabbit reticulocyte cell-free system was performed according to the manufacturer's protocol (Promega, Madison, WI).

DNA Binding Pull-down Assay
To confirm that the bcn-1 element binds to TFE3, 3 l of cell-free system containing 35 S-labeled proteins was incubated in 150 l of FIG. 1. Direct binding of different TFE3 isoforms to the bcn-1 element in vitro. A, diagram of cDNAs used to generate RNAs for cell-free translation: TFE3-full (TFE3-full), TFE3 long (TFE3L), and TFE3 short (TFE3S). B, 35 S-labeled proteins synthesized in cell-free system (58) were incubated with biotinylated synthetic double-stranded oligonucleotide containing wild-type bcn-1 dimer motif. 35 S-Luciferase was used as a negative control. 35 S-Labeled protein⅐DNA complexes were pulled-down using streptavidin-agarose beads, then washed three times with 1 ml of washing buffer. Beads were boiled in SDS-loading buffer, and eluted 35 S-labeled proteins were analyzed by SDS-PAGE and autoradiography (lanes 5-8). 35 S-Labeled translational products used in the binding reactions were run in lanes 1-4 (50% of the amount used). C, fraction of total translational products bound in pull-down assays was calculated from densitometric measurements of the 35 35 S-TFE3L binding was tested in pull-down assays using beads bearing the bcn-1 element in the presence of either no competitor (none), or double-stranded oligonucleotide containing either the wildtype (wt-bcn-1) or mutated (mt-bcn-1) element. Bound proteins were analyzed by SDS-PAGE and autoradiography. 50% of the load is shown in lane 1. The sequences of the oligonucleotide competitors are shown above. B, 35 S-TFE3L bands were quantified by densitometry. The data shown are representative of three experiments.
bcn-1 Element-dependent Activation of the Laminin ␥1 Chain Gene binding buffer for 30 min (4°C) with 0.5 g of wild-type bcn-1 dimer double-stranded oligonucleotide with biotin on the 5Ј-end of the sense strand. After the binding reaction, the DNA⅐protein complexes were pulled-down with streptavidin-agarose beads, and the beads were washed three times with 1 ml of washing buffer (10 mM Tris HCl, pH 7.5, 100 mM KCl, 2 mM MgCl 2 and 0.1%Triton X-100). 35 S-Labeled proteins eluted with SDS-loading buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 5% ␤-mercaptoethanol) and were analyzed by SDS-PAGE and autoradiography.
LAMC1 Promoter-luciferase Reporter Constructs-The LAMC1 promoter fragment spanning the Ϫ1077 to Ϫ20 region of the gene (relative to first ATG codon), designated Ϫ1077/Ϫ20 LAMC1, was constructed by PCR amplification of the LAMC1 Ϫ1104/ϩ35 5Ј-flanking promoter region (13). The deletion constructs of LAMC1 promoter, Ϫ506/Ϫ20 LAMC1, Ϫ293/Ϫ20 LAMC1, Ϫ1077/Ϫ529 LAMC1, were generated by PCR using appropriate primers and the Ϫ1077/Ϫ20 LAMC1 promoter as a template. The PCR products were ligated into pGEM-T Easy vector (Promega) and digested with SacI and BglII. The SacI-BglII fragments were ligated into pGL3 basic vector cut with SacI-BglII using a Rapid DNA Ligation Kit (Roche Molecular Biochemicals, Indianapolis, IN). Ϫ239/Ϫ20 LAMC1 was constructed by digesting the Ϫ1077/Ϫ20 LAMC1 promoter with XhoI and BglII, and the fragments were subcloned into XhoI-and BglII-digested pGL3 basic vector. All constructs were confirmed by DNA sequencing.

Transient Transfections and Luciferase Reporter Gene Assay
SI Ϫ MC cells were plated at 4 ϫ 10 5 cells/well in 6-well plates and incubated in RPMI with 10% fetal bovine serum for 24 h. Cells were transiently transfected with 2.0 g of reporter plasmid, effector expression plasmid (indicated in each experiment), and 0.1 g of pRL-null (Promega) using SuperFect transfection reagent (Qiagen). Cell extracts were prepared 24 h later by passive lysis buffer (Promega), and luciferase activity was assayed by Dual Reporter Assay System (Promega) (27).

Northern Blot Analysis of RNA from Transfected Mesangial Cells
SI Ϫ MC cells were plated at 1 ϫ 10 6 cells/plate in 100-mm plates and grown in RPMI media with 10% fetal bovine serum for 24 h. Cells were transiently transfected with 10.0 g of expression vector containing TFE3L, Smad3, T␤RI-T204D, and complementary empty vector using SuperFect transfection reagent (Qiagen). After 24 h, total RNA was extracted as described before (28) with some modification. Briefly, after lysing cells with 6 M urea/4 M LiCl, total RNA was extracted with phenol/chloroform and 100% ethanol. After denaturing, 10 g of total RNA was electrophoresed on 1% agarose gel with 2.2 M formaldehyde and 20 mM MOPS, pH 7.0. After electrophoresis, RNA was transferred overnight onto a nylon membrane (Nytran-N nylon membrane, Schleicher & Schuell, Dassel, Germany) in 10ϫ standard saline citrate (1ϫ SSC: 150 mM NaCl, 15 mM sodium citrate) by a rapid downward transfer system (Turboblotter, Schleicher & Schuell). After cross-linking of RNA, hybridization was done with 32 P-labeled LAMC1 cDNA probe (14). Membranes were washed, and signals were measured using a phosphorimaging device (Cyclone, Packard Instrument Co., Meriden, CT).

Isolation of TFE3 cDNAs Using the bcn-1 Motif as a Bait in the Yeast
One-hybrid Screen-The rodent and human laminin ␥1 chain gene (LAMC1) promoter contains a transcriptional element, bcn-1, that binds nuclear proteins from mesangial cells (7,13,14). We generated a cDNA fusion library from rat mesangial cells for a screen in yeast one-hybrid system to identify factors that bind the bcn-1 element. One of the cDNA clones isolated in this screen encoded the transcription factor E3 (TFE3) (29). TFE3 is composed of a basic helix-loop-helix region and a leucine zipper (bHLHzip). Both the basic helixloop-helix region and the leucine zipper domains are essential for DNA binding and dimerization (25,29). The members of the bHLHzip family of transcription factors, including TFE3, bind directly the E-box DNA motif. Isolation of the TFE3 was unexpected, because the bcn-1 element (CCCCGCCCACCTCGCGC) does not contain the classical E-box (CACGTG) sequence.
TFE3 Binds to the bcn-1 Motif in Vitro-There are two predominant ubiquitously expressed TFE3 isoforms (25). The two transcripts represent differentially spliced mRNAs that yield long, TFE3L, and short, TFE3S, isoforms. The TFE3L isoform is by far the most abundant accounting for more than 80% of the transcripts, and the rest are mostly in the form of TFE3S (25). There is also another recently described form of TFE3, TFE3-full, that is longer than TFE3L, but its level of expression does not appear to be significant (30,31). TFE3L and FIG. 3. bcn-1 element mediates the LAMC1 promoter response to TFE3 in mesangial cells. A, diagram of the TFE3L and firefly luciferase reporter gene constructs that were used in transfections of mesangial cells. TFE3L was subcloned into pEX mammalian expression vector. The Ϫ1077/Ϫ20 LAMC1 promoter fragment was subcloned into pGL3-basic plasmid. The sequences of wild-type (wt-bcn-1) and mutated (mt-bcn-1) bcn-1 element, located at Ϫ495 to Ϫ479 within the LAMC1 promoter, are shown. B, firefly luciferase reporter gene driven by the LAMC1 promoter containing either wild-type (wt-bcn-1) or mutated (mt-bcn-1) bcn-1 element was co-transfected in mesangial cells with either expression plasmid (vector) or expression plasmid containing the TFE3L cDNA (TFE3L). ␤-Galactosidase reporter gene plasmid was used as a control for transfection efficiency. Relative luciferase activity was calculated as a ratio between the firefly luciferase and ␤-galactosidase activities (mean Ϯ S.D., n ϭ 4).
TFE3-full contain two activation domains, one acidic (AAD) in the N terminus of the protein and the other proline-rich (Pro-AD) C-terminal to the bHLHzip region ( Fig. 1A) (30,31). Although the two activation domains act synergistically, the short spliced variant, TFE3S, lacks the acidic activation domain and its transcriptional activity appears to be diminished (25). To determine if TFE3 binds to the bcn-1 motif directly, the three forms of the protein, 35 S-TFE3-full, 35 S-TFE3L, and 35 S-TFE3S (Fig. 1A) were synthesized in a cell-free system and pull-down assays were done using biotinylated doublestranded oligonucleotide containing the bcn-1 element. 35 S-Labeled luciferase served as a negative control. 35 S-Labeled proteins were incubated with biotinylated double-stranded oligonucleotide containing the bcn-1 element, and then the mix was added to streptavidin beads. After binding, beads were washed, and proteins were eluted by boiling in SDS-loading buffer. 35 S-Labeled translational products (Fig. 1B, lanes 1-4) and 35 S-labeled proteins eluted from the beads (Fig. 1B, lanes [5][6][7][8] were resolved by SDS-PAGE and were analyzed by densitometry (Fig. 1C). All three isoforms of TFE3 (Fig. 1B, lanes  6 -8) bound to the bcn-1 element with apparently similar affinity (Fig. 1C). In contrast, 35 S-luciferase did not bind the oligonucleotide at all (lane 5). These results demonstrate that TFE3 binds to DNA directly. The fact that all three TFE3 isoforms bind the bcn-1 element equally well is to be expected, because all three contain an intact bHLHzip domain that has previously been shown to mediate the binding to the E-box (32,33). This result suggests that all three isoforms may regulate the activity of the LAMC1 promoter.
The bcn-1 motif contains the CAC trinucleotide found in the E-box, suggesting that these nucleotides could mediate TFE3 binding. To test this, we compared binding of 35 S-TFE3L to bcn-1 oligonucleotide in the presence of either no competitor, double-stranded oligonucleotide containing wild-type bcn-1, or double-stranded oligonucleotide mutant bcn-1 in which the AC dinucleotide was mutated to GG. As illustrated in Fig. 2 Ϫ1077/Ϫ20 LAMC1 promoter fragment was subcloned into pGL3 basic. B, firefly luciferase reporter gene driven by the LAMC1 promoter was co-transfected into mesangial cells with either expression plasmid (Ϫ) or expression plasmid containing cDNA (ϩ) of either TFE3L, Smad3, or Smad4. Renilla luciferase reporter gene plasmid was used as a control for transfection efficiency. Relative luciferase activity was calculated as a ratio between the firefly and Renilla luciferase (mean Ϯ S.D., n ϭ 4). C, cells transfected with either empty expression vector, TFE3L, or Smad3 plus Smad4 were used for luciferase assay.
TFE3 binding to bcn-1 was greatly diminished when wild-type bcn-1 competitor was present (compare lane 3 to 2). In contrast, in the presence of a competitor containing the CAC to CGG mutation, there was little or no decrease in the amount of bcn-1-bound 35 S-TFE3 (compare lane 4 to 2). These studies demonstrate that the binding of TFE3 to the bcn-1 element is specific and that the AC dinucleotide found in this motif is important for mediating this interaction.
TFE3 Activates the bcn-1 Element in the Context of the LAMC1 Promoter-To test if TFE3 can activate the LAMC1 promoter and if it is bcn-1-dependent, we carried out reporter gene assays in transient transfections of primary rat mesangial cells. Firefly luciferase reporter gene driven by the LAMC1 promoter was co-expressed with either empty expression vector or expression vector containing cDNA-encoding TFE3L. The ␤-galactosidase gene driven by the SV40 promoter was used as a control for transfection efficiency. These experiments showed a greater than 3-fold activation of the wild-type Ϫ1077/Ϫ20 LAMC1 promoter fragment (subcloned into pGL3-basic plasmid) by TFE3 compared with expression vector (Fig. 3B, wtbcn-1). Mutation of the two nucleotides within the bcn-1 element that are key for TFE3 binding (Fig. 2, B and C), decreased both the constitutive activity of the LAMC1 promoter and its response to TFE3 (Fig. 3B, mt-bcn-1). As in the case of TFE3L, expression of TFE3-full also activated (greater than 3-fold induction) the LAMC1 promoter in a bcn-1-dependent manner (data not shown). These results demonstrate that the LAMC1 promoter is responsive to TFE3 and that the bcn-1 element plays an important role in TFE3-mediated response.
Synergistic Activation of the LAMC1 Gene Promoter by TFE3 and Smad Proteins-The transforming growth factor-␤ (TGF-␤) regulates a host of cellular effects, including cell proliferation, development, and differentiation (34). TGF-␤ activates expression of many genes, including LAMC1 (7). TGF-␤ FIG. 5. Mapping of the LAMC1 promoter regions responsible for its synergistic activation by TFE3 and Smads. A, diagram of firefly luciferase reporter gene driven by the LAMC1 promoter fragments. B, firefly luciferase reporter gene-LAMC1 fragments were co-transfected into mesangial cells with either expression plasmid (Ϫ) or expression plasmid containing cDNA (ϩ) of either TFE3, Smad3, or Smad4. Renilla luciferase reporter gene plasmid was used as a control for transfection efficiency. Relative luciferase activity was calculated as a ratio between the firefly and renilla luciferase (mean Ϯ S.D., n ϭ 4).
bcn-1 Element-dependent Activation of the Laminin ␥1 Chain Gene binds two cell surface serine/threonine kinase receptors, T␤RI and T␤RII, resulting in the activation of the T␤RI kinase, an enzyme that in turn phosphorylates transcription factors such as Smad2 and Smad3 (35). After binding to Smad4, the Smad2⅐Smad4 and Smad3⅐Smad4 complexes translocate to the nucleus where they regulate expression of a diversity of gene targets that account for the pleiotropic effects of TGF-␤ (35). Smad proteins are composed of two Mad homology (MH1 and MH2) domains separated by a linker (26) (Fig. 4A). Although the function of the MH domains has not been fully defined, in the case of Smad3, the MH1 domain mediates its binding to the Smad-binding element (SBE), AGAC (35).
Mesangial cells express TFE3L (data not shown) and several of the Smad proteins, including Smad2, Smad3, and Smad4 (36). It has previously been shown that TFE3L and Smad synergistically activate the plasminogen activator inhibitor-1 (PAI-1) promoter (33). We wondered if there is similar cooperation between TFE3 and Smad proteins in the activation of the LAMC1 promoter in mesangial cells. Expression of either Smad3 or Smad4 alone increased the activity of the Ϫ1077/Ϫ20 LAMC1 promoter fragment by more than 2-fold (Fig. 4B). This result shows the LAMC1 promoter is Smad-responsive. When TFE3 was expressed with either Smad3 or Smad4, the activity of the LAMC1 promoter increased by 8-and 10-fold, respectively (Fig. 4B). Co-expression of TFE3, Smad3, and Smad4 did not further increase the promoter activity beyond that seen with TFE3 and one of the two Smads (Fig. 4C). In agreement with the PAI-1 study (33), these results demonstrate TFE3⅐Smad3 cooperative activation of the LAMC1 promoter. Expression of TFE3 and Smad3 in HeLa cells synergistically activated LAMC1 promoter cloned upstream of luciferase gene (data not shown), thus the cooperation between the two transcription factors is not specific to mesangial cells.
The sequence AGAC is sufficient for high affinity binding of Smad3 and Smad4 to SBEs (35). Previously it has been shown that two closely located SBEs are required for the TFE3⅐Smadmediated synergistic activation of the PAI-1 promoter (26). Inspection of the LAMC1 promoter identified four putative SBEs; two on each side of bcn-1 (position Ϫ495) but several hundred bases away from bcn-1, SBE1 (Ϫ967), SBE2 (Ϫ853), SBE3 (Ϫ318), and SBE4 (Ϫ278). Deletion of the region 5Ј to the bcn-1 element (Fig. 5A), Ϫ506/Ϫ20 LAMC1 fragment, abrogated the TFE3⅐Smad3 and TFE3⅐Smad4 synergism (Fig. 5B), suggesting that one or both of the SBEs 5Ј to the bcn-1 element are required for the synergism. Deletion of a 3Ј region that includes the bcn-1 element, Ϫ1077/Ϫ529 LAMC1 fragment (Fig. 5A), abolished the TFE3 and Smads effects (Fig. 5B) indicating that the presence of the two 5Ј SBEs is not sufficient for the synergism. Also, this result suggests that bcn-1 is an element mediating this synergism.
The above deletion analysis suggests that the bcn-1 element and the two 5Ј SBEs are critical for the synergistic activation of the LAMC1 promoter by TFE3 and Smad proteins (Fig. 5). Site-directed mutagenesis of the bcn-1 element and one or both of the 5Ј SBEs was used to directly test the role of these elements in mediating the TFE3⅐Smad3 synergistic activation of the LAMC1 promoter. Within the bcn-1 element, mutation of two bases (AC 3 GG) that are critical for TFE3 binding (Fig.  2B) abolished the TFE3⅐Smad3 synergism (Fig. 6B). Mutation of either SBE1 or SBE2 (Fig. 6A) did not abrogate the synergistic activation of the LAMC1 promoter by TFE3⅐Smad3, but mutation of both SBE1 and SBE2 did (Fig. 6B). These results demonstrate that the bcn-1 element and at least one of the 5Ј SBEs are necessary for the ability of TFE3 and Smad3 proteins to synergistically activate the LAMC1 promoter in mesangial cells.

Expression of a Constitutively Active TGF-␤ Receptor Stimulates TFE3⅐Smad3-mediated Activation of the LAMC1 Promoter and the Endogenous LAMC1 Gene in Mesangial Cells-It
has previously been shown that TGF-␤ signaling stimulates the synergistic activation of the PAI-1 promoter by TFE3 and Smad proteins (33). We expressed constitutively active TGF-␤ receptor I, TGF-␤ RI(T204D) (26), to test whether or not the TFE3⅐Smad3-mediated activation of the LAMC1 promoter is responsive to TGF-␤ triggered signal transduction pathway (Fig. 7). TGF-␤ RI-T204D increased both the TFE3-and the Smad3-mediated activation of the LAMC1 promoter but the activated receptor had by far the most profound effect when TFE3 and Smad3 were co-expressed (Fig. 7). These results indicate that the TFE3⅐Smad3-mediated activation of the LAMC1 promoter is responsive to TGF-␤ signaling in mesangial cells.
This and previous studies have shown that TFE3 and Smad3 cooperate to activate the promoters in reporter gene assays, but the effects of their combined action on endogenous genes had not previously been examined. Because TGF-␤ treatment activates LAMC1 gene expression (7), it was important to determine if expression of this gene is regulated by the cooperative action of these two transcription factors and, if so, whether their action is responsive to TGF-␤ signaling. Mesangial cells were transfected with either TFE3, Smad3, or TFE3 plus Smad3 with or without TGF-␤RI(T204D). Twenty-four hours after transfection, cells were harvested and total RNA was isolated. Laminin ␥1 chain mRNA level was assessed by Northern blot (Fig. 8) with 32 P-labeled LAMC1 cDNA as a probe. Compared with empty plasmid (Fig. 8A, lane 1)

DISCUSSION
Previous studies have shown that the bcn-1 element plays an important role in the regulation of the LAMC1 promoter (7,14), but until now transcription factors engaged by this element have not been identified. Using the yeast one-hybrid screen, we cloned TFE3 as one of the transcription factors that binds and activates the bcn-1 element (Fig. 3). This was an unexpected finding, because the bcn-1 motif lacks the classic E-box sequence known to bind TFE3. This protein belongs to the bHL-Hzip family of transcription factors whose members are all known to bind the same E-box sequence, CACGTG. The bHL-Hzip domain is the DNA-binding region and is therefore likely to mediate TFE3 binding to the bcn-1 element as well. Besides TFE3, this class of transcription factors includes USF1 (37-39),  1 and 5) or expression vector encoding TFE3 (TFE3, lanes 2 and 6), Smad3 (Smad3, lanes 3 and 7), or both TFE3 and Smad3 expression plasmids (TFE3 -Smad3, lanes 4 and 8). Twenty-four hours after transfection total RNA was isolated ("Materials and Methods"), and RNA was separated by agarose gel electrophoresis. After transfer, nylon membranes were hybridized with 32 P-labeled LAMC1 cDNA probe. Washed membranes were analyzed using a PhosphorImager. 28 S RNA was assayed as loading control, and the intensities of ethidium bromide-stained bands were measured by densitometry. B, graph of the level of the laminin ␥1 chain mRNA normalized to 28 S loading control. The data shown are representative of two similar experiments.
TEFB (40), Mi (41), Myc (37), and Max (37). These proteins bind the E-box, either as homodimers or heterodimers, and serve a diversity of regulatory functions (29, 37, 40 -44). Because these transcription factors share DNA binding specificity, it is likely that other members of this class of transcription factors may also bind to the bcn-1 element. In support of this suggestion is the fact that our yeast one-hybrid screen of human KB cell library identified not only human TFE3 but also the USF1 transcription factor as a bcn-1-binding protein. 2 Thus, the results of these studies show that the E-box is not the only transcriptional element that can be regulated by this important family of transcription factors.
Although TFE3 binds and activates the bcn-1 element in the context of either the LAMC1 (Figs. [3][4][5] or heterologous promoters (data not shown), the level of the TFE3-mediated activation from a single bcn-1 motif is modest. A similar level of activation has been described for other bHLHzip transcription factors acting from a single element (37). The ability of TFE3 to activate the LAMC1 promoter is greatly enhanced by co-expression of Smad proteins, an observation that is in agreement with a previous report about the synergistic activation of PAI-1 by these two classes of factors (33). As in the case of the PAI-1 gene (26,33), the TFE3⅐Smad synergistic activation of the LAMC1 promoter depended on the integrity of the SBE elements. Although there are similarities of these promoters with respect to TFE3⅐Smad3 cooperation, there are also notable differences between the two systems. (i) The bcn-1 element only distantly resembles the TFE3-binding E-box found in the PAI-1 promoter. (ii) In the case of the LAMC1 promoter, one of the 5Ј SBEs seemed sufficient to mediate the synergistic activation compared with two SBEs in the PAI-1 gene. (iii) Unlike the PAI-1 promoter where the TFE3⅐Smad3 synergism depended on a precise three-nucleotide spacer between the E-box and a pair of SBEs, the distance between bcn-1 and the active SBEs was several hundred nucleotides long. This implies that the specific molecular scenarios by which TFE3 and Smad protein cooperate are different and could be promoter-dependent.
Signaling by TGF-␤ is initiated following its binding to the constitutively active serine/threonine kinase type II receptor (T␤RII) (45). The type I receptor (T␤RI), which also contains serine/threonine kinase activity, is then recruited to T␤RII, leading to the formation of an oligomeric complex (Fig. 9). The subsequent phosphorylation and activation of T␤RI by T␤RII leads to phosphorylation of the Smad family of transcription factors, a process that is dependent on adapter proteins such as SARA (46) and Dab2 (47), which recruit Smad proteins to the TGF-␤ receptor.
The Smad class of proteins are divided into three functional groups: (i) the receptor-activated Smads or R-Smads, which include Smad1, Smad2, Smad3, and Smad8; (ii) the co-Smad, Smad4; and (iii) the inhibitory Smads, Smad6 and Smad7. Following phosphorylation, R-Smads form dimers with Smad4, a complex that translocates to the nucleus where it binds to the DNA Smad binding elements (SBE), within target loci leading to transcriptional activation (35), in part, due to recruitment of the paralogous coactivators, CBP or p300 (48).
It has previously been shown that TFE3 is directly engaged by Smad3 and Smad4 through their respective MH1 domains (26). It has also been shown that TFE3 binds the co-activator p300 in vivo (49). It was suggested that the precise E-box⅐TFE3, SBE⅐Smad, and TFE3⅐Smad interaction arrangements on the PAI-1 promoter allow for efficient recruitment of either CBP or p300 coactivator leading to enhanced transcription (26). Binding of TFE3 to DNA induces DNA bending (50,51). Thus, DNA bending mediated by TFE3 together with other factors bound to the LAMC1 promoter may be particularly important for the cooperative activation of the LAMC1 promoter by Smad proteins and TFE3, where their respective DNA binding elements, SBE and bcn-1, are several hundred nucleotides apart (Fig. 6). The binding of TFE3 and Smads to DNA and to each other would generate a constructive promoter topology for an effec-  (33,46,47). Following binding to the constitutively active serine/threonine kinase type II receptor (T␤RII), TGF-␤ triggers recruitment of the type I serine/threonine kinase receptor (T␤RI) leading to the formation of an oligomeric complex (59). This results in the phosphorylation of the T␤RI by T␤RII and the recruitment of Smad3 to the complex by an adapter molecule such as Dab2 (47) or SARA (46). Following phosphorylation, Smad3 dissociates from the receptor complex and associates with Smad4. The heterodimer translocates to the nucleus where it binds one of the 5Ј SBE elements of the LAMC1 promoter that is appropriately bent by TFE3 and other DNA-bending factors. The interaction of TFE3 with the Smad heterodimer that follows generates a constructive topology for an effective recruitment of co-activators, such as CBP (33,49), and basal transcriptional machinery leading to enhanced LAMC1 gene transcription. tive recruitment of CBP/p300 and the basal transcriptional machinery leading to enhanced LAMC1 transcription (Fig. 9).
Regardless of the precise mechanisms, this and the previous PAI-1 gene study (26) show that TGF-␤-triggered activation of Smads would not, by itself, be effective for driving transcription without TFE3. Indeed, several tandem repeats of SBEs are required for a reporter gene to effectively respond to TGF-␤ when Smads are the only activators (52). This suggests a general scenario where Smads require other transcriptional activators, such as bHLHzip factors, to effectively activate their target promoters. Those natural promoters that do not contain classic E-boxes, yet exhibit effective Smad-mediated responses, may contain one or more ambiguous accessory elements that recruit a transcription factor or factors that render(s) the gene TGF-␤-responsive. The yeast one-hybrid screen allowed us to identify TFE3 as a bcn-1-binding factor, which turned out to be an unexpected accessory element to Smad action and TGF-␤ signaling. Results of this study suggest that for many TGF-␤responsive genes there may be other, yet to be identified, nongeneric accessory elements that facilitate Smad-mediated transcription. The TFE3-mediated activation of the bcn-1 element was augmented both by Smad proteins, acting through the LAMC1 SBEs, and by TGF-␤-signaling pathways (Fig. 7). Similarly, TFE3 and Smad3 also cooperatively activated the endogenous LAMC1 gene, action that was responsive to TGF-␤ signaling (Fig. 8). Thus, TGF-␤-activated LAMC1 gene expression (7), is, at least in part, dependent on the cooperative action of TFE3 and Smad3 acting from their respective sites within the LAMC1 promoter, bcn-1 and the SBEs.
Several renal cell carcinomas are associated with chromosomal translocation that result in fusion of TFE3 with a product of another gene (31,53,54). One of the best-studied translocation results is a fusion of TFE3 with PRCC, a gene of unknown function (31). Here, two reciprocal fusion proteins are formed TFE3-PRCC and PRCC-TFE3. In these chimeric proteins the TFE3's transactivation and DNA-binding domains are entirely intact. Both fusion proteins activate the E-box in a reporter gene assay, suggesting that the ability of these fusion proteins to target genes may contribute to the pathogenesis of the papillary renal cell carcinomas. If so, identification of bcn-1 as a TFE3 target that is distinct from E-box, underscores the need to better define the spectrum of TFE3-binding natural elements to elucidate the pathogenesis of this tumor. If the TFE3 chimeric proteins that result from the chromosomal translocation play a direct role in carcinogenesis, these processes could also involve the TGF-␤-Smads protein axis. In this regard, it is interesting to note that the two known targets of the combined action of TFE3 and Smad, PAI-1 (26,33), and LAMC1 (this study), have both been linked to oncogenesis (55)(56)(57).
In summary, the yeast one-hybrid screen allowed us to identify TFE3 as one of the bHLHzip transcription factors that regulates the LAMC1 promoter in a bcn-1 element-dependent manner. Because the bcn-1 element is different from the classic E-box, these studies suggest that the repertoire of DNA sequences that mediate the action of the bHLHzip transcription factors may be broader than originally thought. This and other studies (33,36) suggest that an effective response of gene promoters to TGF-␤ treatment depends on cooperative action of Smad protein recruited to SBEs and other transcriptional activator(s), such as TFE3, bound to an accessory element(s), such as bcn-1. A cooperative action of TFE3 and Smad3 contributes to the TGF-␤-induced LAMC1 gene expression.