Role of E box and initiator region in the expression of the rat follicle-stimulating hormone receptor.

The promoter for the rat follicle-stimulating hormone receptor (FSHR) gene contains a conserved consensus E box sequence and an initiator-like region (InR) sequence. Deletion analysis and transient transfections showed that a 114-base pair region (-143 to -30) that encompasses the E box and the InR was sufficient for greater than 75% of promoter function. DNase I footprint analysis showed that the E box and InR were protected by nuclear proteins from rat Sertoli cells, and the E box region was shown by electrophoretic mobility shift assays (EMSA) to be a site of Sertoli protein interactions. Mutations in the E box disrupted these interactions and reduced FSHR promoter activity. Co-transfection of the inhibitor of DNA binding (Id) with an FSHR/luciferase construct into mouse Sertoli 1 cells reduced FSHR promoter activity. Using EMSA, the upstream stimulatory factor was shown to be a component of the complexes that interacted with the E box in the FSHR promoter. Binding of proteins from rat Sertoli cells to the InR was demonstrated using EMSA. Also, an oligonucleotide that represented the sequence of the terminal deoxynucleotidyltransferase InR displaced the complexes at the FSHR InR. Mutations in the InR resulted in a significant reduction of FSHR promoter activity.

The promoter for the rat follicle-stimulating hormone receptor (FSHR) gene contains a conserved consensus E box sequence and an initiator-like region (InR) sequence. Deletion analysis and transient transfections showed that a 114-base pair region (؊143 to ؊30) that encompasses the E box and the InR was sufficient for greater than 75% of promoter function. DNase I footprint analysis showed that the E box and InR were protected by nuclear proteins from rat Sertoli cells, and the E box region was shown by electrophoretic mobility shift assays (EMSA) to be a site of Sertoli protein interactions. Mutations in the E box disrupted these interactions and reduced FSHR promoter activity. Co-transfection of the inhibitor of DNA binding (Id) with an FSHR/ luciferase construct into mouse Sertoli 1 cells reduced FSHR promoter activity. Using EMSA, the upstream stimulatory factor was shown to be a component of the complexes that interacted with the E box in the FSHR promoter. Binding of proteins from rat Sertoli cells to the InR was demonstrated using EMSA. Also, an oligonucleotide that represented the sequence of the terminal deoxynucleotidyltransferase InR displaced the complexes at the FSHR InR. Mutations in the InR resulted in a significant reduction of FSHR promoter activity.
Follicle-stimulating hormone (FSH) 1 is important for the early development and maintenance of the Sertoli cell function and for normal spermatogenesis in the male and folliculogenesis in the female. FSH exerts its action through the FSH receptor (FSHR), which is a member of a G-protein-coupled receptor subfamily that also includes the luteinizing hormone receptor/chorionic gonadotropin receptor (LHR/CGR) and thyroid-stimulating hormone receptor. The receptors in this subfamily have a large extracellular domain, a transmembrane region, and an intracellular domain (1)(2)(3)(4)(5).
The promoters for this gene in the rat, mouse, and human FSHR are members of a class of promoters that lack a conven-tional TATA and CCAAT box and have multiple transcriptional start sites (6 -8). Several DNA elements have been identified in the 3Ј proximal region of the promoter for the FSHR in the rat, human, and mouse FSHR. These include an initiator-like region (InR) and at least one E box upstream activating sequence, both of which are highly conserved among the rat, human, and mouse FSHR promoters. A consensus AP-1 or cAMP response element (CRE)-like site was also reported in the rat FSHR promoter, but this site was poorly conserved in the mouse and human FSHR promoters (7)(8)(9).
The E box element has the consensus sequence, CANNTG, that interacts with a family of transcription factors known as the basic helix-loop-helix (bHLH) proteins (for a review, see Ref. 10). These proteins are characterized by a basic region that is required for binding of the protein to the DNA and an helix-loop-helix motif required for protein dimerization. A subfamily of the bHLH proteins contains a leucine zipper (bHLHZ) for additional dimerization potential.
The InR is a pyrimidine-rich region that encompasses a transcriptional start site for the initiation of transcription and may or may not function cooperatively with a TATA box (11,12). Several proteins have been shown to interact with the InR, including TFIID (TAF 150), TFII-I, YY1, and the bHLHZ proteins, as well as the upstream stimulatory factor (USF) and c-Myc (11). The InR of the terminal deoxynucleotidyltransferase (TdT) promoter is an example of a well characterized InR that represents the most common consensus sequence observed in InRs.
Previously, we identified a 5-kilobase region upstream of the FSHR gene as its promoter and showed that the first 286 bp of the promoter were able to activate transcription in transient transfection assays (6). In the present study, deletional analysis, site-directed mutagenesis, and transient transfections were performed to show that E box and InR sequences contribute to the activity of the FSHR gene. Additionally, electrophoretic mobility shift assays (EMSA) and DNase I footprint assays were used to characterize the interactions between the consensus E box sequence, the InR, and the nuclear proteins in rat Sertoli cells.

PCR-Directed Mutagenesis/Transient Transfections
Generation of Deletion Constructs-A series of reporter gene constructs, pϪ574/Ϫ30 (from plasmid Ϫ574 to Ϫ30 region of the FSHR promoter), pϪ480/Ϫ30, pϪ383/Ϫ30, pϪ317/Ϫ30, pϪ143/Ϫ30, pϪ117/ Ϫ30, pϪ383/Ϫ1, and pϪ143/Ϫ1 FSHR/luciferase, were generated using PCR. The 5Ј-primers used to generate each construct were as follows: pϪ574/Ϫ30 (Ϫ574 to Ϫ553), pϪ480/Ϫ30 (Ϫ480 to Ϫ457), pϪ383/Ϫ30 (Ϫ383 to Ϫ361), pϪ317/Ϫ30 (Ϫ317 to Ϫ257), pϪ143/Ϫ30 (Ϫ143 to Ϫ123), and pϪ117/Ϫ30 (Ϫ117 to Ϫ98). The 5Ј-primers for the pϪ383/Ϫ1 and pϪ143/Ϫ1 FSHR/luciferase constructs were the same as those used for the pϪ383/Ϫ30 and pϪ143/Ϫ30 FSHR/luciferase constructs. The 3Ј PCR primer used to generate each construct corresponded to nucleotides Ϫ46 to Ϫ30 of the rat FSHR gene. The 3Ј-primers used for the pϪ383/Ϫ1 and pϪ143/Ϫ1 FSHR/luciferase constructs corresponded to nucleotides Ϫ31 to Ϫ1. All constructs were subcloned into the pGL2-* This work was supported by National Institutes of Health Grant HD10808. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Generation of Mutant Constructs-All mutant constructs were generated using a recombinant PCR technique (13). To generate constructs with mutations in the E box, the pϪ383/Ϫ30 FSHR/luciferase construct described above was used as the template. The 5Ј external primer corresponded to nucleotides Ϫ317 to Ϫ297 in the sequence of the rat FSHR promoter. The 3Ј external primer was the GL-2 primer provided with the pGL2-Basic luciferase vector. The internal primers that were used to introduce mutations in the consensus E box corresponded to nucleotides Ϫ129 to Ϫ108 and included the mutations shown in Fig. 5. To generate constructs with mutations in the putative InR, the pϪ383/ Ϫ30 FSHR/luciferase vector was used as a template. The 5Ј external primer was the GL-1 primer, and the 3Ј external primer was the GL-2 primer, both provided with the pGL2-basic vector. The internal primers used to introduce mutations into the InR corresponded to nucleotides Ϫ102 to Ϫ77 in the FSHR promoter and included the mutations shown in Fig. 9.
Transient Transfections and Determination of Luciferase Activity-A mouse Sertoli cell derived line (MSC-1) and primary cultures of rat Sertoli cells were used for transient transfection assays (14). MSC-1 cells have many properties of Sertoli cells but do not express the FSHR, presumably due to methylation of the FSHR gene (6,15). For transient transfections in MSC-1 cells, the cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% bovine calf serum (BCS; Hyclone, Logan, UT). Briefly, 35-cm plates of MSC-1 cells, at 80 -90% confluency, were transfected using liposome-mediated transfer. Liposomes were created by incubating 0.25 g of the FSHR/luciferase constructs and 0.25 g of a ␤-galactosidase expression vector (Promega), which was used to standardize for uptake of DNA by the cells, with 6 l of LipofectAmine (Life Technologies, Gaithersburg, MD) in 200 l of serum-free DMEM for 45 min. The liposomes were added to cells, and after 5 h, 1 ml of DMEM containing 10% BCS was added to each well. The media was changed 24 h after the start of the transfection, and the cells were lysed at 48 h with 150 l of lysis buffer (Promega). All constructs were transfected in triplicate wells, and each sample was assayed in duplicate for the activity of both the luciferase and the ␤-galactosidase.
Luciferase activity was determined by measuring luminescence of 20 l of the cell lysate plus 100 l of luciferase assay reagent for 20 s in a luminometer (LB96P; Wallac-Berthold, Bad Wildbad, Germany). The ␤-galactosidase activity was determined by incubating 10 l of the cell lysate plus 100 l of the Galactolite reagent buffer (Tropix, Bedford, MA) for 1 h in the dark at ambient temperature. Following the incubation, 150 l of accelerator (Tropix) was added and the luminescence was measured for 20 s in a luminometer.
Transient transfections of primary cultures of Sertoli cells were performed as described previously using the calcium phosphate precipitation method with minor modifications (6,16). On day 5 of culture, Ham's F-12 medium with 5% BCS was added 2 h prior to adding the DNA crystals. The DNA crystals were generated using 0.25 g of each of the FSHR/luciferase constructs that were described above. The Sertoli cells were incubated with the DNA crystals for 5 h and then the medium was replaced with fresh Ham's F-12 containing 10% BCS. After 18 h, the cells were lysed and the luciferase activity was measured using luminescence as described above. The replicates were the same as those described above for the MSC-1 transfections. Samples were standardized by determining cellular protein concentration using a bicinchoninic acid (BCA) colorimetric assay (Pierce, Rockford, IL), rather than ␤-galactosidase activity, due to endogenous ␤-galactosidase activity in primary Sertoli cells that contributed to high background levels.
Transient co-transfections in MSC-1 cells with the trans-regulator, inhibitor of DNA binding (Id), were performed as described above with minor modifications. The pϪ317/Ϫ30 FSHR/luciferase construct was added at 0.25 g, along with 0.25 g of a ␤Ϫgalactosidase vector. Id was added at 0.25 ϫ (0.0625 g), 0.5 ϫ (0.125 g), and 1 ϫ (0.25 g) amounts, relative to the concentration of the pϪ317/Ϫ30 FSHR/luciferase. The pMSV vector (the vector into which the Id insert was subcloned) was used to bring all of the combinations of the constructs to equal amounts of DNA. The luciferase and the ␤-galactosidase activities were determined as described above.
Statistical Analysis-Data were presented as a mean Ϯ S.E. and a one-way analysis of variance was performed on the transfection data, followed by a Duncan's multiple range test for all pair-wise comparisons (␣ ϭ 0.05) for the transfections using the pEMut constructs or the Id vector. Fisher's LSD test was performed on the data for the transfections using the deletion/addition and mutated InR constructs. The data for the Id co-transfection and the deletion/addition constructs were obtained from three independent experiments, the data for the InR mutations were obtained from four independent experiments, and the data for the E box mutations were obtained from five independent experiments.

Electrophoretic Mobility Shift Assays (EMSA)
Extraction of Nuclear Proteins-Sertoli cells were isolated and cultured as described above (16). Medium was changed on day 2 of culture, and Ham's F-12 that contained 10% BCS was added on day 4 of culture. Nuclear proteins were harvested on day 6 using a high salt extraction method (17). The concentration of nuclear protein was determined by a colorimetric reaction using the BCA assay.
Preparation of the FSHR Promoter Fragment for EMSA-A 288-bp FSHR promoter fragment was generated using the PCR. This fragment corresponded to the promoter fragment that was subcloned into the pϪ317/Ϫ30 FSHR/luciferase construct and was referred to as the A Ϫ317 / G Ϫ30 FSHR promoter fragment. For amplification of the A Ϫ317 /G Ϫ30 FSHR promoter fragment, the 5Ј-primer corresponded to nucleotides Ϫ317 to Ϫ297 of the FSHR promoter, and the 3Ј-primer was the GL-2 primer. The sequence of the FSHR promoter construct was verified as described above. The generation of the A Ϫ317 /G Ϫ97 FSHR promoter fragment was described previously (6). An 88-bp FSHR promoter fragment was generated using the PCR. This fragment corresponded to the promoter fragment subcloned into the pϪ117/Ϫ30 FSHR/luciferase construct and was referred to as the C Ϫ117 /G Ϫ30 FSHR promoter fragment. The primers for amplification of the C Ϫ117 /G Ϫ30 FSHR promoter fragment corresponded to nucleotides Ϫ46 to Ϫ31 and nucleotides Ϫ118 to Ϫ98.
The C Ϫ117 /G Ϫ30 FSHR promoter fragment was radiolabeled using reverse transcriptase and [␣-32 P]dATP (3000 Ci/mmol). The conditions for the EMSA with the C Ϫ117 /G Ϫ30 FSHR promoter fragment were modified to optimize for specific interactions at the InR (18). The binding reactions were performed in a final volume of 20 l in a solution that contained 1 ϫ binding buffer (described above), 10% glycerol, and 4 g of poly(dA-dT) (Pharmacia). The reactions were incubated for 10 min on ice and resolved by electrophoresis as described above.
Competition EMSA-Specific competition was performed with excess unlabeled synthetic double-stranded oligonucleotides that corresponded to the E box sequence in the FSHR promoter (nucleotides T Ϫ137 to C Ϫ110 ). Nonspecific competition for the A Ϫ317 /G Ϫ30 FSHR promoter fragment was performed using a HincII/EcoRI digest of the 3Ј-end of bovine interleukin-2 cDNA (BLT; kindly provided by Dr. Raymond Reeves, Washington State University, Pullman, WA). The 27-bp doublestranded DNA FSHR oligonucleotide corresponded to nucleotides A Ϫ101 to T Ϫ75 in the rat FSHR promoter. The oligonucleotide that corresponded to the TdT InR was made according to the sequence reported by Smale and Baltimore (12). All oligonucleotides were added to the competition reactions at equimolar concentrations relative to the concentration of the radiolabeled FSHR fragment.
Supershift EMSA with Specific Antibodies-Nuclear extract from Sertoli cells and the radiolabeled A Ϫ317 /G Ϫ97 FSHR fragment were incubated under the same conditions as described above. The A Ϫ317 / G Ϫ97 FSHR fragment was radiolabeled using Klenow enzyme as described above. The binding reactions were incubated in the presence of either 1 l of undiluted polyclonal mouse USF antibody (kindly provided by Dr. Robert Roeder, Rockefeller University, New York, NY), antibody diluted to 1:1 or 1:3 in 1 ϫ phosphate-buffered saline, or 1 l of normal mouse serum (negative control) for 1 h on ice. The reaction was loaded onto a 5% native polyacrylamide gel with 0.5 ϫ Tris borate-EDTA and 10% glycerol. (The polyclonal human c-Myc and Max antibodies used for supershift EMSA were kindly provided by Dr. Robert Eisenman at the Hutchinson Cancer Research Center, Seattle, WA).
DNase I Footprinting-The top strand (sense) of the A Ϫ317 /G Ϫ30 FSHR promoter fragment was radiolabeled using PCR. The 5Ј PCR primer corresponded to nucleotides Ϫ317 to Ϫ297 and was radiolabeled using T4 polynucleotide kinase (Life Technologies, Inc.) and [␥-32 P]ATP (3000 Ci/mmol). The non-radioactive 3Ј PCR primer corresponded to nucleotides Ϫ46 to Ϫ30. Nuclear proteins (60 and 80 g) isolated from Sertoli cells were incubated with the radiolabeled double-stranded A Ϫ317 /G Ϫ30 FSHR promoter fragment using the binding reaction conditions described above in the EMSA experiments. Immediately following the 15 min incubation on ice, 2.5 mM CaCl 2 and 5 mM MgCl 2 were added to the reactions. This was followed by the addition of 200 ng of DNase I (Promega, Madison, WI) in the reactions that contained proteins for footprinting and 100 ng of DNase I in the control reactions. The reactions were incubated at ambient temperature for 1 min and stopped with the addition of 10 mM EDTA and 2% SDS. The DNA was prepared for loading onto an 8% sequencing gel, and the Maxam-Gilbert, G, and A/G chemical sequencing reactions were included as reference ladders.
Northern Blot Analysis-RNA was harvested from Sertoli cells, MSC-1 cells, and the adult rat testis and ovary, and Northern analysis was done as described previously (6,19). The USF-1 and USF-2 cDNA inserts were excised from clones provided kindly by Dr. Michele Sawadogo, The University of Texas (20).

Deletion Analysis of the Promoter for the FSHR Gene-A
series of constructs was generated to determine the regions of the FSHR promoter (Fig. 1) that contributed to maximal promoter activity of the FSHR gene. These constructs were introduced into MSC-1 cells by transient transfection, and the relative luciferase activity was expressed as a percentage of the activity of the pϪ317/Ϫ30 FSHR/luciferase construct. The pϪ574/Ϫ30, pϪ480/Ϫ30, and pϪ383/Ϫ30 FSHR/luciferase constructs showed no significant changes in relative luciferase activity compared with pϪ317/Ϫ30 FSHR/luciferase (p Ͼ 0.05; Fig. 2). As the 5Ј-end of the pϪ317/Ϫ30 FSHR/luciferase was deleted, there was a sequential loss in luciferase activity (Fig.  2). The activity of the pϪ143/Ϫ30 FSHR/luciferase construct was reduced by 27%, while the activity of the pϪ117/Ϫ30 FSHR/luciferase construct was reduced by 72% (both p Ͻ 0.05), relative to the pϪ317/Ϫ30 FSHR/luciferase.
The possible roles of sequences in the 5Ј-untranslated region (5Ј-UTR), from the Ϫ30 nucleotide to the translational start site, were examined using the pϪ383/Ϫ1 and pϪ143/Ϫ1 FSHR/ luciferase constructs. There was no significant change in relative luciferase activity when the pϪ143/Ϫ1 FSHR/luciferase construct was compared with that of the pϪ143/Ϫ30 FSHR/ luciferase (p Ͼ 0.05; Fig. 2). In contrast, the pϪ383/Ϫ1 FSHR/ luciferase construct had a 20% increase in activity over the pϪ383/Ϫ30 FSHR/Luc, and this construct represented the maximum promoter activity (Fig. 2).

Characterization of Protein Interactions with the A Ϫ317 /G Ϫ30 FSHR Promoter Fragment
DNase I Footprint Analysis-DNase I footprint analysis was used to determine the regions of sequence-specific protein interactions with the A Ϫ317 /G Ϫ30 FSHR promoter fragment. DNase I cleavage of naked DNA in the region of interest was impeded somewhat, perhaps by secondary structure, throughout the time course of incubation. However, protection was observed with both 60 and 80 g of nuclear protein in a relatively large region at the 3Ј proximal region of the promoter covering nucleotides Ϫ65 to Ϫ135 (Fig. 3). This region included the E box site, the putative InR, and the transcriptional start sites. We did not observe significant protection of sites upstream from the E box.
Binding and Competition Assays of the pϪ317/Ϫ30 FSHR Promoter Fragment-The radiolabeled A Ϫ317 /G Ϫ30 FSHR promoter fragment was incubated with nuclear proteins extracted from Sertoli cells. The DNA-protein interactions resulted in at least seven shifted bands (Fig. 4A, lane 1). Band 6 appeared to be a doublet, and band 7 could have represented multiple complexes as well. This pattern of shifted bands was similar to results published previously by this laboratory using the A Ϫ317 / G Ϫ97 FSHR promoter fragment (6).
Competitive EMSA was performed using the radiolabeled A Ϫ317 /G Ϫ30 FSHR promoter fragment plus increasing amounts of the T Ϫ137 /C Ϫ110 E box oligonucleotide. Direct binding to this E box oligonucleotide was demonstrated using EMSA (data not shown). Most of the protein complexes that interacted with the A Ϫ317 /G Ϫ30 FSHR promoter fragment were completely displaced by the E box oligonucleotide with the exception of bands 6 and 7 (Fig. 4A, compare lanes 2-5 with lane 1). Band 6 was shown previously to be present only in extracts from primary rat Sertoli cells and MSC-1 cells but not extracts from cells of non-gonadal origin (6). Band 6 was displaced completely by a combination of the E box oligonucleotide and the C Ϫ117 /G Ϫ30 FSHR promoter fragment used in the EMSA of the current study (data not shown). It should be noted that the unlabeled C Ϫ117 /G Ϫ30 FSHR promoter fragment when used alone as a competitor did not appear to disrupt formation of the complexes that interacted with the A Ϫ317 /G Ϫ30 FSHR promoter fragment (data not shown). This apparent non-competition could have been due to masking by the more abundant protein interactions at the E box of the A Ϫ317 /G Ϫ30 FSHR promoter fragment. Band 7 was displaced by competition with an FSHR promoter fragment that contained the region beginning immediately upstream from the E box site and extending to the 5Ј-end of the A Ϫ317 /G Ϫ30 FSHR promoter fragment (Ϫ317 to Ϫ151; Fig. 4B, compare lanes 2-4 with lane 1). Equimolar amounts of BLT cDNA were used as a nonspecific competitor and did not disrupt any of the complexes (Fig. 4A, compare lanes 7-9 with lane  6). It should be noted that the banding pattern in Fig. 4B only appears different due to the use of a nuclear extract preparation that contained less nonspecific binding activity, which resulted in less background, as compared with the nuclear extract that was used for experiments shown in Fig. 4A, C, and D.
Competition with Mutated E Box Oligonucleotides-Muta- The nucleotide sequence of the 3 proximal 574-bp region of the rat FSHR promoter (GenBank/EBI accession number S81117). The translational start site is indicated by ϩ1 and an arrow, while the two transcriptional start sites at Ϫ80 and Ϫ98 were determined previously and are indicated by arrows only (9). The putative InR is underscored, and the relevant DNA elements are underscored and labeled (6,9). tions were introduced into the T Ϫ137 /C Ϫ110 E box oligonucleotide (EMut1 and EMut10), which were then used as unlabeled competitors for protein binding with the radiolabeled A Ϫ317 / G Ϫ30 FSHR promoter fragment. The single base pair mutation (EMut1) has been shown to disrupt binding of c-Myc to the E box consensus (21). In addition to the core of the consensus E box, the two nucleotides flanking the E box have been implicated as binding determinants for bHLH/HLHZ proteins. Therefore, we created the EMut10, which resulted in the mutation of this entire region shown to be involved in bHLH/ HLHZ protein interactions (22). EMut1 disrupted protein complexes at 1000 ϫ excess only (Fig. 4C, compare lanes 1-4 with  lane 5), while the 10-bp mutation (EMut10) failed to disrupt complexes at the same concentration (Fig. 4D, compare lanes  1-4 with lane 5). Additionally, there were no direct interactions detected by EMSA between nuclear proteins from Sertoli cells and the radiolabeled EMut1 and EMut10 oligonucleotides (data not shown).

Characterization of the E Box Consensus Element and the Activity of the FSHR Promoter Using Transient Transfections
Transient Transfections with FSHR Constructs that Contained Mutations in the E Box-Luciferase reporter gene con-structs of the A Ϫ317 /G Ϫ30 promoter region with mutations in the E box were created to determine the role of the E box sequence in FSHR promoter activity. These were the mutations that were shown to disrupt protein interactions in the EMSA described above (EMut1 and EMut10) as well as a 6-bp mutation in the E box core sequence only (EMut6). The constructs were introduced into Sertoli cells or MSC-1 cells, and the relative luciferase activity was expressed as a percentage of the activity of the wild type A Ϫ317 /G Ϫ30 FSHR/luciferase construct. Transfection of the pEMut1, pEMut6, and pEMut10 constructs resulted in a reduction in luciferase activity relative to the A Ϫ317 /G Ϫ30 FSHR/luciferase construct ( Fig. 5; p Ͻ 0.05). A similar reduction in promoter activity was observed when the same constructs were introduced into primary cultures of Sertoli cells (67, 33, and 51%, respectively; Fig. 5; p Ͻ 0.05).
Transient Co-Transfection of the Inhibitor of DNA Binding (Id)-The Id expression vector has been used in previous studies to verify the role of bHLH/HLHZ proteins in transcriptional activation of the promoter for the ␣-glycoprotein hormone gene (23). The over-expression of the Id protein can sequester endogenous bHLH/HLHZ proteins and prevent their interaction with the E box. The transient co-transfection of the Id expression vector with the A Ϫ317 /G Ϫ30 FSHR/luciferase resulted in a dose-dependent reduction of luciferase activity from 70 to 55% as compared with transfection of the A Ϫ317 /G Ϫ30 FSHR/luciferase construct alone ( Fig. 6; p Ͻ 0.05).
Identification of USF in EMSA Complexes Using Mouse Antibody to USF-A sequence similar to the E box site in the rat FSHR promoter was shown in a previously published study to be optimal for the binding of the bHLHZ protein, USF (22). Polyclonal antibody that cross-reacts with mouse USF-1 and USF-2 was used in the EMSA with the radiolabeled A Ϫ317 /G Ϫ97 fragment of the FSHR promoter and nuclear proteins extracted from Sertoli cells (Fig. 7A, lane 1). The antibody against USF-1/2 blocked the formation of all complexes except band 7 and one of the bands in the doublet of band 6 (7A, lanes 2-4). Normal mouse serum had no effect on the complexes (Fig. 7A,  lane 5). Additionally, EMSA using antibodies against c-Myc or Max did not affect any of the complexes (data not shown).
Northern Blot Analysis of USF 1 and USF 2 in the Rat Testis-The mRNA transcript for USF-1 of approximately 2 kilobases in size was detected in RNA from adult rat testis and ovary, Sertoli cells from 20-day-old rats, and MSC-1 cells (Fig.  7B). The transcript for USF-1 appeared to be more abundant in Sertoli cells and MSC-1 cells than in adult rat testis and ovary. A slightly smaller USF-2 transcript of approximately 1.8 kilobases was also detected in all 4 of the tissues that were analyzed (Fig. 7B). The message for USF-2 appeared to be more abundant than that for USF-1 and was equally abundant in the

FIG. 3. DNase I footprinting assays and interactions at the E box and putative initiator region (InR) of the FSHR promoter.
Nuclear proteins from Sertoli cells (60 and 80 g, lanes 2 and 3, respectively, indicated by plus signs) were incubated with radiolabeled A Ϫ317 /G Ϫ30 FSHR promoter fragment. Control reactions did not contain nuclear proteins (lanes 1 and 4, indicated by minus signs).

Characterization of Protein Interactions at the Putative Initiator Region of the FSHR Promoter
Binding and Competition of the C Ϫ117 /G Ϫ30 FSHR Promoter Fragment-Using DNase I footprint analysis, we observed protein protection at the putative InR. The C Ϫ117 /G Ϫ30 FSHR promoter fragment apparently did not disrupt complex formation with the radiolabeled A Ϫ317 /G Ϫ30 FSHR promoter fragment when it was used as an unlabeled competitor (data not shown). Nuclear proteins from Sertoli cells were incubated with the radiolabeled C Ϫ117 /G Ϫ30 FSHR promoter fragment in the presence of excess unlabeled C Ϫ117 /G Ϫ30 FSHR promoter fragment or the nonspecific competitor DNA, BLT. Specific competition of all the bands occurred with the C Ϫ117 /G Ϫ30 FSHR promoter fragment (Fig. 8A, compare lanes 2-6 with  lane 1), while in contrast, the BLT DNA did not disrupt any of the bands at any concentration (Fig. 8A, compare lanes 7-11  with lane 1).
The A Ϫ101 /T Ϫ75 oligonucleotide that contained only the InR and the transcriptional start sites displaced all of the specific complexes (Fig. 8B, compare lanes 2-4 with lane 1). Furthermore, an oligonucleotide that corresponded to the sequence of the TdT InR disrupted the same complexes (Fig. 8B, compare  lanes 5-7 with lane 1).

Mutagenesis of the Putative Initiator Region and Promoter
beled EMut1 oligonucleotide (lanes 2-5). Lane 1 is with no competitor DNA, and lane 6 is probe only. D, nuclear proteins from the Sertoli cells were incubated with radiolabeled A Ϫ317 /G Ϫ30 FSHR promoter fragment in the presence of molar excess amounts of unlabeled EMut10 oligonucleotide (lanes 2-5). Lane 1 is with no competitor DNA, and lane 6 is probe only. Similar results were obtained from five separate experiments. Sequences of the wild-type and mutated oligonucleotides are shown below the EMSA, and the mutations in EMut1 and EMut 10 are underscored.

FIG. 4. Nuclear proteins from primary rat Sertoli cells bind to several regions in the A ؊317 /G ؊30 FSHR promoter fragment, including specific interactions at the consensus E box sequence.
A, nuclear proteins from Sertoli cells cultured from 20-day-old rat testes were incubated with radiolabeled A Ϫ317 /G Ϫ30 FSHR promoter fragment (lane 1) in the presence of a molar excess (-fold molar excess shown above) of unlabeled T Ϫ137 /C Ϫ110 wild-type E box oligonucleotide (Ϫ110 to Ϫ137; lanes [2][3][4][5]. Similar molar amounts of the 3Ј-untranslated region of bovine interleukin-2 (BLT) DNA were used as a nonspecific competitor (lanes 6 -9). Lane 10 is probe only. B, nuclear proteins from similar Sertoli cell preparations were incubated with radiolabeled A Ϫ317 /G Ϫ30 FSHR promoter fragment in the presence of a molar excess of an unlabeled FSHR promoter fragment that did not include the E box or initiator sequences (nucleotides Ϫ124 to Ϫ317; lanes 2-5). Lane 1 is with no competitor DNA, and lane 6 is probe only. C, nuclear proteins from the Sertoli cells incubated with radiolabeled A Ϫ317 /G Ϫ30 FSHR promoter fragment in the presence of molar excess amounts of unla- Activity in Transient Transfections-Mutations were made in three nucleotides that are conserved in the InR among the rat, mouse, and human FSHR promoters. The mutated constructs were introduced into MSC-1 cells by transient transfection, and the luciferase activities were expressed as a percentage of the activity of the pϪ383/Ϫ30 FSHR/luciferase construct. The mutations in both the (A87G) pϪ383/Ϫ30 and (C90A) pϪ383/Ϫ30 FSHR/luciferase constructs resulted in a reduction in luciferase activity of 40 and 65%, respectively, compared with the pϪ383/Ϫ30 FSHR/luciferase construct ( Fig. 9; p Ͻ 0.05). Similarly, transcriptional activities of the (C84A) pϪ383/Ϫ30 and (C84T) pϪ383/Ϫ30 were 30 and 35% less, respectively, than the pϪ383/Ϫ30 FSHR/luciferase construct ( Fig. 9; p Ͻ 0.05).

DISCUSSION
These data comprise the first detailed analysis of DNA elements in the FSHR promoter that influence the expression of the FSHR gene. The promoter of the FSHR gene is in a class of promoters that lack a canonical TATA box. These TATA-less promoters were originally thought to be similar to housekeeping gene promoters that are constitutively and ubiquitously expressed, have multiple start sites spread over a large region, and are GC-rich (24). A second class of TATA-less promoters, which more closely resembles the FSHR promoter, has been identified that includes promoters that are not GC-rich, are regulated during differentiation or development, and initiate transcription at one or only a few tightly clustered transcriptional start sites that are contained within an initiator region (9,12).
The sequence of the InR in the FSHR promoter is distinct from that of other InRs. For example, there is little sequence similarity of the FSHR InR when it is compared with the InR in the promoter of the TdT gene (12). However, the InR of the FSHR promoter has a high degree of similarity with InR of the promoter for the CD2 gene (Fig. 10). CD2 is a glycoprotein expressed in thymocytes and T lymphocytes. The promoter of the CD2 gene contains an E box that interacts with USF and an InR, both of which are required for full transcriptional activation (25). The consensus E box sequence of the CD2 promoter is identical to that seen in the FSHR promoter. The promoters for the FSHR and CD2 genes may be members of a subclass of TATA-less promoters that use an E box and a unique consensus InR for the activation of cell-specific transcription.
The pϪ383/Ϫ1 FSHR/luciferase had the highest activity of the promoter constructs examined. The nucleotides Ϫ30 to Ϫ1, which are downstream from the transcriptional start sites and thus are part of the 5Ј-UTR, have a small positive influence on the full transcriptional activation of the promoter. Several Drosophila genes and the rat LHR gene were shown to require a region downstream from the transcriptional start site for maximum transcriptional activity (26,27). The 5Ј-UTR had a significant affect on FSHR promoter activity in the context of the longer promoter (pϪ383/Ϫ1 FSHR/luciferase) but not the construct that contained the short promoter fragment (Ϫ143/Ϫ1 FSHR/Luc). Thus, it is possible that there were changes in conformation of the longer promoter fragment that influenced promoter activity.
The conclusions from the deletion analyses of the promoter were supported by the protection observed in the DNase I footprint analysis. The entire promoter region including both the E box and putative InR elements were protected by what appeared to be a multi-protein complex. Generally, regions upstream from the E box were not protected. In contrast, a previously published report showed binding of the inducible cAMP early repressor to this site in the rat FSHR promoter (28, 29, 30); we could not demonstrate direct protein interactions with the consensus AP-1 or CRE-like site and rat Sertoli nuclear proteins.
Most of the protein-DNA complexes that interacted with the A Ϫ317 /G Ϫ30 FSHR promoter fragment arose from interactions at the E box. Band 6 probably is a result of at least 2 complexes that co-migrate, one of which interacts at the E box and the other at the InR. Having defined the protein interactions at the E box, we concluded that the reduction in promoter activity in the transient transfections using the mutated E box constructs was most likely due to a loss of protein interaction caused by the E box mutations. Taking the EMSA and transfection data together, the E box is necessary for full transcriptional activa-tion of the FSHR promoter.
Id is a dominant negative regulator of the bHLH/HLHZ proteins as it lacks the basic region for DNA binding but has the helix-loop-helix domain for dimerization. Thus, it can dimerize with other bHLH/HLHZ proteins and prevent their binding to the E box consensus site (31). For example, it was demonstrated that over-expression of Id protein with the promoter sequences of the gene for the ␣-glycoprotein hormone subunit, which contains two E box consensus elements, caused a reduction in promoter activity in transient transfections (23). In addition, it was concluded that the decrease in promoter activity was most likely due to the sequestration of the endogenous USF by Id, which prevented its binding to the E box consensus site. In the present study, the inhibitory effect of Id probably resulted from a similar interaction with a bHLH/ HLHZ protein such as USF.
Few bHLH/HLHZ proteins such as USF have been well characterized in the testis, but the expression of USF 1 and USF 2 has been shown previously only in whole adult mouse testis (20,(32)(33)(34)(35). The ability of USF to form homo-and heterodimers, its involvement in cell-specific gene expression, and its known ability to interact with InR-binding proteins suggested that it was a component of the complexes in the EMSA of the present study (18,23,25,36). It has been shown that USF can trans-activate promoters via the InR and that protein interactions at the E box and InR can result in a cooperative effect on transcriptional initiation (18,25,37). The mechanism for the interaction of the E box and InR is unknown, but it may also involve TFII-I, a protein that has been shown to interact cooperatively with the E box and the InR (18).
The data presented in the study represent one of the first FIG. 8. Nuclear proteins from rat Sertoli cells interact with the InR in the C ؊117 /G ؊30 FSHR promoter fragment and the InR of the TdT. A, nuclear proteins from Sertoli cells purified from 20-day-old rat testes were incubated with radiolabeled C Ϫ117 /G Ϫ30 FSHR promoter fragment (lane 1) in the presence of molar excess amounts of unlabeled C Ϫ117 /G Ϫ30 FSHR promoter fragment (lanes 2-6). Similar amounts of the 3Ј-untranslated region of bovine Interleukin-2 (BLT) DNA were used as a nonspecific competitor (lanes 7-11). The -fold molar excess is shown above the lanes. Lanes 6 and 12 are probe only. B, nuclear proteins from Sertoli cells were incubated with radiolabeled C Ϫ117 /G Ϫ30 FSHR promoter fragment (lane 1) in the presence of molar excess amounts of an unlabeled A Ϫ101 /T Ϫ75 oligonucleotide that corresponded to the InR of the FSHR promoter (Ϫ101 to Ϫ75; lanes [2][3][4] or in the presence of a molar excess amount of an unlabeled oligonucleotide that corresponded to the sequence of the TdT InR (12). The -fold molar excess is shown above the lanes. Similar results were obtained from two separate experiments. efforts in understanding the molecular basis for the regulation of the expression of the rat FSHR gene. The benefits of understanding the transcriptional regulation of the FSHR promoter are both practical and conceptual. 1) The FSHR promoter could be used to target the expression of other genes to the testis in transgenic studies, 2) it would contribute to our basic knowledge of how a promoter without a TATA box initiates transcription, and 3) it defines a sub-class of TATA-less promoters that include a consensus E box and a distinct InR sequence.