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Regular Article |
a Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7
b Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, Quebec, Canada H3A 1A3
c Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada H3T 1J4
ABSTRACT
Expression and activation of follicle-stimulating hormone receptor (FSHR) in the granulosa and Sertoli cells are required for normal development of the ovarian follicles and germ cells. However, little is known regarding the mechanisms by which FSHR expression is regulated. We fused an ovine FSHR promoter to a luciferase gene to understand the promoter regulation in two gonadal cell lines. Deletion studies revealed that the strongest promoter was at -200 to +163 relative to the transcription start site. One of cis-elements protected from DNase I digestion was mapped to between +32 and +54 of the 174-base pair (bp) minimal promoter. Electrophoretic mobility shift assay with a 26-bp probe (+32 to +57) and nuclear extracts from Sertoli (15P1) and granulosa (JC-410) cell lines demonstrated a sequence-specific DNA-protein complex. Southwestern analysis detected a 43-kDa protein bound to the 26-bp probe. Gel supershift with upstream stimulatory factor 1 and 2 (USF-1/2) antibodies revealed that the DNA-protein complex contained these two transcription factors. Mutation within the E-box of the promoter abolished the sequence-specific binding and the minimal promoter activity but also greatly reduced the transcription of the proximal promoters by 49%–70%. These data suggest that the USF-1/2 binding to the promoter is required for the expression of the ovine FSHR in the gonadal cells.
FSH receptor, gene regulation, granulosa cells, Sertoli cells
INTRODUCTION
Follicle-stimulating hormone (FSH) released from the pituitary plays an important role in mammalian reproduction by interacting with its specific receptor in target cells of the ovary and testis [1, 2]. On FSH binding, the FSH receptor (FSHR) was initially thought to couple exclusively with the Gs protein, stimulating the enzyme adenylyl cyclase or other pathways to initiate a cascade of intracellular events leading to the specific biological effects of the gonadotropin [1]. This simplified view now appears to be more complex following the cloning of several alternatively spliced transcripts from the sheep testis [3, 4] and the functional studies of the corresponding proteins [5, 6]. Some of the alternatively spliced transcripts coding for proteins with a novel structural motif of the growth factor type I receptor mediate FSH action through the activation of calcium signaling pathways distinct from the Gs-coupled receptor [5]. Our recent homologous recombination study describing the knockout of the FSHR gene, eliminating all forms of the receptor, has confirmed their critical role in gonadal development and function [7].
Expression of the FSHR appears to undergo a cyclic change, indicating possible transcriptional regulation by hormones and growth factors by mechanisms that are not yet fully defined [8–10]. The promoter regions of the FSHR have been cloned from a number of species, including human, rat, mouse, and sheep [11–14]. By RNase protection or primer extension analysis, a major transcription start site has been localized at -99 in the human and -534 in the mouse relative to the translation start site [11, 14]. In the rat, two major transcription start sites, at positions -80 and -98, have been found [12]. In these three species, additional minor transcription initiation sites have also been observed [11, 12, 14]. However, a unique transcription start site in the ovine FSHR has been mapped at -163 relative to the translation initiation codon [13]. Sequence comparison of the putative promoter regions in these four species has revealed some diversity in the regions upstream of the transcription start site relative to the ovine gene [11–14]. The 5'-flanking regions of the FSHR from the human, rat, and sheep lack typical TATA or CCAAT boxes [11–13], whereas a TATA sequence element is present at 10 nucleotides upstream of the major transcription initiation site of the mouse FSHR promoter [14]. These promoters are of particular interest, because they could contain cell-specific regulatory elements that may be used to direct expression of transgenes with which to target the tissues for gene therapies. However, the precise mechanisms by which the FSHR promoter is regulated are not well known, largely because of a lack of extensive studies in different species.
We have cloned an 8-kilobase (kb) genomic EcoRI DNA fragment containing ovine FSHR 5'-flanking region and the first exon from a sheep genomic library [13]. Previous studies have demonstrated that this promoter contains several interesting regulatory elements, including those that may be specific for the germ cells [13]. In the present study, we have used the ovine FSHR gene and two gonadal cell lines as a model system to investigate the regulatory elements of the promoter. An involvement of two ubiquitous transcription factors, upstream stimulatory factors 1 and 2 (USF-1/2), in mediating promoter activity of the ovine FSHR in the gonadal cell lines was studied by progressive deletions and biochemical assays.
MATERIALS AND METHODS
Construction of Promoter/Luciferase Plasmids
Promoterless plasmid pGL3-basic (Promega, Madison, WI) was used for the preparation of the luciferase fusion reporter constructs. Various truncated ovine FSHR promoters were generated by polymerase chain reaction (PCR) from an 8-kb genomic EcoRI DNA fragment cloned from a sheep genomic library [13]. Seven promoter fragments were amplified, and the sequences of each pair of primers used for PCR are shown in Table 1. All the forward primers were designed with a sequence (5'-CGTAAGATCT) for a BglII site at their 5' ends, whereas the reverse primer was synthesized with a linker (5'-GCATAAGCTT) providing a HindIII site at their 3' ends. The PCR products were digested with BglII and HindIII and then inserted into the pGL3-basic vector at their corresponding sites in front of the luciferase gene. The DNA sequence of each of the promoter fragments was confirmed by sequencing before transfection.
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Site-Directed Mutagenesis
Mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). In brief, a double-stranded plasmid DNA with a wild-type FSHR promoter was used as a template. A pair of synthetic oligonucleotides with a directed mutation was extended by means of Pfuturbo DNA polymerase (Stratagene). The extension product was treated with 10 U of DpnI endonuclease at 37°C for 1 h to digest the parental double-stranded DNA template. The nicked-vector DNA incorporating a mutation was then transformed into Escherichia coli XL1-Blue Supercompetent Cells (Stratagene). Individual clones were isolated, and the DNA was then purified and sequenced to confirm which clones had the correct mutational changes.
DNA Sequence Analysis
The DNA sequence analysis was performed according to the manufacturer's instructions (Amersham Pharmacia Biotech, Piscataway, NJ) using the dideoxy chain-termination method with 
-32P-deoxycytidine triphosphate. The reaction products were resolved on a 7% polyacrylamide denaturing gel. The gel was dried before autoradiography at room temperature with Kodak X-OMAT AR 5 film (Rochester, NY).
Cell Lines and Cell Culture
A mouse Sertoli cell line (15P1) established from testicular cells of adult transgenic mice that express the large T antigen of polyoma virus was kindly provided by Dr. Francois Cuzin (University of Nice, Nice Cedex 2, France) [15]. A stable and spontaneously immortalized porcine ovarian granulosa cell line (JC-410) was a generous gift from Dr. Jorge P. Chedrese (University of Saskatchewan, Saskatoon, Canada) [16]. These cell lines do not appear to express endogenous FSHR, because they fail to respond to FSH stimulation [15, 16]. The cells were routinely maintained in a humidified, 37°C (JC-410) or 32°C (15P1) incubator with 5% CO2 and cultured in F-12/Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin.
Transfections and Luciferase Assay
The 15P1 or JC-410 cells were seeded into 60-mm tissue culture plates (5 x 105 cells/plate) in F12/DMEM medium plus 10% fetal bovine serum. Transient transfections were carried out with 8 µg of FSHR promoter/luciferase reporter and 1 µg of a pCMVlacZ for 18 h using calcium phosphate coprecipitation [17]. The following day, fresh medium was added for an additional 48 h before harvesting. Appropriate aliquots of cell extract were used for both luciferase and ß-galactosidase assays, which were performed as recommended by the manufacturer (Tropix, Bedford, MA). To normalize the transfection efficiency, the light units from the luciferase constructs were divided by the light readings from the ß-galactosidase internal controls.
Preparation of Nuclear Extract
Nuclear extracts were prepared as described previously [18]. Both 15P1 and JC-410 cells were lysed on ice for 5 min in buffer E (0.3% NP-40, 10 mM Tris-HCl [pH 8.0], 60 mM NaCl, 1 mM EDTA, 1 mM 1,4-dithiothreitol [DTT], and 1 mM phenylmethylsulfonylfluoride [PMSF]). Nuclei were pelleted by spinning for 5 min at 2500 rpm at 4°C. The pellet was washed in buffer E lacking NP-40 and resuspended in buffer C (20 mM Hepes [pH 7.9], 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 2 mM EGTA, 2 mM DTT, 20% glycerol, and 1 mM PMSF). Next, NaCl was added to a final concentration of 0.4 M, and the nuclei were gently shaken for 20 min at 4°C, then spun for 10 min at 12 000 rpm at 4°C. Nuclear extracts were stored at -80°C.
Electrophoretic Mobility Shift Assay
The oligonucleotides footprinting 1 (FP1) and mutated FP1 (FP1mt) were synthesized by a local supplier:
FP1: 5'-GGGCGGGTCACGTGACCCTACCAGCT
3'-CCCGCCCAGTGCACTGGGATGGTCGA
[rpFP1mt:[sp[fy21,1] 5'-GGGCGGGTCaaTTGACCCTACCAGCT
3'-CCCGCCCAGttAACTGGGATGGTCGA
The electrophoretic mobility shift assays (EMSAs) were performed as described previously [17]. Briefly, double-stranded oligonucleotides were labeled at the 5' ends using T4 polynucleotide kinase and 
-32P-ATP. Nuclear extracts (5 µg) from 15P1 and JC-410 cells or purified recombinant USF-1 protein (10 ng; a gift of Drs. D. Steger and J. Workman, Pennsylvania State University, University Park, PA) [17, 19] were incubated in a binding buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 3 mM DTT, 10% glycerol, 0.05% NP-40, 0.1 mM ZnCl2, 50 µg/ml poly(dI-dC), and 20 fmol of labeled DNA probe. After addition of the radiolabeled probe, the mixture was incubated at room temperature for another 15 min. Excess unlabeled DNA competitors were added 5 min before adding the radiolabeled DNA probe. The reaction mixture was analyzed using a 5% nondenaturing polyacrylamide gel in 1x TBE buffer (50 mM Tris-borate-EDTA, pH 8.0). For the gel supershift assay, 1 µg of USF antibody or preimmune control immunoglobulin (Ig) G (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the reaction mixture and incubated at room temperature for an additional 15 min. Gels were then dried and visualized by autoradiography.
Southwestern Analysis
The Southwestern analyses were performed as described previously [20]. Briefly, 120 µg of nuclear extracts were loaded on to a SDS-PAGE denaturing gel, and electrophoresis was carried out at 15 mA for 5 h. Proteins were then transferred to a nitrocellulose membrane and incubated at room temperature for 60 min in a buffer containing 5% dry skim milk, 25 mM NaCl, 5 mM MgCl2, 25 mM Hepes (pH 7.9), and 0.5 mM DTT. The binding reaction was performed in the same buffer with 106 cpm/ml of 32P-labeled, double-stranded DNA probe in the presence of 5 µg/ml of salmon sperm DNA at room temperature for 18 h. Before autoradiography, blots were washed four times at room temperature utilizing the same buffer.
DNase I Footprinting
Coding strand probes were prepared by linearizing proper plasmids (P174, p363, and P363mt) with HindIII. Noncoding strand probes were prepared by cutting the same plasmids with BglII and 32P-5' end labeled with T4 polynucleotide kinase. Labeled DNA was run through a ProbeQuant G-50 column (Amersham Pharmacia Biotech) to remove free nucleotides. The coding and noncoding probes were released from the plasmids by digesting with BglII and HindIII, respectively, and were purified by 1% agarose gel electrophoresis. The DNase I footprinting reactions were carried out as described previously [21]. Binding reactions containing 25 mM HEPES (pH 7.6), 5 mM MgCl2, 84 mM KCl, 5% glycerol, 1 mM DTT, 1 µg of poly(dI-dC), 20 µg of nuclear extract or 40 ng of purified USF-1, and 1 µl of labeled template DNA (
20 000 cpm) were incubated at temperature for 15 min, followed by 5 min of DNase I digestion on ice. The footprint reactions were terminated by the addition of 80 µl of a stop buffer (20 mM Tris-HCl [pH 8.0], 250 mM NaCl, 20 mM EDTA, 0.5% SDS, 50 µg/ml of salmon sperm DNA, and 250 µg/ml of proteinase K). The samples were incubated at 45°C for 1 h and then extracted twice with phenol/chloroform. Finally, after ethanol precipitation, the samples were loaded on a 7% sequencing gel along with A, C, G, and T DNA sequence tracks, followed by autoradiography.
RESULTS
Deletion Analysis of the Ovine FSHR Promoters
To map regulatory elements and to analyze the truncated promoter function, portions of the ovine FSHR promoter were amplified and cloned into the promoterless pGL3-basic vector (Fig. 1). Promoter activity was analyzed in transiently transfected 15P1 and JC-410 cells (Fig. 1). All constructs were able to transcribe a luciferase reporter gene in JC-410 cells, whereas five of seven (i.e., P1268, P807, P468, P363, and P174) were active in 15P1 cells. Generally, the luciferase activity of the same construct was 1.6-to 14.0-fold higher in JC-410 cells than in 15P1 cells (Fig. 1). The longest construct P1268, containing nucleotides from -1105 to +163 relative to the transcription start site, showed 25-fold higher activity in JC-410 cells and 7-fold higher activity in 15P1 cells compared to the pGL3-basic vector (Fig. 1). Deletion from -1105 to -644 (P807) resulted in a reduction of promoter activity by 24% in JC-410 cells but an increase by 70% in 15P1 cells (Fig. 1). Further deletion of 286 base pairs (bp) from the 5'-flanking end (P519) did not alter the level of expression in JC-410 cells but reduced its expression to near basal level of the promoterless vector in 15P1 cells (Fig. 1). Further stepwise deletions from -356 to -305, or -200, greatly increased in the luciferase activity in both cell lines, suggesting the possible presence of inhibitory sequences in these two regions (Fig. 1). The construct of P363 containing the fragment from -200 to +163 conferred the strongest promoter activities, which were 138-fold higher in JC-410 cells and 42-fold higher in 15P1 cells than in pGL3-basic vector level (Fig. 1). Additional removal of 94 bp from the 5' end of P363 (P269) reduced the luciferase expression by 84% in JC-410 cells and by 96% in 15P1 cells, suggesting the possible presence of strong enhancer(s) in this region. Interestingly, a construct only containing nucleotides from -11 to +163 (P174) led to 32-fold higher activity in JC-410 cells and fivefold higher activity in 15P1 cells than in the pGL3-basic vector (Fig. 1).
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Identification of the Regulatory Element and Its Binding Protein
Promoter deletion analysis has localized several positive and negative regulatory regions (Fig. 1). To map the cis-elements within the ovine FSHR promoter, we performed a DNase I footprinting assay using P519, P363, and P174 plasmid as templates (Fig. 1). At least five cis-elements within a 519-bp length of FSHR promoter are recognized by the nuclear proteins and are protected from DNase I digestion (Fig. 2). The FP1 was mapped from nucleotides +32 to +54 of the 174-bp minimal promoter (Fig. 2, A and B). It may be noted that sites which are hypersensitive to DNase I digestion can also be seen above the footprinting regions.
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To identify transcription factors that bind to the FP1 region, we synthesized oligonucleotides that encompass sequences from +32 to +57 of the ovine FSHR promoter (see Materials and Methods). The FP1 sequence overlapped a GC (GGGCGGG) box, a half-hormone response element (5'-TGACC), and a consensus E-box (5'-GGTCACGTGACC) as potential transcription factor-binding sites. Using this double-stranded DNA probe, our EMSAs detected a single retarded protein-DNA complex with the nuclear extracts from both 15P1 and JC-410 cells (Fig. 3). This binding appeared to be sequence specific, because it was competed by the unlabeled FP1 probes in a concentration-dependent manner, but not by an unrelated DNA sequence, Sp-1, Egr-1, and consensus estrogen-responsive elements (Fig. 3).
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To characterize the nuclear protein(s) that bind the FP1 probe, we performed a Southwestern blot analysis. The FP1 probe specifically recognized a protein with an apparent molecular weight of 43 kDa in nuclear extracts from both 15P1 and JC-410 cells (Fig. 4). To test whether USF-1 and USF-2, whose molecular weights are approximately 43 and 44 kDa, respectively, among E-box-binding proteins bind to that region, we turned to antibody supershift assay with anti-USF-1 and anti-USF-2 antibodies. The DNA-nuclear protein complexes were supershifted in the presence of either USF-1 or USF-2 antibodies but were unaffected by control rabbit IgG (Fig. 5). This would be consistent with a USF-1/2 heterodimer at the promoter, because both antibodies shifted the same DNA-protein complex. Further experiments confirmed that the interactions include USF-1 and USF-2 by demonstrating that the retarded band with purified USF-1 displayed a migration similar to that of the DNA-protein complex as in 15P1 and JC-410 nuclear extracts (Fig. 5). Moreover, USF-1-DNA complex was supershifted by USF-1 antibody in the same way as the DNA complex with the nuclear proteins from 15P1 and JC-410 cells (Fig. 5).
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The protein-DNA complex detected in EMSAs may contain one or more classes of proteins bound to the same region of promoter context, as predicted by computer-based sequence analysis. To rule out the participation of other transcription factors in the complex, we compared purified USF-1 protein with the nuclear extracts from 15P1 and JC-410 cells by DNase I footprinting assays (Fig. 6). This experiment is also more precise in mapping the protein-DNA binding with a single nucleotide resolution. The DNase I footprinting with a purified USF-1 protein demonstrated the same protected region within the FSHR promoter as in the nuclear extracts from either 15P1 cells or JC-410 cells (Fig. 6). These data are in agreement with the results of EMSA and supershift assays, suggesting that USF-1 and USF-2 are the predominant factors bound to the E-box region of the promoter.
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E-Box Mutation Reduces USF Binding and Promoter Activities
Previous studies have shown that in the E-box enhancer sequence, the central nucleotides of CG are the most important for USF binding [17]. To understand the contributions of the E-box to the promoter activity of the ovine FSHR, we mutated the central CG to AT (leaving the E-box consensus sequence, CAatTG) and used the FP1mt to test the USF binding. The EMSA showed that the FP1mt probe was not bound by the nuclear proteins from either 15P1 or JC-410 cells (Fig. 7). In addition, the mutated probe failed to compete for the binding to wild-type probe (FP1), even at 200-fold molar excess (Fig. 7). To further test whether the E-box mutation within the promoter region would completely abolish the protein binding, we incorporated the same mutations as the FP1 into a FSHR promoter/luciferase constructs. Our DNase I footprinting assay in vitro failed to detect any protein binding to the mutated E-box sequence, because the promoter template was digested in the presence of nuclear extracts or USF-1 protein (Fig. 8). In contrast, the nuclear extract from JC-410 cells as well as the purified USF-1 effectively bound to the corresponding region of the wild-type promoter, therefore protecting it from DNase I digestion (Fig. 8).
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To test the contribution of USF binding to the promoter's function, we made three constructs containing the same mutated E-box within a 807-, 363-, and 174-bp length of FSHR promoters (Fig. 1). We transiently transfected those reporter genes into 15P1 and JC-410 cells to analyze the luciferase activities (Fig. 9). In agreement with the USF-binding experiments shown in Figures 5 and 8, luciferase assays demonstrated that the E-box mutation completely abolished the promoter activities in P174mt transfected cells (Fig. 9A). Interestingly, the same mutation within the 363-bp length of promoter from -200 to +163 had a moderating effect in reducing the promoter activities by 49% in JC-410 cells and 63% in 15P1 cells (Fig. 9B). Similar results were found for the P807mt construct (Fig. 9C), in which luciferase expression was diminished by approximately 70% in both cell lines. Finally, whereas the expression of luciferase from the mutated promoters (P363mt and P807mt) was significantly less than that seen with the wild types, the mutated promoters still represented significant induction over the basal level of promoterless vector (Fig. 9, B and C). In contrast, the mutated minimal promoter (P174mt) was actually below the level of the vector control (Fig. 9A). These elevations of P363mt and P807mt constructs are attributable to the presence of regulatory elements upstream of the transcription start site. Because the identity of the upstream factors is still under investigation, it is not yet possible to provide data on the interaction between USF and other factors. However, the notable reduction in the promoter activity of the constructs P363mt and P807mt could highlight the importance of the USF-E-box interaction in the activation of ovine FSHR promoter.
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DISCUSSION
In the male and female, selective FSHR expression in the gonads seems to be stage dependent, which coincides with the different maturational status of the germ cells or follicles [22–24]. This expression pattern of the FSHR appears to be regulated by many paracrine/autocrine factors and gonadotropic hormones by either affecting mRNA stability or modulating the regulatory elements in the promoter region [25–30]. Therefore, identification of the cis-elements within the 5'-flanking region of the FSHR gene is important, because this inducible, tissue-specific promoter could direct a transgene in the ovary and the testis. In our studies, we used the ovine FSHR promoter/luciferase gene as a model system to examine the promoter regulation because of the extensive information available regarding the promoter sequence and the interesting regulatory elements described previously [13]. We made a series of promoter deletions and assayed their activities in 15P1 and JC-410 cells (Fig. 1). Our transfection studies revealed that the promoter constructs were more active in JC-410 cells than in 15P1 cells after normalizing the transfection efficiency (Fig. 1). The expression profiles of the promoter constructs were generally similar in the two cell lines, except for P807, which gave rise to an opposite result after a deletion from -1105 to -644 (Fig. 1). However, deletion and transfection studies suggest that at least two positive (-200 to -107 and -11 to 163) and three negative regulatory regions (-356 to -306, -305 to -199, and -106 to -10) have been roughly localized within the 519-bp length of promoter (Fig. 1). Our analyses demonstrated that the minimal promoter is from -11 to +163 relative to the transcription start site, which confers 23% and 12% of the strongest promoter activity in JC-410 and 15P1 cells, respectively (Fig. 1, P174). The strongest promoter identified in the present study is located from -200 to +163, which should contain both negative and positive cis-elements (Fig. 1, P363). Further DNase I footprinting studies with the 174-bp minimal promoter revealed a positive regulatory element of 26-bp DNA from +32 to +54 (Fig. 2, A and B). Subsequent biochemical experiments with a probe and nuclear extracts demonstrated that the binding proteins correspond to two transcription factors, USF-1 and USF-2 (Figs. 3–6). A site-directed mutation within the E-box of the promoter prevented the sequence-specific binding as detected by gel shift and DNase I footprinting assays (Figs. 7 and 8). Incorporation of the same mutant E-box into the wild-type promoter/luciferase reporters completely blocked the minimal promoter activity (Fig. 9A) but greatly diminished the transcriptions of the proximal promoters by 49%–70% (Fig. 9, B and C). These experiments suggest that one of the regulatory elements, an E-box downstream of the transcription start site, is required for the FSHR expression.
Direct studies of the ovine FSHR promoter regulation in a homologous system would be ideal but, currently, are difficult, requiring the use of primary cultures from the ovary and testis. The alternative of using immortalized granulosa or Sertoli cell lines from the same species is also precluded, because these are not yet available. However, that the rat FSHR promoter was tissue-specifically active in a transgenic mouse [8] and the human FSHR promoter displayed its activities in the same way in the rat Sertoli cell line as in primary human granulosa cells [11] suggests that target cells from heterologous species can be used for dissecting the promoter. Moreover, rat FSHR promoter was equally active in a mouse Sertoli cell line (MSC-1) and primary rat Sertoli cells [31, 32]. These studies indicate that the transcription factors that are important for the FSHR expression are well conserved among the mammalian species. Based on these considerations, we utilized the 15P1 and JC-410 cell lines that have been recently established and characterized [15, 16] as a first step and model system to localize the regulatory elements for the ovine FSHR promoter and to identify the potential binding proteins. These two cell lines exhibit many features that are characteristic of granulosa and Sertoli cells [15, 16, 33, 34], including transcription of a detectable mRNA [35], suggesting that the endogenous promoters, though very weak, are nevertheless active. By transfecting the nonchromatin promoters into these cells, avoiding repression by the chromatin structure and DNA methylation, we have demonstrated here that ovine FSHR promoters are active in both cell lines. The great difference of luciferase activity from the same construct in the two cell lines (Fig. 1) could be cell-line dependent, because they were derived from different sexes as well as from different species. The 15P1 cells came from the testis of the adult male mouse, whereas the JC-410 cells were derived from the pig ovary. Therefore, further studies need to be carried out in primary cell culture from sheep. Notwithstanding these caveats, our experiments suggest that the JC-410 cell line would be a better in vitro granulosa cell model for the transcriptional studies of FSHR gene and, perhaps, of other genes as well.
The consensus E-box in the FSHR promoter is located 110 bp upstream of the translation start site [13]. In fact, the similar sequence of an E-box overlapped with a GC box and a half-estrogen responsive element is conserved in the comparable regions of the mouse, rat, and human FSHR promoters [13]. This suggests that transcription factors binding to this region play an important role in FSHR gene expression. Our experiments have demonstrated that major E-box-binding components contain USF-1/2 transcription factors rather than other transcription factors, such as Myo D, c-myc, and USF-like binding proteins, that are predicted to recognize this sequence [31, 32, 36–39]. In fact, the portion of promoter from -15 to +163 of the ovine FSHR gene is highly homologous to those of the other species, and some regions were thought to be important for rat FSHR transcription [13, 32]. However, our in vitro DNase I footprinting assays detected a unique protein-DNA interaction within the 174 bp of the minimal promoter (Figs 2, 6, and 8). This technique enabled us to identify the proteins that actually interact with a promoter from potential binding sites predicted through computer-based analysis or simple deletion studies [11, 13, 14, 32]. Indeed, our experiments suggest that the presence of a number of potential regulatory elements does not necessarily mean that their proteins potentially bind to them. Therefore, the sequence adjacent to the E-box may function through positioning an E-box at a proper space in the architecture of promoters and determine which factor activates via this site, because block replacements of the flanking sequences significantly affected the USF function in the rat FSHR model system [32].
The mechanisms of tissue-specific gene expression include the interaction of regulatory factors, DNA methylation, chromatin structure and remodeling, modification of chromatin assembly proteins, and mRNA or protein turnover. Previous studies have shown that a 5-kb rat FSHR promoter could direct a tissue-specific expression of a reporter gene in vivo [8]. However, a series of constructs containing a portion of promoter from 280 bp to 5 kb fused to a reporter gene failed to demonstrate Sertoli cell-specific expression in transiently transfected cell lines [32]. Moreover, human FSHR promoter is active in the Chinese hamster ovary cell line, and rat promoter activity could be detected in the Leydig cell line [8, 11, 32]. Recent studies have shown that methylation within the regulatory region of the promoters represses the endogenous gene expression in nongonadal cells of the transgenic mice and the mouse Sertoli cell line (MSC-1) [8]. These studies suggest that chromatin architecture and DNA methylation might play an important role in the cell-specific regulation of FSHR in vivo and the gene silencing in vitro. Previous studies have demonstrated that USFs not only participate in remodeling chromatin to allow tissue-specific transcription factors to establish a preinitiation complex but also are directly involved in recruitment of the general transcription machinery to the promoter [31, 40–44]. Therefore, studies on the interactions of USFs with their upstream regulators on the chromatin template of the ovine FSHR and their contributions to the cell-specific expression as well as initiation would be of interest for future investigations.
ACKNOWLEDGMENTS
We are grateful to Drs. P.J. Chedrese (University of Sasketchwan, Saskatoon, Canada) and F. Cuzin (University of Nice, Nice, France) for providing the cell lines used in this study. The secretarial assistance of Odile Royer is greatly appreciated.
FOOTNOTES
1 Supported by grants from the Canadian Institutes of Health Research (CIHR). W.X. is the holder of a doctoral scholarship award from the CIHR. 
2 Correspondence: M. Ram Sairam, Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7. FAX: 514 987 5585; sairamm{at}ircm.qc.ca
Accepted: September 11, 2000.
Received: June 5, 2000.
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