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Biology of Reproduction 65, 1142-1149 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Role of CACC-Box in the Regulation of Ovine Follicle-Stimulating Hormone Receptor Expression1

Weirong Xinga,b, and M. Ram Sairam2,a,b,c

a Molecular Reproduction Research Laboratory, Clinical Research Institute of Montréal, Montréal, Québec, Canada H2W 1R7 b Division of Experimental Medicine, Department of Medicine, McGill University, Montréal, Québec, Canada H3A 1A3 c Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3T 1J4

ABSTRACT

Tissue-specific and stage-specific expression of follicle-stimulating hormone receptor (FSH-R) in granulosa and Sertoli cells is required for normal development of ovarian follicles and germ cells. However, little is known of the transcription factors that regulate the FSH-R gene and its promoter. Using an ovine FSH-R promoter as a model system, we have identified a second DNase I footprinting 2 (FP2) region from -46 to -67 of the strongest ovine FSH-R promoter (-200 to +163) relative to the transcription start site. Electrophoretic mobility shift assay with a 22-base pair DNA probe (-46 to -67) and nuclear extracts from Sertoli (15P1) and granulosa (JC-410) cell lines demonstrated a sequence-specific DNA-protein complex. Further Southwestern and UV cross-linking analyses detected three predominant proteins of molecular weights 87, 60, and 50 kDa present in both Sertoli and granulosa cells bound to a 32P-labeled DNA probe as a complex. Gel competition experiments with DNA probes containing known Krupple-like factor binding sites revealed that the testis-specific zinc finger protein, ZNF202-like factor, Ras-responsive element binding protein-like factor, or both, may be among the potential candidate regulators. Mutation within the CACC box of the promoter abolished Krupple-like factor binding and significantly diminished promoter activity in both gonadal cells. These data suggest that Krupple-like transcription factors may play a role in the regulation of ovine FSH-R expression.

follicle-stimulating hormone, follicle-stimulating hormone receptor, gene regulation, granulosa cells, Sertoli cells

INTRODUCTION

Follicle-stimulating hormone plays an important role in mammalian reproduction by interacting with its specific receptor in ovarian granulosa cells and testicular Sertoli cells [1–3]. Upon FSH binding, the FSH receptor (FSH-R) is believed to couple with the Gs protein, stimulating adenylyl cyclase or other pathways to initiate a cascade of intracellular events, leading to specific biological effects of the gonadotropin [1, 4, 5]. Recent gene disruption studies of either the FSHß subunit or FSH-receptors have not only reinforced their critical role in spermatogenesis and folliculogenesis but also assisted in understanding their indirect effects in controlling lipid metabolism, bone remodeling, and tumorigenesis [3, 6–10].

The expression of FSH-R seems to be stage-specific during follicular maturation and the spermatogenic cycle, suggesting a possible transcriptional regulation by mechanisms that have not been fully defined [11–16]. The promoter regions of FSH-R have been cloned from a number of species, including human, rat, mouse, and sheep [17–20]. Sequence comparison of the putative promoter regions in four species has shown that they have the same characteristics as many "housekeeping" genes. The 5'-flanking regions of the FSH-R lack typical TATA or CCAAT boxes, and are believed to have a few known-conserved regulatory elements, including half ERE, AP-1, and SF-1 like binding sites; an E-box; and an initiator region [17–23]. The core promoter regions could be located within the first 400 base pairs (bp), which have shown high homology among the human, rat, mouse, and ovine FSH receptors [17–20, 24]. Recent in vitro studies suggest that a region containing SF-1 binding sites may be involved in cell-specific regulation of mouse FSH-R expression [22]. However, these and other in vitro results based on transient transfection studies need to be further examined in vivo, wherein the promoter template is wrapped around histones as a nucleosome. Such packed chromatin architecture under normal physiological conditions controls accessibility of transcription factors to their regulatory elements. Chromatin remodeling induced by hormones or other transcription factors renders the nucleosome "open" or "closed," leading to activation or repression of target genes. Tissue-specific and stage-specific expression of genes is dependent on cellular context of transcription factors as well as their selective activation by hormones.

Previous in vivo footprinting studies also demonstrated that the conserved E-box was occupied by upstream stimulatory factors in Sertoli cells but not in other nongonadal cells [25]. More recent studies have demonstrated that multiple site-specific cytosine methylation processes within the rat FSH-R promoter may be involved in the tissue-specific repression of the FSH-R gene [26–28]. Thus, demethylation of these sites could reactivate transcription [26] as necessary. These studies suggest that full activation of promoters containing recognition sequences for many different transcription factors require simultaneous binding of an entire set of transcription factors that may bind cooperatively to their sites or act synergistically by other mechanisms. Therefore, as a first step, it is necessary to identify the regulatory elements and binding proteins required for the expression of the FSH-R gene within the core promoter regions. Moreover, characterization and identification of regulatory elements in the FSH-R promoters are particularly of interest because they may contain tissue-specific and stage-specific cis-elements that can be used for gene therapy of ovarian and testicular diseases. Our previous studies on the ovine FSH-R promoter have mapped several positive and negative regulatory regions. The strongest promoter is localized from -200 to +163 relative to the transcription start site. In the present paper, we report the identification and characterization of a second regulatory element, a CACC-box within the strongest ovine FSH-R promoter. By DNase I footprinting assay, the repeated CACC-motifs, were mapped at -46 to -67 upstream of the transcription start site, to which a number of transcription factors can potentially bind. We have also studied the potential role of CACC-box in the regulation of ovine FSH-R expression.

MATERIALS AND METHODS

Plasmids, Cell Lines, and Oligonucleotides

Promoterless plasmid pGL3-basic (Promega, Madison, WI) and ovine FSH-R promoter/luciferase constructs were described previously [23]. A mouse Sertoli cell line (15P1) established from testicular cells of adult transgenic mice that express the large T antigen of Polyomavirus was kindly provided by Dr. Francois Cuzin (University of Nice, Nice, France) [29]. 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, SK, Canada) [30]. All oligonucleotides were commercially synthesized and were from either Gibco-BRL (Gaithersburg, MD) or BioCorp Inc. (Montréal, PQ, Canada).

Cell Culture and Transient Transfections

The cells were routinely maintained in a humidified, 37°C (JC-410 cells) or 32°C (15P1 cells) incubator with 5% CO2, and cultured in F-12/Dulbecco modified Eagle medium (DMEM; Gibco-BRL) containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Transient transfections were carried out using electroporation as described previously [31]. Cells (5 x 106) were resuspended in 500 µl of serum-free and antibiotics-free F-12/DMEM medium containing 5 µg of luciferase reporter and 1 µg of a pCMVlacZ. After a 10-min incubation at room temperature, the cells were electroporated in a 0.4-cm-gap-width electroporation cuvette with 960 µF at 220 V, using a Gene Pulser (Bio-Rad, Hercules, CA). The cells were incubated at 37°C for 10 min and then cultured on 60-mm Petri dishes. After 48 h, cells were lysed in a lysis buffer containing 100 mM Tris-HCl, 0.5% NP-40, and 1 mM dithiothreitol (DDT). Appropriate aliquots of cell extract were used for both luciferase and ß-galactosidase assays as described previously [23].

Preparation of Nuclear Extracts

Nuclear extracts were prepared as described previously [32]. 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, 60 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 5 µg/ml leupeptin, 1 µg/ml pepstatin A, and 4 µg/ml aprotinin). Nuclei were pelleted by spinning for 5 min at 2500 x g 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, 1 mM PMSF, 5 µg/ml leupeptin, 1 µg/ml pepstatin A, and 4 µg/ml aprotinin). 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 x g at 4°C. Nuclear proteins were stored at -80°C.

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assays (EMSAs) were performed as described previously [33]. Briefly, double-strand oligonucleotides were labeled at the 5' ends using T4 polynucleotide kinase and [{gamma}-32P]ATP. Nuclear extract (10 µg) from 15P1 or JC-410 cells was incubated in a binding buffer containing 25 mM Hepes pH 7.9, 100 mM NaCl, 3 nM 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 radiolabeled probe, the mixture was incubated at room temperature for another 20 min. Excess unlabeled DNA competitors were added 5 min before the radiolabeled DNA probe was added. Gels were dried and visualized by autoradiography.

Southwestern Analysis

The Southwestern analyses were performed as described previously [23, 34]. Briefly, 120 µg of nuclear extracts was separated on an SDS-PAGE denaturing gel. Proteins were then transferred to nitrocellulose and the membrane was incubated at 4°C overnight in a buffer containing 5% dry skim milk, 25 mM NaCl, 5 mM MgCl2, 25 mM Hepes pH 7.9, and 1 mM DTT. The binding reaction was performed in the same (but milk-free) buffer with 106 cpm/ml of 32P-labeled double-stranded DNA probe in the presence of 5 µg/ml salmon sperm DNA at room temperature for 18 h. Prior to autoradiography, blots were washed four times at room temperature using the same buffer.

Ultraviolet Cross-Linking

Ultraviolet cross-linking was carried out as described previously [33]. Protein-DNA binding was carried out at room temperature for 20 min with 15 µg of nuclear extract in EMSA binding buffer. The vials were then placed on ice and irradiated in a Genelinker 250 (Stratagene, La Jolla, CA) for 20 min. Subsequently, samples were heated at 100°C for 5 min and electrophoresed on 10% SDS-PAGE. The gel was dried prior to autoradiography.

DNase I Footprinting

Noncoding strand probes were prepared by cutting the plasmid P363 with BglII and 32P-5' end-labeled with T4 polynucleotide kinase [23]. Labeled DNA was run through a ProbeQuant G-50 column (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) to remove free nucleotides. The probe was released from the plasmids by digesting with HindIII and was purified by 1% agarose gel electrophoresis [23]. DNase I footprinting reactions were carried out as described previously [23]. Binding reactions containing 25 mM Hepes pH 7.6, 5 mM MgCl2, 84 mM KCl, 5% glycerol, 1 mM DTT, 1 µg poly(dI-dC), 20 µg nuclear extract, and 1 µl labeled template DNA (~20 000 cpm) were incubated at room 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 salmon sperm DNA, and 250 µg/ml of proteinase K). The samples were incubated at 45°C for 1 h, and extracted twice with phenol/chloroform. Finally, after ethanol precipitation, the samples were analyzed on a 7% sequencing gel along with A, C, G, and T DNA sequence tracks, followed by autoradiography.

Site-Directed Mutagenesis

Mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). In brief, a P363 construct containing a wild-type FSH-R promoter fused to a luciferase gene was used as a template [23]. A pair of synthetic oligonucleotides (5'-CCCTCTACCTCTCaCACCaCACCaCCACCAAAGTCACTGC) with a directed mutation was extended by means of Pfu turbo DNA polymerase. The extension product was treated with 10 units of DpnI endonuclease at 37°C for 1 h to digest the parental, double-strand DNA template. The nicked vector DNA incorporating a mutation was then transformed into E. coli XL1-Blue Supercompetent cells. Individual clones were isolated, and the purified DNA was sequenced as described previously to confirm which clones had the correct mutational changes [23].

RESULTS

Nuclear Proteins Bind to a CACC-Box of the Ovine FSH-R Promoter

Previous promoter deletion analysis has localized several positive and negative regulatory regions [23]. To further map cis-elements within the ovine FSH-R promoter, we performed a DNase I footprinting assay using the previously reported P363 plasmid as a template [23]. Two cis-elements from +95 to -121 relative to the transcription start site of the ovine FSH-R promoter are recognized by nuclear proteins and are protected from DNase I digestion (Fig. 1). In this species the single transcription start site is located 163 bases 5' from the translation codon. The region FP1 has been identified as an E-box to which the USF-1/2 binds as a heterodimer and contributes to minimal and proximal promoter activities [23]. Footprinting 2 (FP2) was localized from -46 to -67 of the strongest promoter (i.e., 209–230 bp upstream of the first translation initiation codon). This 22-bp region contained classical GT/CACC-boxes to which a number of zinc finger proteins can potentially bind [35–42]. Two sites hypersensitive to DNase I digestion can also be seen (Fig. 1).



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  		FIG. 1. In vitro DNase I footprinting with nuclear extract from JC-410 or 15P1 cells. The 363-bp DNA fragment was 5' end-labeled on the noncoding strand. The DNA templates were then subjected to DNase I digestion in presence or absence of nuclear extract from the indicated cell lines as described in Materials and Methods. The digested DNA was analyzed on a 7% polyacrylamide denaturing gel, followed by autoradiography. The regions of protection from DNase I digestion are indicated by brackets, and the arrowheads indicate DNase I hypersensitive sites. The protected sequence against DNase I digestion in the presence of nuclear extracts is shown on the left side of DNA sequence tracks. Lanes 1–4, DNA sequence tracks; lane 5, DNase I digestion without nuclear extract (-NE); lanes 6 and 7, DNase I digestion with 20 µg of nuclear extracts as indicated (+NE)

To identify transcription factors that bind to the FP2 region, we synthesized oligonucleotides that encompass sequences from -46 to -67 of the ovine FSH-R promoter. Using this double-stranded DNA probe, our EMSAs detected a retarded protein-DNA complex with the nuclear extract either from 15P1 or JC-410 cells (Fig. 2). This binding appeared to be sequence-specific because it was competed by the unlabeled FP2 probe and slightly competed away by the sequence-homologous Sp1 probe, but not by an unrelated, nonspecific competitor (Fig. 2). Further gel competition experiments with unlabeled DNA probes containing known DNA binding sites for the zinc finger proteins demonstrated that the FP2-protein complex was not at all competed by early growth response protein-1 element (Egr-1) [43, 44], but partially by the RIN ZF protein binding site (RZFE) at high concentrations of 200-fold molar excess (Fig. 3). Moreover, the DNA-protein complex of Egr-1 migrated faster than the complex of FP2-protein (Fig. 3), suggesting that the proteins seen in the EMSAs may not be the same as Egr-1. However, unlabeled FP2 probe could partially compete for the formation of Egr-1/protein complex at high concentrations of 200-fold molar excess compared with the nonspecific competitor and the control without any competitors (Fig. 3). These data suggest that the zinc finger family may be involved in the binding to the FP2 probe. Therefore, we chose two known Krupple-like factor binding sites whose sequences are highly homologous to FP2, and performed a cross-competition experiment (Fig. 4). In fact, the FP2-protein complex was competed both by the Ras response element (RRE) and the ZNF202 element (RZF202E) (Fig. 4) [35, 41]. Not only were the proteins binding to RRE and ZNF202E competed by the unlabeled FP2 probe, but interestingly, they also migrated in almost the same way as the FP2-protein complex (Fig. 4).



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  		FIG. 2. Sequence-specific protein binding to FP2 double-strand oligonucleotides. EMSA was conducted using nuclear extract (10 µg) from JC-410 and 15P1 cells with a 32P-labeled FP2 probe. The binding reactions were analyzed on a 5% polyacrylamide nondenaturing gel, followed by autoradiography. Lane 1, probe control without nuclear extracts; lanes 2 and 6, probe with nuclear extract from JC-410 and 15P1 cells, respectively; lanes 3 and 7, probe with nuclear extract from JC-410 and 15P1 cells, respectively, plus 200-fold molar excess of unlabeled double-strand FP2 oligonucleotides; lanes 4 and 8, probe with nuclear extract from JC-410 and 15P1 cells, respectively, plus 200-fold molar excess of unlabeled double-strand Sp1 oligonucleotides; lanes 5 and 9, probe with nuclear extract from JC-410 and 15P1 cells, respectively, plus 200-fold molar excess of unlabeled, nonspecific, double-strand oligonucleotides. The sequences of oligonucleotides used are given at the bottom



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  		FIG. 3. Zinc finger proteins bind to FP2 probe. Cross-gel competition experiments were carried out with nuclear extract (10 µg) from 15P1 cells and double-strand oligonucleotides containing known binding sites for zinc finger proteins. Lane 1, radiolabeled FP2 probe and 15P1 nuclear extract without any DNA competitors; lane 2, radiolabeled FP2 probe and 15P1 nuclear extract in the presence of 200-fold molar excess of unlabeled FP2 double-strand oligonucleotides; lanes 3–9, radiolabeled FP2 probe and 15P1 nuclear extract in the presence of unlabeled DNA binding sites for early response growth 1 (Egr-1), RIN ZF protein (RZFE), and nonspecific competitor (NSC), respectively, as indicated in the Figure; lane 10, radiolabeled Egr-1 double-strand oligonucleotides and 15P1 nuclear extract without any DNA competitors; lanes 11–15, radiolabeled Egr-1 double-strand oligonucleotides and 15P1 nuclear extract in the presence of unlabeled Egr-1, FP2, and NSC double-strand oligonucleotides as indicated. Sequences of the oligonucleotides used are given at the bottom



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  		FIG. 4. Krupple-like transcription factors bind to FP2 probe. Cross-gel competition experiments were carried out with nuclear extract (10 µg) from 15P1 cells and double-strand oligonucleotides containing known Krupple-like transcription factor binding sites. Lane 1, radiolabeled FP2 probe and 15P1 nuclear extract without any DNA competitors; lanes 2–5, radiolabeled FP2 probe and 15P1 nuclear extract in the presence of 50-fold molar excess of unlabeled double-strand FP2, Ras responsible element (RRE), ZNF202 element (ZNF202E), and nonspecific competitor (NSC), respectively; lane 6, radiolabeled RRE probe and 15P1 nuclear extract without any DNA competitors; lanes 7–9, radiolabeled RRE probe and 15P1 nuclear extract in the presence of 50-fold molar excess of unlabeled FP2, RRE, and nonspecific competitor (NSC); lane 10, radiolabeled ZNF202E and 15P1 nuclear extract without any DNA competitors; lanes 11–13, radiolabeled ZNF202E and 15P1 nuclear extract in the presence of unlabeled FP2, ZNF202E, and NSC. Sequences of the oligonucleotides used are given at the bottom

Previous studies have characterized the RRE binding protein-1 (RREB-1) as having 755 amino acids [35], whereas testis-specific ZNF202 has two isoforms of 424 and 648 amino acids, respectively [41, 45]. To characterize and compare nuclear proteins that bind the FP2 probe, we performed a Southwestern blot. The FP2 probe recognized proteins with apparent masses of 87, 60, 55, and 50 kDa in nuclear extracts from JC-410 cells, but also masses of 87, 60, and 50 kDa in nuclear extracts from 15P1 cells (Fig. 5). RZFE probe bound predominantly to a protein with a mass of 60 kDa, although weak binding to 87- and 50-kDa proteins was visible (Fig. 5). RRE and ZNF202E probes likely detected the same proteins as FP2 but with two extra bands with masses of 32 and 30 kDa for RRE and three extra bands of 110, 32, and 30 kDa for ZNF202E. Because slightly different regulatory elements derived from different genes detected the same classes of proteins, we may predict that Krupple-like factors, the RREB-1-like and ZNF202-like proteins, may be candidate proteins involved in the protein binding to the FP2 probe [41, 45].



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  		FIG. 5. Southwestern analysis of FP2 DNA-binding proteins. An aliquot (120 µg) of nuclear extracts from 15P1 and JC-410 cells were separated on a 10% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane and probed with 32P-labeled double-strand DNA probe as indicated in the figure, followed by autoradiography. Lane 1, nuclear proteins from JC-410 cells probed with radiolabeled FP2; lanes 2–5, nuclear proteins from 15P1 cells probed with radiolabeled FP2, RZFE, RRE, and ZNF202E, respectively

To test whether proteins seen in the EMSA and Southwestern analysis actually interact with the FP2 probe in a binding reaction under nondenaturing conditions, we turned to UV cross-linking experiments. In agreement with data seen in the Southwestern blot, we were able to demonstrate cross-linking of the FP2 probe to polypeptides of 87, 60, and 50 kDa, as well as some small proteins with molecular weights ranging from 35 to 26 kDa in nuclear extracts from both 15P1 and JC-410 cells (Fig. 6). As seen in the EMSA, interaction of the FP2 probe with 87-, 60-, and 50-kDa proteins was effectively displaced by an excess of unlabeled FP2 probe, but was unaffected by an equivalent excess of unlabeled Sp1 oligonucleotides (Fig. 6). In contrast, a 100-fold molar excess of unlabeled FP2 probe did not affect the binding of small molecular proteins, suggesting that these latter interactions may be nonspecific. Taken together, the results indicate that the 87-, 60-, and 50-kDa proteins actually participated in sequence-specific interaction with FP2, as seen in biochemical assays (Figs. 2–6).



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  		FIG. 6. UV cross-linking analysis of FP2 DNA-binding proteins. An aliquot (15 µg) of nuclear extract either from 15P1 or JC-410 cells was incubated with a 32P-labeled FP2 probe at room temperature for 20 min, then irradiated for 0 and 20 min, respectively. The bound proteins were analyzed by 10% SDS-PAGE prior to autoradiography. Lanes 1 and 5, complete binding reaction control containing nuclear extract from JC-410 and 15P1 cells, respectively, without UV irradiation; lanes 2 and 6, complete binding reaction control containing nuclear extract from JC-410 and 15P1 cells, respectively, with 20-min UV irradiation; lanes 3 and 7, 20-min UV irradiation of complete binding reaction control containing nuclear extract from JC-410 and 15P1 cells, respectively, plus 100-fold molar excess of unlabeled FP2 probe; lanes 4 and 8, 20-min UV irradiation of complete binding reaction control containing nuclear extracts from JC-410 and 15P1 cells, respectively, plus 100-fold molar excess of unlabeled Sp1 probe

CACC-Box Mutation Reduces Krupple-Like Factor Binding and Proximal Promoter Activity

To understand the contribution of the CACC-box to promoter activity of ovine FSH-R, we made a series of mutations by replacing C with A in the CACC-box and used the mutated FP2 oligonucleotides (FP2m1 to FP2m5) to test for protein binding. Gel competition experiments demonstrated that FP2m1 and FP2m2 oligonucleotides were able to partially compete for binding to wild-type probe (FP2) at 200-fold molar excess (Fig. 7). FP2m3 oligonucleotides were also able to do this, but they could not completely compete for protein binding at 200 molar excess, compared with unlabeled FP2 probe at equivalent molar excess (Fig. 7, compare lanes 10 and 14). However, FP2m4 and FP2m5 failed to compete for nuclear factor binding (Fig. 7). In addition, EMSA found that the labeled FP2m5 probe was also not bound by nuclear proteins from 15P1 cells (data not shown). These data suggest that at least two C residues in the CACC-box were required for high-affinity DNA-protein binding. To completely eliminate potential CACC-box binding protein binding to the FP2 region of FSH-R promoter, we incorporated the same mutations as FP2m5 into a 363-bp length of FSH-R promoter fused to a luciferase gene (P363CACCmt). We transiently transfected this reporter gene together with the control reporters into 15P1 and JC-410 cells to analyze the luciferase activities (Fig. 8). Consistent with the EMSA binding experiments shown in Figure 7, luciferase assays demonstrated that the CACC-box mutation diminished the proximal promoter activities by 76% in JC-410 cells and by 58% in 15P1 cells (Fig. 8). These data suggest that the CACC-box and its binding proteins may play a role in the regulation of ovine FSH-R expression.



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  		FIG. 7. CACC-box mutation abolishes Krupple-like transcription factor binding. An EMSA was performed as described in Figure 2. A competition experiment was conducted by adding a 10-, 50-, and 200-fold molar excess of unlabeled oligonucleotides, respectively, to the binding reaction. Lane 1, FP2 probe with nuclear extract from 15P1 cells; lanes 2–4, FP2 probe with nuclear extract from 15P1 cells, plus 10-, 50-, and 200-fold molar excess of unlabeled FP2m1 oligonucleotides, respectively; lanes 5–7, FP2 probe with nuclear extract from 15P1 cells, plus 10-, 50-, and 200-fold molar excess of unlabeled FP2m2 oligonucleotides, respectively; lanes 8–10, FP2 probe with nuclear extract from 15P1 cells, plus 10-, 50-, and 200-fold molar excess of unlabeled FP2m3 oligonucleotides, respectively; lanes 11–13, FP2 probe with nuclear extract from 15P1 cells, plus 10-, 50-, and 200-fold molar excess of unlabeled FP2m4 oligonucleotides, respectively; lane 14, FP2 probe with nuclear extract from 15P1 cells, plus 200-fold molar excess of unlabeled FP2 oligonucleotides; lanes 15–17, FP2 probe with nuclear extract from 15P1 cells, plus 10-, 50-, and 200-fold molar excess of unlabeled FP2m5 oligonucleotides, respectively. Sequences of the wild-type FP2 probe and the mutated FP2 probes are given at the bottom



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  		FIG. 8. CACC-box mutation reduces promoter activity. A luciferase assay was carried out as described previously [23] using a reporter gene construct with either wild-type promoter or the promoter with a mutated CACC-box. JC-410 and 15P1 cells were transfected with a luciferase gene fused to 363 bp of wild-type promoter (P363) [23] or a similar promoter containing a mutant CACC-box (P363 CACC-mt) or a control, promoterless vector (pGL3-basic). Relative luciferase activity was normalized with ß-galactosidase activity and expressed as fold induction over the activity of the pFL3-basic as described previously [23]

DISCUSSION

We used the ovine FSH-R promoter and cell lines from the pig granulosa and Sertoli cells as a model system to investigate transcription factors binding to the promoter. Our DNase I footprinting studies mapped a regulatory element, CACC-box, from -46 to -67 upstream of the transcription start site (Fig. 1). Subsequent EMSA, Southwestern blot, and UV cross-linking experiments with double-strand DNA probes and nuclear extracts revealed that Krupple-like factors were potential candidate binding proteins with molecular weights of 87, 60, and 50 kDa (Figs. 4 and 5). Site-directed mutations within the CACC-box of the promoter either reduced binding affinity or blocked sequence-specific binding as detected by gel shift assays (Fig. 7). Incorporation of a completely mutated CACC-box into the wild-type promoter context reduced promoter activities by 76% in JC-410 cells and by 58% in 15P1 cells (Fig. 8). These data suggest that the CACC-box upstream of the transcription start site and its binding proteins are required for expression of ovine FSHR in these two gonadal cell lines.

As discussed previously [23], it is difficult to perform direct characterization of the ovine FSH-R promoter in granulosa and Sertoli cells from the same species. However, the use of two immortalized cell lines from other species has provided a feasible alternative because of conservation of not only transcription factors but also most regulatory elements of the FSH-R promoters among the species [17–23]. In the present study, we used our model system and identified a CACC-box that is located upstream of the previously reported E-box (Fig. 1) [23]. After a series of mutations, we found that a part of this repeating CACC-box sequence (CCCCCACC or CCCACCCC) is sufficient for protein binding with a relatively lower affinity (Fig. 7, and data not shown). This is also consistent with observations found in other gene promoters in which Krupple-like transcription factors bound to the CACCC/CCCTC motif [37–40, 46–50]. Similar motifs may be also found in FSH-R promoters in other species. For example, two GT-boxes are located from -777 to -784 and -1058 to -1065, respectively, relative to the first nucleotide of the translation initiation codon of the human FSH-R promoter [17]. A GT/CACC box from -1261 to -1272 in the mouse FSH-R promoter and multiple CACC-boxes in a region from -479 to -542 of the rat FSH-R promoter appeared to be conserved [18, 20]. These observations further indicate that the GT/CACC box may play an important role in the regulation of FSH-R promoters in many species.

G-Rich elements, including GC-box and GT/CACC boxes, are widely distributed in promoters, enhancers, and locus control regions of housekeeping genes as well as many tissue-specific genes [37, 42, 46, 49, 51–54]. These motifs are believed to be important cis-acting elements because not only are they involved in gene-specific localization, cell cycle regulation, hormonal activation, and developmental patterning, but also in maintaining the methylation-free active status of the gene leading to tissue-specific gene expression [37, 42, 46, 53–55]. Recent molecular cloning and characterization of GC and GT/CACC box binding proteins has allowed them to be classified as a family, including 1) Sp transcription factors; 2) the close relatives, basic transcription element binding protein (BTEB) and TGFß-inducible early genes (TIEGs), also named EGRs (for early growth response genes); and 3) the Krupple-like factors (XKLFs) [42]. Our competition experiments with probes of Sp-1, Egr-1, and ZNF202E, to which three corresponding subfamily proteins supposedly bind, have demonstrated that the proteins seen in gel shift assays are neither SP members nor those of the EGR family, but likely belong to Krupple-like transcription factors (Figs. 2–5). These binding proteins with molecular weights of 87, 60, and 50 kDa present in the granulosa and Sertoli cells seem to bind to FSH-R promoter as a complex, because we detected a single DNA-protein complex in the gel shift assay but three binding peptides in the UV cross-linking analysis (Figs. 2, 6, and 7). The repeating pattern of the CACC-box sequence suggests that two molecules of Krupple-like factors may be necessary for strong binding. This would be consistent with the results of the EMSAs described above (Fig. 6), in which mutation of bases in either repeat within FP2 probes interfered with binding. Moreover, earlier observations showing the purified recombinant protein-DNA complex migrating faster than the nuclear protein-DNA complex in gel shift assays [35, 38, 45], and the presence of Krupple-associated box and protein interaction modules, which can interact with corepressor/coactivator, further support the hypothesis of Krupple-like factor dimerization [36, 39–41, 45].

Although RRE and ZNF202E could complete for FP2-nuclear factor binding, and their corresponding binding proteins are normally expressed in both the testis and ovary, the predicted molecular weights of RREB-1 and ZNF202 seem to be slightly different from the proteins detected in our Southwestern blot and UV cross-linking analyses (Figs. 5 and 6) [41, 45]. In addition, ZNF202 proteins have been reported as transcription repressors, whereas RREB-1 has been reported as an activator [35, 41, 45]. However, we cannot exclude the possibility that these proteins may be involved in DNA-protein binding, because proteins detected on SDS-PAGE showed variable apparent molecular weights, depending on the experimental conditions and protein modifications. These two members of Krupple-like factors have been reported as having potential phosphorylation sites, resulting in bipartite functions [35, 41, 45]. The binding proteins present in the granulosa and Sertoli cells could also be other members of Krupple-like factors that potentially bind to probes containing CACC motifs. Whether RREB-1 and ZNF202 or other Krupple-like factors present in granulosa and Sertoli cells are involved in the FP2-protein interaction requires further investigation.

The multiple binding proteins detected by Southwestern blot and UV cross-linking (Figs. 5 and 6) may result from different genes or the same gene by alternative splicing, such as ZNF202 proteins [41, 45]. They could act in either a positive or negative manner, depending on the target cells. However, repressor or activator functions may compete with each other for the same cis-element, providing mechanisms by which expression of target genes may be regulated by competition among the entities [49, 56]. Because the identity of proteins and chromatin-based models of FSH-R promoters in cell lines and transgenic mice have not yet been established, caution may be exercised in drawing firm conclusions as to whether Krupple-like factors contribute to cell-specific and stage-specific expression of the FSH-R gene. Nevertheless, for the first time, our data provide a basis for further characterization of these factors in Sertoli and granulosa cells.

ACKNOWLEDGMENTS

We are grateful to Dr. P.J. Chedrese, University of Saskatchewan, Saskatoon; and Dr. F. Cuzin, University of Nice, Nice, France, for providing the cell lines used in this study. We also thank Dr. B.D. Nelkin, Oncology Center, Johns Hopkins Medical Institutions, Baltimore, Maryland; Dr. L.G. Tillotson, the Division of Digestive Diseases and Nutrition, Department of Medicine, University of North Carolina, Chapel Hill, North Carolina; and Dr. M. Crossley, Department of Biochemistry, University of Sydney, Australia, for generously providing various antisera, expression vectors, and recombinant proteins tested in this study. The secretarial assistance of Odile Royer is greatly appreciated.

FOOTNOTES

First decision: 6 April 2001.

1 This investigation was supported by grants from the Canadian Institutes of Health Research (CIHR). W. X. holds a doctoral scholarship award from CIHR. Back

2 Correspondence: M. Ram Sairam, Molecular Reproduction Research Laboratory, Clinical Research Institute of Montréal, 110 Pine Avenue West, Montréal, PQ, Canada H2W 1R7. FAX: 514 987 5585; sairamm{at}ircm.qc.ca Back

Accepted: May 11, 2001.

Received: March 14, 2001.

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