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Testis |
Division of Reproductive Biology,4 Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene regulation, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The exact molecular mechanisms responsible for stage-specific gene expression in Sertoli cells are unknown. Unraveling these mechanisms is crucial not only to understand the regulation of gene expression in Sertoli cells but also the regulation of male fertility. A requirement for defining how male germ cells modulate stage-specific expression in Sertoli cells is the identification of a promoter that confers accurate, stage-specific gene expression in vivo. Gene transcription in Sertoli cells has been predominantly studied by transient transfection of reporter constructs into established Sertoli cell lines or primary cultures of Sertoli cells. Results from these studies have allowed the identification of promoter regions that potentially play important roles in vivo. However, in vivo, spermatogenesis occurs in a complex physiological environment, the seminiferous epithelium. Since the complex interactions between Sertoli cells and male germ cells cannot be fully replicated in vitro, it is crucial to establish that the promoter regions characterized in vitro are able to confer accurate, stage-specific gene expression in vivo. Unfortunately, to date attempts to demonstrate this requirement have not been successful [8–12].
Cathepsin L is a cysteine protease synthesized in a proenzyme form that requires proteolytic processing in order to be active (reviewed in [13]). Cathepsin L appears to play an important role during spermatogenesis, as mice that express catalytically inactive cathepsin L exhibit a 10-fold increase in seminiferous tubule atrophy and quantitatively reduced spermatogenesis in normal, nonatrophic tubules [14]. Levels of cathepsin L mRNA in murine and rat Sertoli cells are highly regulated with respect to the stages of the cycle of the seminiferous epithelium. In rodents, cathepsin L transcripts are expressed at high levels in mature Sertoli cells in tubules at stages VI–VIII, and at low or undetectable levels at all other stages [5, 14–16]. In these studies, mature Sertoli cells are isolated from testes that exhibit complete spermatogenesis. Recently, we reported that a 3-kilobase (kb) genomic fragment immediately upstream of the rat cathepsin L translation start site directs in vivo expression of the reporter gene, ß-galactosidase, only in Sertoli cells [17]. The expression pattern of the reporter gene recapitulated that of the endogenous gene in Sertoli cells (i.e., high levels of expression were seen in mature Sertoli cells of stage VI–VIII tubules). Thus the 3-kb genomic fragment located immediately upstream of the cathepsin L translation start site contained all of the regulatory elements required for accurate stage-specific gene expression in vivo in Sertoli cells.
It has been proposed that stage-specific expression of the cathepsin L gene results in part from the sequential activation and repression of transcription [15, 16]. Such a model predicts the presence of domains that mediate stimulatory and inhibitory effects within the cathepsin L promoter region identified in the transgenic analysis. The promoter region tested in the transgene contained 2065 base pairs (bp) of sequence upstream of the transcription start site of the rat cathepsin L gene, the first exon (74 bp), the first intron (891 bp), and the first 11 bp of exon 2. To begin to dissect the putative regulatory domains within this 3046-bp DNA fragment, we examined the function of the DNA region upstream of the transcription start site in transient transfection assays using Sertoli cells isolated from sexually mature rats. Reporter gene activity in mature Sertoli cells transfected with a construct that contains the region of the rat cathepsin L gene spanning -2060 to +33 was reduced by 30% in the presence of pooled male germ cells, but was unchanged when Sertoli cells were transfected with a construct that contains the region spanning -244 to +33 [18]. In view of the activation/repression model explaining the stage-specific expression of the cathepsin L gene, regulatory elements residing between -2060 and -244 of the rat cathepsin L gene were postulated to mediate, in part, inhibitory effects of male germ cells.
The present study focuses on identifying regulatory domains that activate transcription of the cathepsin L gene in Sertoli cells. A refined analysis of the region upstream of the transcription start site of the rat cathepsin L gene was undertaken to characterize the regulatory elements contained within this region. The promoter activity of a compact 120-bp DNA fragment that spans the transcription start site of the rat cathepsin L gene was shown to be identical to that of the 
2-kb region upstream of the transcription start site. Within this 120-bp promoter region, a GC-rich motif (GC-box) that binds the transcription factor Sp3 was essential for promoter activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sexually mature (60- to 70-day-old) Sprague Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). The use of animals in the experiments described in this manuscript was approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University.
Cathepsin L Promoter Constructs
The assembly of the luciferase construct cath L (-2060/+33)-Luc that contains 2060 bp upstream of the transcription start site of the rat cathepsin L gene and 33 bp of downstream sequence has been described [18]. The nucleotide sequence of this genomic fragment can be found in the GenBank database under accession AF025476. This construct served as a template to generate reporter gene constructs that contain sequential 5' deletions of the 2060-bp DNA region. Deletions in the 5' flanking region of the rat cathepsin L gene were generated by PCR using forward and reverse primers. The nucleotide sequences of the primers used are listed in Table 1. The amplified DNA fragments were digested with KpnI and HindIII, purified, and subcloned into the vector pGL2-Basic (Promega Corp., Madison, WI) cut with KpnI and HindIII. The plasmid pGL2-Basic contains the Firefly luciferase reporter gene but lacks eukaryotic promoter or enhancer elements. To assemble the construct cath L (mut -87/+33)-Luc, two DNA fragments were generated. The first fragment was a synthetic double-stranded oligonucleotide (Integrated DNA Technologies, Inc., Coralville, IA)5'-CATGCAGGCACAGCCAATGACAGGCACAGCTGCA-3'3'-CATGGTCCGTGTCGGTTACTGTCCGTGTCGTGCA-5'that contains a 5'-KpnI overhang and a 3'-PstI overhang (the mutated nucleotides are indicated in boldface). The second DNA fragment (spanning -57 to +33 of the rat cathepsin L gene) was amplified by PCR using the forward primer -57/PstI/F and the reverse primer +33/HindIII/R (Table 1). This fragment was then cut with PstI and HindIII. A three-way ligation between these two DNA fragments and the plasmid pGL2-Basic cut with KpnI and HindIII yielded the construct cath L (mut -87/+33)-Luc in which the nucleotides from position -70 to -58 were mutated. The mutated sequence is identical to the one found in the oligonucleotide m -79/-51 (Table 2). The nucleotide sequence of the cathepsin L fragment in each construct was verified by DNA sequencing. The reporter gene constructs were purified using the EndoFree Plasmid Maxi Kit (Qiagen, Inc., Chatsworth, CA) followed by CsCl2 centrifugation.
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Cell Culture and DNA Transfection
Sertoli cells were isolated from sexually mature (60- to 70-day-old) rats as previously described [18, 19] and plated at a density of 1.5 x 105 cells/cm2 on 30 mm Millicell-HA culture chambers (Millipore Corp., Bedford, MA) coated with 280 µl of Matrigel (Becton-Dickinson Laboratories, Bedford, MA). Cells were cultured in Ham's F-12/DMEM medium supplemented with human transferrin (5 µg/ml), insulin (10 µg/ml), epidermal growth factor (1 ng/ml), retinol acetate (3.5 x 10-8 M), testosterone (10-7 M), human recombinant or highly purified ovine FSH (50 ng/ml), 2.1 µM vitamin E, and 200 µM vitamin C. Transient transfections were performed as previously described [18]. Lipofectamine (Invitrogen Corp., Carlsbad, CA) was used to transfect mature Sertoli cells with 0.5 pmol of each promoter construct. Cotransfection with 0.09 pmol of the plasmid pRL-CMV (Promega) that contains the Renilla luciferase reporter gene was performed to correct for variations in transfection efficiency. Cells were lysed in passive lysis buffer (Promega), frozen in dry ice, and stored at -80°C. All experiments were carried out in triplicate and independently performed at least three times.
Luciferase Assays
Firefly and Renilla luciferase activities were measured in 5–20 µl of cell extracts using the Dual-Luciferase Reporter Assay System (Promega). Light emissions were measured for 10 sec using a Monolight 3010 luminometer (Analytical Luminescence Laboratory, Sparks, MD). Luciferase activity was defined as the ratio between Firefly luciferase activity and Renilla luciferase activity. Titration of extracts demonstrated that Firefly and Renilla luciferase activities measured in cell extracts were in the linear range of the assays.
Preparation of Nuclear Extracts
Sertoli cells were isolated from sexually mature rats (60 to 70 days old), cultured overnight on matrigel-coated tissue culture plates [18], and washed 3–4 times the next day to remove male germ cells. Nuclear extracts were prepared as described [20], with slight modifications. Okadaic acid was added to buffers NE1 and NE2 (final concentration 10 nM), and 0.2% NP-40 (Calbiochem-Novabiochem Corp., La Jolla, CA) was added to buffer NE1. Protein concentrations of the nuclear extracts ranged from 3 to 7 mg/ml. Nuclear extracts were frozen in liquid nitrogen and kept at -80°C until use.
Oligonucleotides
PAGE-purified, complementary, single-stranded oligonucleotides (Integrated DNA Technologies, Inc.) were annealed, end-labeled with [32P]dCTP using the Klenow fragment of DNA polymerase (Invitrogen), and purified using Sephadex G-25 columns. The oligonucleotides used for electrophoretic mobility shift assay (EMSA) are listed in Table 2.
Electrophoretic Mobility Shift Assays
EMSAs were performed by incubating for 15 min on ice 5–10 fmol 32P-labeled double-stranded oligonucleotides (20 000 cpm) with 20 µg of nuclear extracts in a reaction solution containing 20 mM HEPES (pH 7.6), 5 mM MgCl2, 1 mM EDTA, 50 mM KCl, 1 mM dithiothreitol, 10% glycerol, 4% ficoll, and 1 µg poly(dI-dC). Competition analysis was conducted by preincubating the nuclear extracts with a 100-fold molar excess of unlabeled oligonucleotides for 10 min on ice. The labeled double-stranded oligonucleotide was then added, and the reaction was incubated for another 15 min on ice. For the supershift experiments, antibodies (2 µg) were incubated with nuclear extracts on ice for 30 min before adding the labeled oligonucleotide for 15 min on ice. The antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) used for supershift experiments were anti-Sp1 (SC-59X), anti-Sp3 (SC-644X), anti-Egr1 (SC-189X), and anti-WT1 (SC-192X). The antibody to Egr3 was kindly provided by Dr. Jay Baraban (Johns Hopkins University). DNA-protein complexes were separated on 5% nondenaturing polyacrylamide gels in 40 mM Tris-acetate, pH 8.3, 1 mM EDTA at 10 V/cm at room temperature. The gels were dried and complex formation was visualized using the Typhoon 9200 Imager (Amersham Biosciences, Piscataway, NJ) and analyzed with the ImageQuant software. Results from EMSAs were reproduced using five different nuclear extract preparations.
Statistical Analyses
Data from transient transfection assays were analyzed by ANOVA and differences between individual means tested by Fisher test using StatView 5.0.1 (SAS Institute, Inc., Cary, NC). Differences were defined as significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify the regulatory elements required for transcriptional control of the cathepsin L gene in mature Sertoli cells, we have created a series of 5' deletion constructs of the 2060-bp region located upstream of the transcription start site. The transcriptional activity of these promoter constructs was analyzed by transient transfection in Sertoli cells isolated from sexually mature rats (60 to 70 days old). As previously reported [18], luciferase enzymatic activity in mature Sertoli cells transfected with the construct cath L (-2060/+33)-Luc was 100-fold higher than that measured in cells transfected with the promoterless vector pGL2-Basic (Fig. 1). Similar levels of luciferase activity were also measured in cells transfected with reporter gene constructs that contain 1250, 933, 737, 450, 241, 155, or 87 bp of sequence upstream of the transcription start site (Fig. 1). The differences in luciferase activity between each of the promoter constructs tested were not statistically significant. These results established that the promoter activity of a compact 120-bp DNA fragment (-87/+33) that spans the transcription start site of the rat cathepsin L gene is identical to that of the 2-kb region upstream of the transcription start site. To further delineate the cis-acting elements present on the DNA fragment spanning -87 to +33, a construct that contains the region spanning -47 to +33 of the cathepsin L gene was generated. As shown in Figure 1, luciferase activity in cells transfected with the reporter construct cath L (-47/+33)-Luc is not statistically different from that measured in cells transfected with the vector pGL2-basic (Fig. 1). Thus it appears that the relatively compact region of the rat cathepsin L gene that spans -87 to -48 contains essential regulatory element(s) required for promoter activity in mature Sertoli cells. Since the DNA region that spans -87 to +33 was sufficient to mediate transcription in mature Sertoli cells, we termed this 120-bp DNA fragment the proximal promoter region.
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Identification of the Regulatory Element(s) Located in the Region Spanning -87 to -48 of the Rat Cathepsin L Gene
Putative binding sites for transcription factors within the nucleotide sequence spanning -87 to -48 of the rat cathepsin L gene were identified by searching the TRANSFAC database using the program AliBaba 2.1 (available at http://www.gene-regulation.com/pub/programs/alibaba2/index.html). Results from this analysis revealed a 17-bp GC-rich motif 5'-CGGGGCGGGGGCGGGCC-3' (spanning -71 to -55) that represents a potential binding site for transcription factors such as AP-2
, ETF, Sox-2, WT1, and members of the early growth response (Egr) and Sp/XKLF families. The presence of functional Egr- and Sp1-binding motifs had been suggested in a previous study [21]. To assess if nuclear proteins from mature Sertoli cells bind to this 17-bp GC-rich motif (GC-box), the double-stranded oligonucleotide -79/-51 (Table 2) that contains the GC-box was used in EMSAs (Fig. 2). Two complexes (labeled C1 and C2) were obtained following incubation of nuclear extracts from mature Sertoli cells with the labeled oligonucleotide -79/-51 (Fig. 2, lane 2). A third complex that migrates below complex C2 was sometimes observed. The specificity of these interactions was demonstrated by the ability to efficiently compete formation of both complexes with a 100-fold molar excess of the unlabeled double-stranded oligonucleotide -79/-51 (Fig. 2, lane 3). However, competition with the double-stranded oligonucleotide m -79/-51 in which the 17-bp GC-box was mutated (Table 2) did not prevent complex formation (Fig. 2, lane 4). Consistent with this observation, complexes C1 and C2 were not formed when nuclear extracts from mature Sertoli cells were incubated with the labeled oligonucleotide M-79/-51 (data not shown). Hence formation of complexes C1 and C2 was dependent on the presence of an intact 17-bp GC-box.
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Four Guanosines Are Crucial for Binding Within the 17-bp GC-Box
To determine the location of the binding motif(s) within this 17-bp GC-box, four oligonucleotides that contain overlapping groups of 4- or 5-bp mutations were designed and the effects of these mutations on DNA-protein complex formation was monitored using EMSA (Fig. 3). Complex formation equivalent to that seen with the oligonucleotide -79/-51 (Fig. 3, lane 2) was observed when mutated oligonucleotides M1 and M4 were used (Fig. 3, lanes 4 and 10, respectively). However, the mutated oligonucleotides M2 and M3 greatly reduced formation of both complexes C1 and C2 (Fig. 3, lanes 6 and 8, respectively). A complex that migrates below the position of complex C2 was observed with oligonucleotides M2 and M3. As stated above, in some experiments this complex was also detected with the oligonucleotide -79/-51. From this analysis, it appears that the four guanosines (position -65 to -62) are the core of the GC-box and are crucial for protein binding to the 17-bp GC-box.
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Sp3 Predominantly Binds to the GC-Box
The results presented above indicate that the GC-box binds nuclear proteins from Sertoli cells isolated from sexually mature rats. To establish the identity of the protein(s) binding to this DNA motif, competition assays were designed in which the binding of nuclear proteins to the labeled double-stranded oligonucleotide -79/-51 was challenged with oligonucleotides that contain the consensus binding sites for transcription factors capable of binding GC-boxes (Table 2). As shown in Figure 2 (compare lane 2 with lane 5), complex formation was not altered by the addition of a 100-fold molar excess of an oligonucleotide that contains a consensus Egr/WT1-binding site [21]. However, a 100-fold molar excess of unlabeled double-stranded oligonucleotides that contain a consensus Sp1-binding site [22] abrogated the formation of the two DNA-protein complexes (Fig. 2, compare lane 2 with lane 6). Although this result suggests that Sp1 could be the protein binding to the GC-box, it is important to emphasize that any member of the Sp/XKLF family has the potential to bind the oligonucleotide that contains the Sp1 consensus motif.
Based on the results of the competition studies, supershift analyses were performed. Antibodies to transcription factors capable of binding GC-boxes were added to the nuclear extracts before the binding reaction. Confirming the results from the competition experiments, antibodies to Egr1, Egr3, and WT1 did not disrupt complex formation (Fig. 4, lanes 5, 6, and 7, respectively). The addition of antibodies to Sp1 did not give rise to a detectable supershift, nor did they abrogate complex formation (Fig. 4, lane 3). However, complexes C1 and C2 were not detected when nuclear extracts were preincubated with an antibody to Sp3. Rather, a slow migrating complex (labeled SC) was observed (Fig. 4, lane 4). The results obtained from the competition and supershift studies suggest that Sp3 or a Sp3-related protein binds predominantly to the GC-rich motif that spans -71 to -55, relative to the transcription start site.
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Sp1-Binding Activity Is Not Detected in Nuclear Extracts of Sertoli Cells Isolated from Mature Rats
Results from EMSAs indicated that the GC-box in the proximal promoter region of the rat cathepsin L gene primarily bound Sp3 and not Sp1 (Fig. 4). To establish if nuclear extracts from mature Sertoli cells contain Sp1-binding activity, nuclear extracts from mature Sertoli cells were incubated in the presence of an oligonucleotide that contains an Sp1 consensus binding sequence. Two DNA-protein complexes, labeled S1 and S2, were detected (Fig. 5, lane 2). The banding pattern was identical to the one observed with the oligonucleotide -79/-51. Addition of an antibody specific to Sp1 did not perturb complex formation (Fig. 5, lane 3). In the presence of an Sp3 antibody, both complexes were shifted to a slowly migrating complex labeled SS (Fig. 5, lane 4). These results strongly suggest that the Sp3-binding activity in nuclear extracts from mature Sertoli cells is substantially greater than that of Sp1.
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Functional Analysis of the Contribution of the GC-Box to the Activity of the Proximal Promoter Region of the Rat Cathepsin L Gene
Taken together, the data presented above indicate that interactions between nuclear proteins from Sertoli cells from sexually mature rats and the 17-bp GC-box give rise to two major complexes and that the transcription factor Sp3 (or an Sp3-related protein) is part of these complexes. To assess the functional relevance of the GC-box on the activity of the 120-bp proximal promoter region, the GC-box in the sequence spanning -87 to +33 was mutated. The mutations introduced in the GC-box were those tested in the double-stranded oligonucleotide m -79/-51 (Table 2), which was shown not to compete for complex formation in EMSAs (Fig. 2, lane 4). The mutant construct cath L (mut -87/+33)-Luc was transfected into mature Sertoli cells, and luciferase activity in cells transfected with this construct was compared with that measured in cells transfected with the constructs cath L (-87/+33)-Luc, cath L (-47/+33)-Luc, and pGL2-Basic, respectively. Figure 6 shows that the promoter activity of the construct cath L (mut -87/+33)-Luc was not statistically different from that of construct cath L (-47/+33)-Luc or pGL2-Basic. Therefore, these results indicate that the GC-rich motif is essential for the high transcriptional activity of the cathepsin L proximal promoter region in mature Sertoli cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The goal of the present study was to identify a regulatory domain that activates transcription of the cathepsin L gene in Sertoli cells isolated from sexually mature rats. To this end, luciferase-based constructs were used to analyze the promoter activity of DNA fragments derived from the 2060-bp upstream region of the rat cathepsin L gene. Results from transient transfection experiments established that most of the transcriptional activity was retained in the construct that contains only a 120-bp DNA fragment that spans -87 to +33 (Fig. 1). Furthermore, removal of the region between -87 and -48 abrogated promoter activity in mature Sertoli cells (Fig. 1). These results indicate that the region between -87 and -48 contains essential regulatory element(s) required for promoter activity in Sertoli cells isolated from sexually mature rats. Since the DNA region that spans -87 to +33 was sufficient to mediate transcription in mature Sertoli cells, this 120-bp DNA fragment was termed the proximal promoter region. Of interest, the nucleotide sequence that defines the proximal promoter region of the rat cathepsin L gene is highly conserved in the mouse gene (93% identity) but weakly conserved in the human gene (64% identity), as established using computer-assisted sequence analysis (LALIGN; available at http://fasta.bioch.virginia.edu/fasta/lalign2.htm).
Structural Features of the Proximal Promoter Region of the Rat Cathepsin L Gene
We have demonstrated that the GC-box located in the proximal promoter region of the rat cathepsin L gene was crucial for promoter activity in Sertoli cells isolated from mature rats. The essential role of the GC-box in mediating transcription was demonstrated by showing that promoter activity was abolished when mutations were introduced in this DNA binding motif (Fig. 6). It has been reported that GC-boxes are required for the appropriate expression of several genes expressed in a ubiquitous, tissue-specific, and developmental manner (reviewed in [23]). The 17-bp GC-box found in the minimal promoter region of the rat cathepsin L gene is present in the upstream sequence of the human [24] and mouse [25] cathepsin L gene, located approximately 50-bp upstream of the transcription start site in each case. The fact that this regulatory element is present in the upstream region of the human cathepsin L gene is interesting in view of the weak nucleotide sequence identity between the proximal promoter of the human and rodent cathepsin L genes. This observation suggests that the GC-box is a regulatory element with an evolutionarily conserved function in the transcription of the cathepsin L gene.
In addition, the 120-bp proximal promoter region of the rat cathepsin L gene is highly GC-rich (70%) and lacks a classical TATA-binding motif, usually located 25- to 30-bp upstream of the transcription start site (reviewed in [26]). These features are typical of promoters found in housekeeping genes [27, 28]. In the absence of a TATA motif, the nucleotides surrounding the transcription start site are likely to function as an initiator (Inr) element [29]. The Inr element, conserved among vertebrates and invertebrates, has a consensus sequence PyPyA+1NA/TPyPy [30]. Inspection of the nucleotide sequence surrounding the transcription start site of the rat cathepsin L gene (5'-TTT
CTC-3') does reveal a putative Inr motif (underlined sequence).
Sp3-Binding Activity in Sertoli Cells of Mature Rats
Sp3 is a member of the Sp/XKLF (specificity protein/Kruppel-like factor) family of transcription factors (reviewed in [31]), characterized by a DNA binding domain that contains three conserved zinc fingers. Sp3 is a ubiquitously expressed protein [22, 32] and binds to GC-binding motifs [32]. Sp3 has been shown to act either as a transcriptional activator or repressor (reviewed in [33]). The cellular context, the structure and arrangement of the binding sites, determine the positive or negative activity of Sp3.
We have established that Sp3 (or an Sp3-related protein) is present in the two protein complexes C1 and C2 formed following incubation of nuclear extracts with the oligonucleotide that contains the 17-bp GC-box (Fig. 4). As such, it appears that Sp3 is necessary for promoting the transcription of the cathepsin L gene in Sertoli cells of mature rats. The banding pattern obtained following incubation of the oligonucleotide -79/-51 with nuclear extracts from mature Sertoli cells is identical to the profile observed using in vitro translated Sp3 proteins and an oligonucleotide that contains a GT-box [22]. Two major complexes are observed because translation of the Sp3 gene gives rise to three protein isoforms (110-, 80-, and 78-kDa proteins) due to internal translation initiation [34]. Based on the banding pattern observed in other studies [22, 33], it appears likely that complex C1 contains the full length Sp3 protein, whereas complex C2 contains the 80- and 78-kDa Sp3 isoforms.
It had been previously reported that the GC-box found in the proximal promoter region of the rat cathepsin L gene bound a member of the Egr family and Sp1 using nuclear extracts from rat fibroblast SR-3Y1-2 and SR-3Y1 cells, respectively [21]. However, the identity of the proteins that bound the GC-box was inferred by competition analysis and not verified using antibodies. In our assays, complex formation was not altered by the addition of a 100-fold molar excess of an oligonucleotide that contains a consensus Egr/WT1-binding site (Fig. 2, lane 5). In another study, both Sp1 and Sp3 were shown to bind to the GC-box located 50-bp upstream of the transcription start site of the human cathepsin L gene, as demonstrated by EMSA using nuclear extracts from two human melanoma cell lines [24]. However, mutations in this binding site only reduced promoter activity by 30%.
A striking result was obtained when Sp1- and Sp3-binding activity in nuclear extracts from mature Sertoli cells were analyzed (Figs. 4 and 5). EMSAs demonstrated that Sp1-binding activity was not detected in these nuclear extracts. This result was surprising in view of the fact that Sp1 is expressed at high levels in most tissues [32] and that high Sp1-binding activity has been detected in nuclear extracts prepared from Sertoli cells [35–37]. However, it should be emphasized that the nuclear extracts used in these earlier studies were prepared from TM4 Sertoli cells or from primary Sertoli cells isolated from sexually immature (15-day-old) rats. Since the TM4 cell line was originally derived from Sertoli cells isolated from immature mice [38], it is interesting to speculate that Sp1- and Sp3-binding activity may fluctuate depending on the stage of maturation of the Sertoli cells.
Further analysis will be required to examine if Sp3 can stimulate transcription of other genes in mature Sertoli cells. Mice that lack a functional Sp3 gene would provide insights into this question. Unfortunately, these mice show embryonic growth retardation and die at birth due to respiratory failure, precluding the analysis of later developmental stages [39]. Conditional disruption of the Sp3 gene in Sertoli cells will be required for such an analysis.
Transcriptional Activation and Repression Are Mediated by the 2060-bp DNA Region Located Upstream of the Transcription Start Site of the Rat Cathepsin L Gene
Two regulatory domains that mediate opposite effects have now been identified in the 2060-bp genomic fragment located upstream of the transcription start site of the rat cathepsin L gene. It is important to emphasize that their biological function has been validated by the fact that the transcriptional activators and repressors that regulate the stage-specific expression of the rat cathepsin L gene in Sertoli cells in vivo are contained within a 3-kb promoter region that includes both of these domains ([17]). The first domain appears to be responsible for the repressive effects mediated by male germ cells [18]. Reporter gene activity in mature Sertoli cells transfected with a construct that contains the region of the rat cathepsin L gene spanning -2060 to +33 was reduced by 30% in the presence of pooled male germ cells. However, reporter gene activity was unchanged when Sertoli cells were transfected with a construct that contains the region spanning -244 to +33. Coupled with the observation that transcription of the cathepsin L gene in mature Sertoli cells is repressed at most stages of the cycle of the seminiferous epithelium, this result is consistent with the hypothesis that male germ cells modulate transcription of the cathepsin L gene in Sertoli cells via regulatory elements that reside between -2060 and -244. Further characterization of this domain will be needed to delineate the regulatory elements that are responsible for the repression mediated by male germ cells. We have now characterized a second functional domain within the 2060-bp DNA region located upstream of the transcription start site of the rat cathepsin L gene. The current study has allowed the identification of a 120-bp proximal promoter region. Within this region, a single 17-bp GC-box, which binds the transcription factor Sp3 (or an Sp3-related protein) was shown to be a critical regulatory element for promoter activity in mature Sertoli cells. In view of the stage-specific expression of the cathepsin L gene in mature Sertoli cells, a future goal will be to establish whether or not the GC-box (alone or in combination with other regulatory elements) plays a direct role in this process.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: William W. Wright, Johns Hopkins University Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, Room 3508, 615 North Wolfe St., Baltimore, MD 21205. FAX: 410 614 2356; wwright1{at}jhem.jhmi.edu
3 Current address: Department of Microbiology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030
Received: 14 October 2002.
First decision: 8 November 2002.
Accepted: 22 November 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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