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Biology of Reproduction 63, 1341-1351 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Clusterin Gene in Rat Sertoli Cells Is Regulated by a Core-Enhancer Element

Elena S. Lymar2,a, Ann M. Clark3,a, Raymond Reevesa, and Michael D. Griswold1,a

a School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660

ABSTRACT

Clusterin is a ubiquitous glycoprotein that is promiscuously expressed at a low basal level but can be highly induced by a variety of stress conditions. In contrast, in some secretory cells associated with tissue-fluid interfaces such as the Sertoli cells in the testis, clusterin demonstrates high constitutive expression. In this study, we address the mechanisms that regulate the constitutive expression of the clusterin gene by using primary cultures of immature rat Sertoli cells. We have identified a region of the rat clusterin gene promoter that activated transcription only in Sertoli cells and that mapped between positions -426 and -311. Sequence analysis of this region revealed a high concentration of potential regulatory elements. Using gel-shift assays combined with hydroxyl radical footprinting, we identified the elements recognized by the Sertoli cell nuclear factors. Comparison of the interactions with this region of the nuclear factors from different cell types demonstrated that recognition of the core-enhancer element is specific for the Sertoli cells, and in vitro, the core region was recognized by the transcription factor CBF. Transient transfections showed that a core enhancer is responsible for more than a half of the total promoter activity and is an essential element for the cell-specific activity of the Sertoli-specific region. In addition to the core enhancer, tandem Sp1 sites are also required for maximal activity of this region.

Sertoli cells, testes

INTRODUCTION

Clusterin is a highly conserved 74-kDa glycoprotein composed of two subunits that are the result of the proteolytic processing of a precursor polypeptide encoded by a single-copy gene. Clusterin has been independently isolated from many sources and characterized under a dozen different names: TRPM2, SGP2, CLI, ApoJ, and others (for review, see [1]). Despite the fact that this protein is very abundant, its physiological function remains unclear. The most convincing evidence suggests that clusterin can be an extracellular version of the heat-shock protein that may function as a membrane cytoprotector and/or as a clearing agent for extracellular damage [2–5].

The ubiquitous expression of clusterin is tightly regulated, and the level of expression is normally low. In the human, relatively high constitutive expression of clusterin can be seen in certain secretory cells of some fluid-epithelial interfaces [5] or as a result of environmental and pathophysiological stresses. For example, clusterin is a component of atherosclerotic plaques and is expressed by arterial smooth muscle cells in the vicinity of these plaques but not in normal arterial stroma [6]. Clusterin is also a component of senile plaques of Alzheimer disease [7]. In addition, intracellular amounts of clusterin are positively correlated with tumor aggressiveness in prostate cancer [8], and clusterin protein and mRNA are increased in meningiomas, astrocytomas [9], and renal clear-cell carcinomas [10]. At the fluid-epithelial interface, constitutive clusterin expression is also high in Sertoli cells of the testis; so high that clusterin comprises up to 70% of the total protein mass secreted by these cells in primary culture [3, 4].

These patterns of expression are evidence of a very strong promoter for the clusterin gene that can be either inducible or constitutive, depending on the cell type. Understanding the regulation of this promoter, and thereby gaining a better understanding of the function of clusterin itself, is important to understanding the direct involvement of clusterin in various cancers and pathological diseases. Because of the high constitutive expression of clusterin by Sertoli cells, we believe that these cells provide an ideal model to study the regulation of the clusterin gene promoter. Using transient transfection assays, we show here a 120-base pair (bp) region of the rat clusterin gene promoter that is active only in Sertoli cells, binds Sertoli cell nuclear proteins, and contains a core-enhancer site as the major functional element.

MATERIALS AND METHODS

Cell Culture

Sertoli cells were isolated from 20-day-old Sprague-Dawley rats and maintained in culture in serum-free Ham F12 media for 3 days, then in the media containing 10% bovine calf serum (BCS) [11]. MSC1 mouse Sertoli [12], NIH 3T3, mouse fibroblast, and COS7 monkey kidney cell lines were maintained in Dulbecco modified Eagle medium supplemented with 5% BCS for MSC1 and 3T3 and with 10% fetal calf serum for COS7 cells.

Plasmids

The plasmids pLLTRPM2+, pLL48, pLL311, pLL426, and pLL776 were obtained from Dr. Martin Tenniswood (Lake Placid, NY) and contained a region of the clusterin promoter and a fragment of the first exon (bases from +57 to -1302, -48, -311, -426, and -776, respectively) linked to the luciferase reporter gene. The other plasmids used in this study represent promoter constructs generated by polymerase chain reaction (PCR) mutagenesis using pLLTRPM2+ as a template. All PCR primers were designed with XhoI or HindIII sites, and PCR-amplified fragments were subcloned in the XhoI/HindIII sites of the pGL2-basic vector (Promega, Madison, WI) upstream of the firefly luciferase reporter gene. The following constructs were made: pWT, p412, and p308 containing a clusterin gene fragment from +57 to -480, -412, and -308, respectively; pSPmut, which had the same fragment as pWT but with four G-to-A substitutions of the underlined bases in the region (bases -378/-366); and pENHmut, containing the same fragment as pWT but with a single G-to-C transversion at position -317. The sequence fidelity of all constructs was verified by DNA sequencing. The plasmids were propagated in Escherichia coli DH5{alpha} and purified using the Endo-Free Plasmid purification kit (Qiagen, Valencia, CA).

Transient Transfection Assays

Transient transfection of primary Sertoli cells was performed using Ca3(PO4)2-DNA coprecipitation as described elsewhere [13], with some modifications. For the deletion constructs, cells were transfected with 3.5 µg of appropriate reporter vector and 0.35 µg of the coreporter vector pRL-SV40 (Promega) per dish. The coreporter vector encoded Renilla luciferase and was used as an internal standard of transfection efficiency unless otherwise specified. Primary Sertoli cells were plated onto 60-mm Petri dishes and transfected on Day 5 of culture. A fresh serum-free media was added 2 h before transfection, DNA was incubated with the cells for 6 h, and after that, the medium was changed and BCS added. The cells were harvested 24 h after transfection, and luciferase activity was assayed using the Dual-Luciferase Reporter Assay System (Promega) according to the protocol provided by the vendor. Fluoresence was quantified using a luminometer (MicroLumat LB96P, Berthold). For the data in Figure 1, cell lines at approximately 80% confluency were placed in serum-free media and transfected with 250 ng of an appropriate reporter vector and of pSV-ß-Gal coreporter vector encoding ß-galactosidase using LipofectAmine (Life Technologies Inc., Rockville, MD) and according to the recommendations of the supplier. DNA was incubated with the cells for 5 h, and serum was added and cells were harvested 43 h later. Luciferase activity was measured using Luciferase assay reagent (Promega); ß-galactosidase activity, using the Galacto-Light reporter assay system (Tropix, Bedford, MA) according to the protocols of the manufacturers. Luminescence was measured manually with the TRI-CARB liquid scintillation analyzer (Hewlett Packard, Palo Alto, CA). Each transfection experiment was done in triplicate and repeated at least three times. Data are presented as a mean ± SEM.



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  		FIG. 1. Activity of transfected 5' deletion constructs of the clusterin promoter in Sertoli cells and in NIH3T3, COS7, and MSC1 cell lines. Activity is given as a percentage of maximal and expressed as mean ± SEM

Electrophoretic Mobility Shift Assay

Extraction of nuclear proteins was performed according to the established procedures [14]. Nuclear proteins were extracted from the Sertoli cells on the sixth day of culture and after a 24-h incubation with 10% BCS unless otherwise specified. For the analysis of the effect of serum on nuclear protein binding, cells were maintained in serum-free media for 5 days, then 10% BCS was added and nuclear proteins were extracted after 1, 2, 4, 6, 8, 12, 16, and 24 h of incubation. Serum-free control extracts were made from the cells either immediately before the serum addition (control 1) or after 24 h of the parallel incubation of the cells without serum (control 2). The protein concentration in the extracts was determined by the micro-BCA method (Pierce, Rockford, IL).

A probe bracketing the Sertoli-specific region of the clusterin promoter (-436/-302) was obtained by Ksp 623I digestion of pWT. The resulting 135-bp DNA fragment was purified from an agarose gel, labeled using reverse transcriptase RT-Superscript (Life Technologies) and [{alpha}-32P]dATP (3000 Ci/mmol; NEN Life Science Products, Boston, MA), purified on a native 6% polyacrylamide gel in 0.5x Tris borate, EDTA buffer (TBE), extracted with dH2O and 20 000–40 000 cpm of probe (0.2–0.4 ng DNA) were used per binding reaction.

Oligonucleotide probes were synthesized in the Laboratory for Bioanalysis and Biotechnology (Washington State University) using phosphoramidate technology and were purified on 16% polyacrylamide gel containing 7 M urea. Radiolabeled probes were made with T4 polynucleotide kinase (Life Technologies) and [{gamma}-32P]ATP (3000 Ci/mmol, NEN Life Science Products), with subsequent annealing of strands and gel purification of the DNA duplex. The single-stranded oligonucleotide probes were also labeled using T4 kinase and gel purified. The following oligonucleotides were used in this study:


For simplicity, only the sense strand-derived oligonucleotides are shown. Positions of substitutions are given in bold in the mutant oligonucleotides and are underlined in a wild type.

Binding reactions with crude nuclear extracts were performed in 15 µl total volume in buffer containing 12.5 mM Hepes (pH 7.9), 60 mM KCl, 2.5 mM dithiothreitol (DTT), 0.05% Triton X-100, 10 mM MgCl2, 10% glycerol, 3 µg synthetic polynucleotide, and 1 µg E. coli DNA that had been heat denatured immediately before its addition to the binding reactions. Different types of synthetic polynucleotides (poly(dA)-poly(dT), poly(dG)-poly(dC), and poly(dI-dC); Pharmacia Biotech Inc., Uppsala, Sweden) were used to block nonspecific binding to the probes and to optimize detection of sequence-specific binding in each particular case. Binding reactions contained 2 µg of crude nuclear proteins and were performed on ice for 20 min. The resulting complexes were resolved on 6.5% native polyacrylamide gel in 0.5x TBE at room temperature or at 15°C if single-stranded probes were used. Competition electrophoretic mobility shift assay (EMSA) was performed in the presence of an indicated molar excess of the specific oligonucleotide competitor added before addition of the probe. For EMSA with specific antibodies, the extracts were incubated with antibody for 2 h on ice in the buffer containing 20 mM Hepes (pH 7.9), 100 mM KCl, 10 mM MgCl2, 8% glycerol, 0.5 mM DTT, and 0.2 mM PMSF, centrifuged for 10 min at 4°C, and used for EMSA. As a control, extracts were incubated with an equivalent amount of BSA or preimmune serum. Antibodies to Sp1, WT1, and early growth response factor 1 (Egr1) transcription factors were supplied by Santa-Cruz Biotech, Inc. (Santa Cruz, CA). For EMSA with recombinant proteins, reaction buffer contained 25 mM Tris-HCl (pH 7.9), 60 mM KCl, 6 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, 10% glycerol, and 4 µg BSA. Complexes were loaded under running conditions and resolved on 5% polyacrylamide gel in 0.5x TBE at 4°C. Recombinant murine CBF{alpha}2 (14–214 aa) was kindly provided by Dr. N.A. Speck (Dartmouth Medical School, NH) and Dr. B.J. Graves (University of Utah). Recombinant murine Ets-1, 12-kDa DNA-binding domain and 95-kDa full-length version, were also provided by Dr. B.J. Graves. Recombinant Sp1 was obtained from Promega.

DNase I Footprinting

These experiments were performed according to the standard protocol with minor modifications [15]. The probe was the XhoI/KpnI promoter fragment (bases -480/-265) of pWT labeled on a sense strand using T4 polynucleotide kinase and [{alpha}-32P]ATP. Nuclear proteins were extracted as described for EMSA and were dialyzed against a buffer containing 20 mM Hepes (pH 7.9), 60 mM KCl, 10 mM MgCl2, 20% glycerol, 1 mM DTT, and 0.2 mM PMSF at 4°C for 2 h. Binding reactions were carried out in 50-µl volume under the same conditions as for EMSA except that nonspecific DNA competitor was 5 µg poly(dA)-poly(dT) and 1 µg denatured E. coli DNA and contained 2 ng of radiolabeled probe and 15 µg of nuclear proteins or BSA (Pierce).

Hydroxyl Radical Footprinting

Nuclear proteins were purified as described for DNase I footprinting, except that glycerol was omitted from both extraction and dialysis buffers, and proteins were used immediately after preparation. The probes were the XhoI/KpnI promoter fragments of pWT (bases -480/-265) or p412 (bases -412/-265; Fig. 3 and data not shown) labeled on the sense strand using T4 polynucleotide kinase and [{alpha}-32P]ATP. Binding reactions were performed as described for DNase I footprinting, except that glycerol was absent. After a 15-min incubation with 5, 10, or 20 µg of the nuclear proteins on ice, DNA was subjected to the HR cleavage for 90 sec at ambient temperature as previously described [16]. Reactions were stopped by the addition of 50% glycerol in the amount of 1/10 of the reaction volume. Concentrations of H2O2 were 0.03% and 0.09% in the reaction mixture without and with nuclear proteins, respectively. Proteins were then digested with proteinase K (100 µg/ml) for 3 h at 45°C. DNA was phenol/chloroform extracted and ethanol precipitated. Samples were analyzed on 8% polyacrylamide-7 M urea gel, and equal loading was ensured by measuring Cherenkov counts in the samples. Data were analyzed using PhosphorImager and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).



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  		FIG. 3. Hydroxyl radical footprinting of 3' half of SER identifies multiple sites of interactions between Sertoli nuclear proteins and the SER. The positions of the specific sites recognized by Sertoli nuclear proteins are denoted by lines. F = free DNA; P = protein in decreasing amounts; G/A = sequencing ladder. The probe was the -412/-265 promoter fragment labeled on the 5' end of the sense strand; 5, 10, and 20 µg of Sertoli cell nuclear protein were used. The inset shows a larger view of the putative CBF-binding region

Computational Analysis

Sequence analysis was performed by using the Wisconsin Package, version 9.1 (Genetics Computer Group, Inc., University of Wisconsin, Madison). Pairwise comparison of the nucleotide sequences was performed using the Gap function. The local alignments were improved using the BestFit function, which was run with the randomization option (random = 100) to estimate the specificity of alignments calculated as a Z score. A search for transcription factor-binding sites was assisted by the Findpatterns function. Additional information was obtained with MaInspector, release 2.1, which was run with the following solution parameters: core similarity > 0.9 and matrix similarity > 0.85 [17]. All findings were further analyzed for the conservation of important bases within the sites using a compilation of the vertebrate-encoded transcription factors [18], reference information, and recent publications containing updated information relative to the transcription factor consensus sites in each particular case.

RESULTS

Transcriptional Activation by the Sertoli Enhancer Region (SER)

Primary Sertoli cells and the MSC1, NIH3T3, and COS7 cell lines were transiently transfected with 5'-deleted clusterin promoter fragments linked to the luciferase reporter gene (Fig. 1). The MSC1 cells express clusterin at a lower level than primary Sertoli cells but at a higher level than most other cell lines. We found that in MSC1 cells and all cell lines that normally express clusterin at a low level (e.g., NIH3T3 and COS7), the proximal 311 bp of the promoter were sufficient for maximal expression of the reporter gene ([19] and unpublished data). Consistent with our results was the finding that proximal 266 bp of the clusterin promoter were also sufficient to drive maximal expression of the reporter gene in the MA-10 cell line derived from the mouse Leydig cells normally expressing clusterin at a low level [20]. In contrast, in primary Sertoli cells, the upstream fragment from -426 to -311 was also required for the maximal promoter activity, and absence of this fragment decreased transcription of the reporter gene by 50 to 70% (Fig. 1). We subsequently refer to this region of the promoter as the Sertoli enhancer region (SER).

Computer-assisted analysis demonstrated that the clusterin promoter contains multiple, redundant, and often overlapping potential regulatory elements that are concentrated in two clusters. One cluster is located within the -40- to -210-bp proximal promoter, and another is located in the SER (-426 to -311; Fig. 2A). These potential sites within the SER include two each of high mobility group protein I(Y) (HMG-I[Y]), Sp1, Ets, and CBF sites and one each of Pets-like, Sry-related HMG-box protein/Sry, Egr1, and C/EBP sites. In addition, by using EMSA, we identified a site for the sequence-specific, single-stranded, purine-binding protein that we called PuBP because its identity remains unknown (unpublished results). A sequence comparison of the rat and mouse clusterin promoters shows that the promoter is highly conserved in rodents: there is 86% identity within the proximal 440 bp and 79% in the region containing the SER (bases from -436 to -302). Interestingly, within the SER, the islands of conservation are restricted to the putative transcription factor-binding sites, suggesting their potential functional importance (Fig. 2B).



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  		FIG. 2. Sequence analysis of the rat clusterin gene promoter. A) The nucleotide sequence of the rat clusterin gene promoter (GenBank accession number M64733). The nucleotides are numbered relative to the transcription initiation site. Potential transcription factor binding sites are given in bold and labeled. B) Sequence alignment of the rat and mouse promoter fragments bracketing the Sertoli enhancer region demonstrates conservation of transcription factor binding sites. Binding sites present in both promoters are shown in boxes and labeled

Hydroxyl Radical Footprint Analysis Identified Multiple Sites of the DNA-Protein Interactions Within SER

The sites of interactions of the Sertoli cell nuclear proteins with the SER region were analyzed by both DNase I and HR footprinting. DNase I footprinting revealed three broad, protected regions (data not shown). Because each of these regions contained two to four adjacent or overlapping potential transcription factor-binding sites, it was not possible to determine the exact sites of interactions. HR footprinting pinpointed the bases that interact with proteins and, thus, helped to identify the exact sites of interactions. The HR footprinting (Fig. 3) defined more details of the protected regions. In region 1 of Figure 3, protein-DNA interactions occur within the core-enhancer site with the following pattern of protection: C (hypersensitive bases shown in bold; protected bases underlined). Protected bases are those that may directly interact with a protein, whereas hypersensitive bases most probably result from the DNA distortion and increased sensitivity to cleavage caused by protein binding. This pattern of protection is consistent with a pattern of the DNA protection by CBF shown by the methylation interference assay [21] and suggests that CBF-related protein may be a core enhancer-binding protein in Sertoli cells. In region 2, interactions take place exactly within the Pets-like site. The Pets (peri-Ets) site is a GT-rich sequence without a clearly defined consensus located next to the Ets site and represents a site for binding of a potential Ets partner [22]. Unfortunately, our experimental conditions did not allow for definitive conclusions about possible interactions within the Ets site that characteristically demonstrates a very weak protection. Data shown in Figure 3 and additional HR footprints (data not shown) demonstrated that downstream interactions take place within an unidentified site AATGTGAAG (-397 to -386), Sp17/Egrl4 composite site (-383 to -373), and within PuBP and in two HMG-I(Y) sites (-446 to -402). For HMG-I(Y) sites, an extensive protection along the minor groove is consistent with binding of HMG-I(Y) that is known to recognize a minor groove of DNA [23].

Nuclear Protein-DNA Interactions Were Analyzed by EMSA

We investigated further the Sertoli-specific DNA-protein interactions within the SER in vitro by excising a 135-bp fragment (-436/-302) from the plasmid and using it as a probe in EMSA (Figs. 4–6). Electrophoretic mobility shift assay with proteins that were extracted from the Sertoli cells cultured in the presence of serum detected at least 10 different DNA-protein complexes manifested as shifted probe bands. This is consistent with detection of multiple protected regions by footprint analysis. This significant binding was observed with as little as 2 µg of protein and in the presence of the 5000-fold excess of the nonspecific competitor E. coli DNA added to the commonly used synthetic polynucleotides. DNA binding by Sertoli cell nuclear proteins was compared to DNA binding of proteins extracted from the several cell lines (Fig. 4). The primary difference in binding between nuclear extracts from Sertoli cells and other cell lines was a band referred to as the Sertoli-specific band (marked with an arrow). All the other bands detected using Sertoli nuclear proteins were present in at least one other cell extract.



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  		FIG. 4. EMSA: Sertoli cells contain a unique set of nuclear factors that interact with the Sertoli enhancer region. Nuclear proteins were extracted from different cell types (shown above), and their binding to the Sertoli enhancer region (-436/-302) was compared. Binding reactions were performed in the presence of 3 µg poly(dI-dC) and with 5000-fold excess of E. coli DNA as an additional nonspecific competitor. The arrow indicates the most prominent band obtained with Sertoli cell extracts (Sertoli-specific band or band 4)

Assuming that the factor essential for the constitutive expression is continuously expressed itself, we tested whether the Sertoli-specific band is the result of constitutive protein binding. Bovine calf serum was used as an inducing factor. The Sertoli cells were cultured in serum-free media, then serum was added and nuclear proteins isolated at various time points following stimulation (from 1 to 24 h), and their binding to the SER was compared by EMSA (Fig. 5). We found that about half of the EMSA-shifted bands were constitutive, including Sertoli-specific band (band 4). Band 4 was represented by a doublet, and the relative intensity of the two band complexes was not changed by serum treatment.



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  		FIG. 5. EMSA: Effect of serum on nuclear protein binding to the Sertoli enhancer region. Comparison of binding to the SER (-436/-302) of Sertoli cell nuclear proteins extracted after a defined period of incubation with BCS (time is indicated above). Binding reactions were performed in the presence of E. coli DNA and poly(dI)-poly(dC). The bands that result from constitutive binding are marked with arrows and specified by numbers according to the pattern in Figure 6A

Competition EMSA with specific competitor DNA corresponding to the different double- or single-stranded fragments of SER (see Materials and Methods) was used to identify the site of Sertoli cell-specific recognition (Fig. 6). Of all the competitors used, only the wtEnh oligonucleotide was able to compete for the proteins in the Sertoli-specific band, band 4 (Fig. 6A). This oligonucleotide represents a composite element containing the core-enhancer site (shown in bold) and three potential Ets sites (two reverse sites are underlined, and one direct site coinciding with a core-enhancer is shown in bold). A similar competition with wtEnh for 3T3 and MSC1 extracts did not result in displacement of any band, proving that recognition of a core element can be specific for the Sertoli cells in a tested cell range (data not shown). We also examined the ability of mutant oligonucleotides (mEnh-1 to -4) to compete for binding. The mutant oligonucleotides had different combinations of C/G to G/C transversions (Fig. 6B). All oligonucleotides (mEnh-1, -3, and -4) that contained the G-to-C mutation that disrupted the superimposed core/Ets sites failed to compete for binding (Fig. 6A, lanes 4, 6, and 7). Moreover, a mutant oligonucleotide competitor with a single mutation in the core/Ets site (mEnh-1) is sufficient for eliminating competition at the enhancer core. The mutant competitor mEnh-2 did compete for binding, although somewhat less efficiently than did wtEnh (compare lanes 3 and 5 in Fig. 6A). Therefore, we conclude that Sertoli-specific band 4 is a result of binding to the core/Ets site.



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  		FIG. 6. Competition EMSA of Sertoli cell nuclear factor binding to the Sertoli enhancer region identifies a Sertoli-specific band as a result of recognition of the core-enhancer element. A) Nuclear proteins were assayed for binding to the Sertoli enhancer region (-436/-302) in the presence of 5000-fold excess of E. coli DNA to minimize nonspecific interactions. Binding was challenged by the addition of the double- or single-stranded oligonucleotides corresponding to different regions within this fragment (shown above). The shifted bands are specified by numbers. Binding reactions were performed in the presence of 3 µg poly(dA)-poly(dT) (lanes 1–9) or poly(dG)-poly(dC) (lanes 10–14). Each oligonucleotide competitor was added in a 250-fold molar excess. B) The sequences of the oligonucleotides are presented; specific binding sites are underlined and labeled. Oligonucleotides were designed as wild type or mutant, in which specific base substitutions were made to disrupt a specific potential binding site. For the mutant oligonucleotides, positions of substitutions are shown; - indicates the unchanged bases

Next, we tested whether Ets proteins can bind to the core/Ets site and determined the base requirements for such recognition. The wild-type and mutant Enh-oligonucleotides described in Figure 6B were used as probes for binding of a recombinant 12-kDa DNA-binding domain of the Ets-1. This DNA-binding domain is conserved in all members of the Ets family, has similar DNA-binding characteristics as the full-length protein, and recognizes the Ets consensus sequence GGAW [24]. Of the three potential Ets-elements, only the middle one was recognized by Ets DNA-binding domain (Fig. 7A). The full-length recombinant Ets-1 protein also bound to this site (Fig. 7B). In contrast, mutation of the G residue (mEnh-1) that is essential for the Sertoli-specific recognition of the core/Ets site did not affect binding of either 12-kDa and full-length Ets-1 (Fig. 7A and data not shown). From these results, we conclude that this is a core-enhancer element rather than a coinciding Ets site that is specifically recognized by Sertoli nuclear proteins.



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  		FIG. 7. EMSA: Transcription factor Ets-1 binds specifically to the core element in the Sertoli enhancer region. A) Binding of the 12-kDa Ets-1 to the wt and mutant oligonucleotide probes identifies Ets binding site. Concentration of 12-kDa Ets-1 in binding reactions was 25 nM. The arrows indicate the Ets-shifted bands; n.s., nonspecific. The sequence of wild-type oligonucleotide is shown below; potential Ets sites are underlined. The sequences of mutant oligonucleotides are given in Figure 6B. B) the full-length Ets-1 protein binds to the wtEnh oligonucleotide. Concentration of the protein in the binding reactions is indicated above

Recombinant murine CBF{alpha}2 was also capable of binding to the core/Ets site within SER (Fig. 8A). In contrast to the binding of Ets, the effect of the core site mutations on binding of CBF (Fig. 8B) was similar to the effect of the core site mutations on Sertoli nuclear protein binding demonstrated in the competition experiments. By comparing complex formation on the wtEnh probe to that on mEnh-1, mEnh-2, and mEnh-3, we conclude that it is the core/Ets site within the SER that is recognized by CBF (Fig. 8B). Titration with increasing amounts of protein showed that half-saturation of a wtEnh probe occurred at CBF concentration between 2.5 x 10-8 and 2.5 x 10-9 M (Fig. 8C). This titration gives a rough estimate of the dissociation constant about 10 nM for the CBF protein, which is within an expected physiological range. Because Ets proteins are known as CBF accessory factors [25–27], we also tested whether Ets-1 and CBF can bind to wtEnh probe simultaneously. Unexpectedly, their binding was mutually exclusive (data not shown).



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  		FIG. 8. EMSA: The core element in the Sertoli enhancer region is recognized by core binding factor CBF{alpha}2 in vitro. Concentration of recombinant murine CBF in binding reactions was 25 nM unless otherwise specified. The arrows indicate CBF-shifted bands. A) Binding of recombinant CBF to the SER probe (-436/-302). B) Binding of CBF to the oligonucleotide probes containing different base substitutions demonstrates similarity with binding of the Sertoli-specific band-4 factor. The probes used are defined above. The sequences of the probes are given in Figure 6B. C) Titration with an increasing amount of CBF. The probe is wtEnh oligonucleotide. Concentration of CBF in the binding reaction is shown above

Finally, competition with wtSp, but not mutant mSp, oligonucleotide demonstrated that bands 6 and 9 result from protein binding to Sp1 sites (Fig. 6A, lanes 8 and 9). Because only the Sp17 (see Fig. 2A for designation of sites) site was protected in footprinting, we believe that only this site is recognized. Unfortunately, identification of proteins interacting in this region was complicated by the fact that the Sp17 site overlaps a noncanonical Egr1 site (; Sp1 site is underlined; Egr1 site is shown in bold). Mutations that disrupt an Egr1 site may also affect binding of Sp proteins. Although Egr1 consensus contains one deviation of the 5'-flanking base A from the consensus base G, this is a bona fide Egr 1-binding site [28]. Supershift analysis with anti-Egr1 demonstrated that it bound Egr 1 present in 3T3 and COS7 nuclear extracts (data not shown). Recombinant Sp1 was also able to bind to this site (data not shown). Nevertheless, our attempts to identify the proteins that bind to these sites in the Sertoli extracts by using specific antibodies to Sp1, Egr1, and WT1 (a member of the Egr1 family expressed in Sertoli cells) have not been successful (data not shown), and identity of these proteins is yet to be determined.

Core-Enhancer Element Is Essential for the Function of SER

Transient transfection experiments using wild-type and mutant promoter constructs were used to examine the function of the cis elements in transcription (Fig. 9). The relative luciferase activity of the mutant constructs is given as a percentage of activity of the wt promoter construct. Deletion of the SER fragment upstream of position-412 that contains HMGI/Y and PuBP sites that were protected in the footprinting resulted in the loss of only 20.8 ± 5.9% of the promoter activity (p412). Disruption of Sp1 sites by introducing the mutations that prevent protein binding to these sites in EMSA decreased promoter activity by 47.4 ± 4.9% (pSPmut). Finally, a single-base pair mutation that has been shown to eliminate binding to the core-enhancer site in EMSA decreased promoter activity by 55.8 ± 6.1% (pENHmut). This single mutation was essentially equivalent to the removal of the entire SER that decreased promoter activity by 62.0 ± 4.3% (p308). Thus, the core enhancer is an essential element of SER, but the presence of functional Sp1 sites is also required for its activity.



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  		FIG. 9. Transient transfection of primary Sertoli cells identifies the core-enhancer site as a necessary but not sufficient element for transcriptional activation by the Sertoli enhancer region. The lines on the left are schematic representations of the clusterin promoter constructs used in the transfection experiments. Numbers indicate the positions of the ultimate 5'-nucleotides in the promoter fragment; the position of the ultimate 3' nucleotide was +57. Location of the base substitutions introduced in SER is indicated by an x. The p412 construct had a deletion of the fragment containing HMG-I(Y), Sox/Sry, and PuBP binding sites. The pSPmut construct contained two disrupted Sp1 sites. The pENHmut construct contained a single G to C transversion that knocked-out the core-enhancer site. The p308-constructs had the entire SER deleted. The firefly luciferase activity of the reporter gene was normalized to the Renilla luciferase activity that was used as a coreporter. The bars on the right represent normalized luciferase activity of the 5'-deleted or mutant constructs relative to the wt construct. Results are given as mean ± SEM

DISCUSSION

We have identified a 120-bp region (an SER) of the rat clusterin gene promoter that plays a significant role in Sertoli cell-specific constitutive expression of the clusterin gene. We showed that within this 120 bp, a core-enhancer element is required for cell-specific transcription. Binding of Sertoli cell nuclear proteins to this element was demonstrated by the HR footprint assays and further substantiated by EMSA. The binding of nuclear proteins to the clusterin core-enhancer element was constitutive and specific for Sertoli cells. Moreover, the binding was greatly reduced by a single base substitution of a to C in the core sequence . Interestingly, this mutation had basically the same effect on the promoter activity as the removal of the entire 120-bp SER in the transient transfection experiments.

The core enhancer element is an essential component of many mammalian viral enhancers. It is commonly found in many papovaviruses (e.g., simian virus 40 [SV40], polyoma) and type C retroviruses (e.g., Moloney murine leukemia virus [MoMLV], MLV, murine sarcoma virus [MSV]) [24, 29, 30]. The core element can contribute to constitutive expression and is one of the primary determinants of the cell specificity in the virus enhancers [27,31]. A core-enhancer consensus was defined as GTGTGG(A/T)(A/T)(A/T)G [29]. However, for a particular enhancer, the function of this site was very sensitive to base substitutions, even to those permitted by the consensus. A single-base pair substitution rendered it nonfunctional for transcriptional activation [32]. Similarly, mutations in the core element involving the first G in the dinucleotide GG attenuate transcription from MoMLV and SL3-3 MLV promoters in a cell-specific manner and abolish cell-specific function of the complete 200-bp transcriptional enhancer of the T cell receptor (TCR){gamma} gene in T cells [25, 33, 34].

Similar "core" elements have also been characterized in the promoters of many cellular genes that demonstrate cell-specific expression, including interleukin 3 and TCR{alpha}, ß, {delta}, and {gamma} in T lymphocytes [25, 34–38]; myeloperoxidase, neutrophil elastase, macrophage colony-stimulating factor receptor in myeloid cells [39]; osteocalcin in osteoblasts [40]; and ß-myosin heavy chain in myoid cells [41]. The clusterin gene in Sertoli cells can be now added to this list. Several transcription factors have been shown to bind to the core depending on the exact sequence of the site, including transcriptional enhancer factor, TEF-1; CAAT/enhancer-binding protein, C/EBP; and core binding factor, CBF [21, 42–46]. CBF (PEPB2/AML1/SEF-1) recognizes the consensus YGYGGTY, binds to polyoma, MoMLV, and SL3–3 MLV virus core sequences and regulates expression of the aforementioned genes in lymphoid and myeloid cells [21, 44]. A related protein NMP2 has been found in osteoblasts [40]. C/EBP binds to SV40 GT-I, polyoma, and MSV core-enhancers and has a consensus site TKNNGYAAK [43, 45]. TEF-1 binds to the SV40 GT-II core and belongs to the TEA/ATTS family, whose DNA-binding consensus sequence is WRRWATGY [46]. Because both C/EBP and TEF-1 consensus sites are dissimilar to the clusterin core element TGTGGATTC and because their binding does not seem to depend on the G residue in the fourth position, C/EBP and TEF-1 proteins are probably not involved in clusterin gene expression.

Families of transcription factors are represented by a rapidly increasing number of protein members, making it difficult to assess which specific factor is involved in the in vivo gene regulation, especially when primary cells are used rather than well-characterized cell lines. The presence of transcription factors that are encoded by multiple alternatively spliced mRNAs makes analysis even more complicated because alternative splicing can remove epitopes recognized by a specific antibody [28]. Although our study identifies a core enhancer as an essential element, it does not establish the identity of the protein or proteins that interact with it. However, the dependence of binding in the clusterin core sequence on a single G residue, a recognizable pattern of protection using hydroxyl radical footprinting, and the binding of recombinant CBF{alpha}2 to this site strongly suggest that this protein may belong to the CBF family or has similar DNA-binding characteristics.

The core element and its binding proteins (e.g., CBF and TEF-1) have an essential role in the formation of the cell-specific complexes on several promoters, but activation of transcription requires a specific context or specific cofactors [26, 32, 38, 46]. Therefore, the core element binding proteins are thought to function rather as transcriptional organizers. Cooperation between Sp1 and CBF has been previously reported, and the members of Sp1 family are expressed in the testis and known to regulate the constitutive expression of many genes [47–49]. Because the Sertoli cell nuclear proteins recognize Sp1 sites in SER, we tested the effect of Sp1 sites on clusterin gene transcription. We found that Sp1 sites were also required and that mutations that disrupt them decrease promoter activity about twofold. Therefore, in the clusterin promoter, the core site seemed to be necessary but not sufficient for full transcriptional activation by SER in transfection assays. The effects of mutations in the core and Sp sites were not additive, suggesting that the proteins that bind these sites affect the same process.

The transient transfection method has limitations as a gene transcription assay system. Although plasmid DNA introduced into cells by transient transfection becomes assembled into chromatin, its structure is generally looser and different from the physiological chromatin [50]. This chromatin structural difference may be even more dramatic for nondividing cells such as Sertoli cells, because there may not be a significant pool of free histones in the cells, and DNA introduced into them may remain essentially naked. This situation may distort the results of the transfection assays. First, sites that may be inaccessible and inactive in physiological chromatin may become accessible for the transcription factors to bind in such transient transfection assays. In our experiments, this situation would create an enormous background as a clusterin proximal promoter contains multiple sites for ubiquitous transcriptional activators (Fig. 2A). Second, some transcriptional activators, such as CBF, are especially active on chromatin templates and may be only moderate activators on naked DNA templates [51]. Thus, the presence of transfected DNA as a naked species may explain why the core site stimulated transcription only 2.5 times; however, the use of the dividing Sertoli cell line (MSC1) would not allow detection of either Sertoli-specific sites or proteins (Fig. 4).

In a search for potential transcriptional activators, we identified numerous binding sites for the nuclear factors interacting with SER. These were detected as multiple bands in EMSA and as multiple regions of protection in footprint analysis and were recognized by the recombinant proteins. This multiple binding was specific because it was detected in the presence of a large excess of E. coli DNA in the binding reactions (see Materials and Methods). The physiological significance of this multiplicity remains to be determined. The sites may be differentially used in different cells in vivo. Within one particular cell type, this multiplicity may be beneficial in the context of chromatin where transcription factors have to compete with nucleosomes for binding to DNA. Because binding of transcription factors is intrinsically cooperative in the chromatin context [52], the presence of the multiple sites recognized by many different proteins will increase the ability of factors to compete with nucleosomes and to target chromatin-remodeling mechanisms to the particular sites within a promoter [53]. This may result in efficient nucleosomal displacement and inefficient repositioning, higher occupancy of a promoter with transcriptional activators, and establishment of a constitutive enhancer within SER. Furthermore, the core element within this enhancer may nucleate the formation of the complex in a cell-specific manner.

These studies describe an element of the clusterin gene promoter that is involved in the constitutive synthesis of clusterin in the Sertoli cells of the testis. This element presumably accounts for the relatively high basal level of clusterin synthesis in the testis. The physiological meaning behind the high levels of testicular clusterin remain unknown at this time but may relate to the relatively high frequency of germ cell apoptosis and the need to remove and clear debris from dead cells.

ACKNOWLEDGMENTS

We thank Dr. Gerhard Munske for the synthesis of oligonucleotides, Alice Karl and David Marsh for the preparation of Sertoli cell primary cultures, and Dr. Tamara L. Goetz for technical assistance. We also thank the people who provided us with important reagents: Dr. Nancy A. Speck (Dartmouth Medical School, NH) for recombinant CBF{alpha}2, Dr. Martin P. R. Tenniswood (Lake Placid, NY) for TRPM2+ promoter constructs, and Dr. Barbara J. Graves (University of Utah) for recombinant Ets-1 and for the opportunity to work in her laboratory. We are also very grateful to Dr. Walter A. Tribley and Dr. Daniel S. Johnston for the critical reading of the manuscript.

FOOTNOTES

First decision: 24 March 2000.

1 Correspondence. FAX: 509 335 9688; griswold{at}mail.wsu.edu Back

2 Current address: The Rockefeller University, New York, NY 10021. Back

3 Current address: Ontogeny, Inc., Cambridge, MA 02138-1118. Back

Accepted: June 8, 2000.

Received: February 4, 2000.

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