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Originally published In Press as doi:10.1074/jbc.M409616200 on December 1, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4462-4468, February 11, 2005
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Repression of the Human Sex Hormone-binding Globulin Gene in Sertoli Cells by Upstream Stimulatory Transcription Factors*

David M. Selva, Kevin N. Hogeveen{ddagger}, and Geoffrey L. Hammond§

From the Department of Obstetrics & Gynaecology, University of British Columbia and the B. C. Research Institute for Children's and Women's Health, Vancouver, British Columbia V5Z 4H4, Canada

Received for publication, August 20, 2004 , and in revised form, November 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of the sex hormone-binding globulin gene (SHBG) in the liver produces SHBG, which transports sex steroids in the blood. In rodents, the SHBG gene is also expressed in Sertoli cells giving rise to the testicular androgen-binding protein, which is secreted into the seminiferous tubule where it presumably controls testosterone action. Evidence that the SHBG gene functions in this way in the human testis is lacking, and mice containing a human SHBG transgene (shbg4) under the control of its own promoter sequence are characterized by SHBG gene expression in the liver but not in the testis. A potential cis-element, defined as footprint 4 (FP4) within the human SHBG promoter, is absent in SHBG promoters of mammals that produce the testicular androgen-binding protein, and we have produced mice harboring a shbg4 transgene in which FP4 was deleted to evaluate its functional significance. Remarkably, these mice express the modified human SHBG transgene in the testis as well as the liver. Human SHBG transcripts were found within their Sertoli cells, primary cultures of which secrete human SHBG, and this was increased by treatment with follicle-stimulating hormone, retinoic acid, and estradiol but not testosterone. We have also found that the upstream stimulatory factors (USF-1 and USF-2) bind FP4 in vitro by electromobility shift assay of Sertoli cell nuclear extracts and in vivo by chromatin immunoprecipitation assay and conclude that USF transcription factors repress human SHBG transcription in Sertoli cells through an interaction with FP4 within its proximal promoter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasma sex hormone-binding globulin (SHBG)1 is produced by hepatocytes, and it transports sex steroids in the blood and regulates their access to target tissues (1). A protein with similar physiochemical and steroid-binding characteristics as plasma SHBG was originally identified in the testis and epididymis of rats and rabbits (2–4). This protein, which is generally referred to as the testicular androgen-binding protein, has since been shown to be the product of the same transcription unit that encodes plasma SHBG (5). A substantial body of work has established that androgen-binding protein production by Sertoli cells in several animal models is regulated by FSH and retinoids but not testosterone (2, 6). Because this protein is secreted preferentially into the seminiferous tubules (7) and migrates to the caput epididymis where it is internalized by epithelial cells (8), it is thought to regulate androgen access to target cells within the male reproductive tract and thereby influence androgen-dependent mechanisms of sperm maturation (5).

The identification of a protein in human testis extracts with essentially the same biochemical properties as plasma SHBG (9, 10) led to the widely held assumption that SHBG expression and function in the human testis mirrors that in other mammals. However, human testis extracts are invariably contaminated by plasma SHBG, and it has been virtually impossible to determine whether the SHBG in these extracts is really testicular in origin. In fact, although SHBG transcripts can be readily detected in human testis (11), conclusive evidence that human Sertoli cells actually produce SHBG is lacking. Moreover, SHBG transcripts in the human testis are distinct from those in rat testis in that the majority of human transcripts comprise an alternative exon 1 sequence (11, 12). These alternative human SHBG transcripts are products of a distinct transcription unit, which appears to be under the control of a promoter whose activity is confined in the testis to germ cells (13), and they appear to encode an isoform of SHBG that accumulates in the acrosome of sperm (13).

In transgenic mice, a 5.5-kb rat SHBG transgene is expressed appropriately in Sertoli cells during puberty and in sexually mature animals (15). By contrast, the corresponding human SHBG transgene is expressed in mouse hepatocytes throughout development (16) but is not expressed at all in the testis (13, 14). This suggested to us that the human SHBG promoter must differ in some way from the corresponding promoters in other mammals that produce an SHBG homologue (i.e. androgen-binding protein) in Sertoli cells, and a phylogenetic comparison showed that the human SHBG promoter contains a potential cis-element (footprint 4) identified previously by DNase 1 footprinting (17), which is not present in the SHBG promoters of subprimate species. Removal of this footprint 4 (FP4) from a human SHBG transgene permits its expression in Sertoli cells in vivo, and the modified transgene responds to hormone treatment in a manner consistent with that of the rat SHBG in Sertoli cells. Because we have also found that the upstream stimulatory factors (USF-1 and USF-2) bind to FP4 in the human SHBG promoter we conclude that this likely represses its activity in Sertoli cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—Transgenic mice (line shbg 4-a) containing a 4.3-kb region of the human SHBG gene have been characterized previously (14, 16). The FP4 sequence within the SHBG promoter was removed from the 4.3-kb human SHBG transgene construct using the following mutagenic oligonucleotide (5'-CTTGACCCCTGCCCAGGGGCTCACCCCTCTGGGGATCAAT) in the Altered Sites® mutagenesis system (Promega, Madison, WI) and was introduced into the mouse genome by pronuclear injection. Injected embryos were implanted into pseudo-pregnant recipient mice using a standard protocol (18). Two independent lines were established from founders, and progeny were screened by PCR amplification of human SHBG transgene sequences using primers spanning exon 7 (forward, 5'-GGAAGAATTCGGCCACAGGCAGTAGGC; reverse, 5'-GGTTGAATTCCGCCTCCCTTGAGCTG).

Animals were housed under standard conditions and provided with food and water ad libitum.At ~10 weeks of age, mice were sacrificed for the isolation of Sertoli cells and/or germ cells for RNA analysis and primary cell cultures (see below). All procedures were approved by the Animal Use Subcommittees of the University Council on Animal Care (University of Western Ontario and University of British Columbia).

Testicular Cell Isolation and Primary Sertoli Cell Culture—Sertoli cells and germ cells were isolated from the testes of wild-type and transgenic mice using an established method (19, 20) and frozen for RNA analysis (see below). For primary Sertoli cell cultures, a mixed population of Sertoli and germ cells was cultured at 33 °C for 10 h in Dulbecco's modified Eagle's medium (Invitrogen) containing 2% fetal bovine serum, and germ cells were removed by washing and aspiration of the adhered Sertoli cells. Sertoli cells isolated in this way were cultured for up to 8 days in Dulbecco's modified Eagle's medium containing 2% fetal bovine serum. For experiments involving treatments with hormones, cells were treated every 2 days with different concentrations of FSH, retinoic acid, estradiol, and testosterone, and culture medium samples were collected for human SHBG measurements by time-resolved immunoassays (see below).

RNA Analysis—Total RNA was extracted from mouse liver, testes, and isolated testicular cells using TRIzol reagent (Invitrogen). Poly(A)+ RNA was extracted using the PolyATtract mRNA isolation system III (Promega). The RNA was separated by electrophoresis on a 1% agarose gel in the presence of formaldehyde and transferred to a Zeta-Probe nylon membrane (Bio-Rad). The membrane was hybridized with a 32P-labeled human SHBG cDNA (21). Murine 18 S ribosomal RNA and cyclophilin A cDNAs were also used as an additional control for RNA loading and transfer.

To confirm the presence or absence of human SHBG transcripts in separated testicular cell types we also performed a standard reverse transcription-PCR based assay (13) using total RNA extracts with human SHBG-specific primer pairs (forward, 5'-GTTGCTACTACTGCGTCACAC; reverse, 5'-AAGAGGTGGAAGAGTCTTCTC) as well as mouse cyclophilin A-specific primer pairs (forward, 5'-CAGATGGGGTAGGGACG; reverse, 5'-ATGGTCAACCCCACCACCGTG) as an internal control. The PCR was performed for 35 cycles at 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 45 s.

Time Resolved Immunofluorometric Assay—Culture medium samples (100 µl) from primary Sertoli cultures were added to the wells of a Nunc MaxiSorpTM (Nalge Nunc International, Rochester, NY) plate previously coated with a rabbit polyclonal anti-serum against human SHBG and blocked with a buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl) containing 1% casein. After three washes with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% Tween-20, the wells were incubated with 100 µlof europium-labeled monoclonal antibody against human SHBG (22) diluted 1/500 in assay buffer (PerkinElmer Life Sciences) for 1 h at room temperature. After three washes, the wells were incubated with shaking in 100 µl of enhancement solution (PerkinElmer Life Sciences) at room temperature for 10 min in the dark. Time-resolved fluorescence was then measured in a Victor-3 plate reader (PerkinElmer Life Sciences).

Electrophoretic Mobility Shift Assays—Nuclear extracts were prepared from cultures of TM4 (obtained from the American Type Culture Collection) and MSC-1 (kindly provided by Dr. M. Griswold) mouse Sertoli cell lines as described (23). The Sertoli cell nuclear extracts (10 µg) were used in an electrophoretic mobility shift assay (EMSA), as described previously (17, 24). Complementary pairs of oligonucleotides used in the EMSAs included overhanging extensions (lowercase letters) to facilitate labeling with Klenow fragment of DNA polymerase I and [{alpha}-32P]dCTP (24). The same oligonucleotide pairs were also used as unlabeled competitors in the EMSA reactions. They included a sequence spanning FP4 (5'-tcgacTGATAGCTGAGTCTTGTGACTGGGCCCCTc, 5'-gACTATCGACTCAGAACACT GACCCGGGGAgagct), a similar FP4 sequence in which the core element within the putative USF binding site was mutated (5'-tcgacTGATAGCTGAGTCCCGCGACTGGGCCCCTc, 5'-gACTATCGACTCAGGGCGCTGACCCGGGGAgagct), and a DNA sequence (5'-tcgacGATCTGTAGGCCACGTGACCGGc, 5'-gCTAGACATCCGGTGCACTGGCCgagct) from the adenovirus major late promoter (MLP) containing a known USF binding site (25).

The corresponding end-labeled oligonucleotide probes were added to the nuclear extracts, and the binding reaction was allowed to proceed for 15 min at room temperature. For antibody supershift EMSA experiments, nuclear extracts were incubated on ice for 10 min before and 15 min after the addition of the radiolabeled oligonucleotide. Aliquots (1 µg) of antiserum against USF-1 (C-20, catalog number sc-229, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), USF-2 (C-20, catalog number sc-862, Santa Cruz Biotechnology), or normal rabbit serum were then added, and the complexes were further incubated at room temperature for 15 min. In EMSA and supershift EMSA experiments, protein-DNA complexes were separated from free probe by 6% PAGE, and the gel was dried and exposed to Biomax MR film (Eastman Kodak Co.) against an intensifying screen at –80 °C.

Chromatin Immunoprecipitation Assay—Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP-ITTM kit (Active Motif, Inc., Carlsbad, CA) following the manufacturer's instructions. Unless otherwise stated, all reagents, buffers, and supplies were included in the kit. Briefly, Sertoli cells and germ cells from transgenic mice were cross-linked with 1% formaldehyde for 10 min at room temperature. After washing and treatment with glycine Stop-Fix solution, the cells were re-suspended in lysis buffer and incubated for 30 min on ice. The cells were homogenized and nuclei were re-suspended in shearing buffer and subjected to optimized ultrasonic disruption conditions to yield 100–400-bp DNA fragments. The chromatin was precleared with protein G beads and incubated (overnight at 4 °C) with 1 µg of each of the following specific antibodies: negative control mouse IgG and positive control anti-TFIIB (provided in the ChIP-ITTM kit), anti-USF-1 (Santa Cruz Biotechnology) anti-USF-2 (Santa Cruz Biotechnology), anti-COUP-TF II (T-19, catalog number sc-6578, Santa Cruz Biotechnology), and anti-HNF4{gamma} (C-18, catalog number sc-6558, Santa Cruz Biotechnology). Protein G beads were then added to the antibody/chromatin incubation mixtures and incubated for 1.5 h at 4 °C. After extensive washings, the immunoprecipitated DNA was removed from the beads in an elution buffer. To reverse cross-links and remove RNA, 5 M NaCl and RNase were added to the samples and incubated for 4 h at 65 °C. The samples then were treated with proteinase K for 2 h at 42 °C, and the DNA was purified using gel exclusion columns. The purified DNA was subjected to PCR amplification (1 cycle of 95 °C for 2 min, 40 cycles of 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 30 s) of the SHBG promoter using specific forward (5'-GATCCCCAGAGGGGTGATAGC) and reverse (5'-GGGTAAAGGAAACAGGGGCAC) primers designed to amplify a region (–130 to –48 nucleotides in relation to the transcription start site) spanning FP3 and FP4 in the human SHBG promoter (17). As a control, PCR amplification (1 cycle of 95 °C for 2 min, 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s) of the murine GAPDH promoter was performed using speciesspecific forward (5'-AGTGCCAGCCTCGTCCCGTAGACAAAATG) and reverse (5'-AAGTGGGCCCCGGCCTTCTCCAT) primers (26). The PCR products were resolved by electrophoresis in a 6% acrylamide gel and visualized after ethidium bromide staining.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Phylogenetic Comparison of SHBG Promoter Sequences— Alignment of human (NCBI accession code M31651 [GenBank] ), sheep (NCBI accession code AY838765 [GenBank] ), rabbit (NCBI accession code AF144711 [GenBank] ), rat (NCBI accession code M19993 [GenBank] ), mouse (NCBI accession code AL731687 [GenBank] ), and chimpanzee (NCBI accession code AADA01179238) SHBG promoter sequences available in the public data base (www.ncbi.nlm.nih.gov), showed that a footprinted region (FP4) in the human SHBG proximal promoter (17) is present in the chimpanzee SHBG promoter but is absent in the SHBG promoters of all the other species. An alignment of the human, chimpanzee, rabbit, sheep, and rat SHBG promoters illustrates this difference (Fig. 1). Analysis of this region using a public data base (wwwmgs.bionet.nsc.ru) indicated that it contains a potential USF binding site (underlined in Fig. 1). The FP3 region in the human SHBG promoter (Fig. 1) is known to interact with COUP-TF and HNF4 (17), and the binding sites for these related transcription factors comprise a consensus GGTCA repeat separated by a variable number of nucleotides (27). Thus, although there are two nucleotides between the GGTCA repeat within the human SHBG FP3 sequence, these repeats in the rat and rabbit SHBG promoters have one and three nucleotides between them, respectively (Fig. 1).



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  		FIG. 1.
Phylogenetic comparison of SHBG promoter sequences. The human SHBG sequence from nucleotides –57 to +1 relative to the transcription start site in the liver (17) is aligned with corresponding regions of the chimpanzee SHBG (NCBI accession code AADA01179238), sheep SHBG (NCBI accession code AY838765 [GenBank] ), rat SHBG (NCBI accession code M19993 [GenBank] ) and rabbit SHBG (NCBI accession code AF144711 [GenBank] ) sequences. Footprinted regions (FP1–FP4) identified previously within the human SHBG promoter (17) are shown. A putative USF binding site within the FP4 region is underlined. FP1 and FP3 have been shown previously to interact with COUP-TF and HNF-4 (17).

 
Deletion of FP4 Permits Expression of Human SHBG in Sertoli Cells—As reported previously (14), human SHBG transcripts are not present in testis RNA extracts from mice harboring the 4.3-kb human SHBG transgene. However, they are detectable in the testis extracts of mice containing the same region of the human SHBG in which FP4 has been deleted (Fig. 2A). By contrast, deletion of the FP4 sequence within this transgene has essentially no effect on its expression in the liver as measured by Northern blotting of SHBG mRNA (Fig. 2A) or serum SHBG levels (data not shown). The same observations were made using samples from two independent lines of mice containing the modified human SHBG transgene lacking FP4 (data not shown).



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  		FIG. 2.
Removal of the FP4 region from the human SHBG proximal promoter results in expression of a 4.3-kb human SHBG transgene in the testis as well as the liver of transgenic mice. A, human SHBG transcripts were detected by Northern blotting in total RNA extracted from the liver of 4.3-kb human SHBG transgenic mice that contained (FP4+) or lacked (FP4–) the FP4 region within the human SHBG proximal promoter. By contrast, human SHBG transcripts were only detected in poly(A)+ RNA extracts from the testes of mice containing the 4.3-kb human SHBG transgene lacking FP4. No signals were detectable in the liver or testes RNA extracts from wild-type mice (WT). Mouse cDNAs for 18 S RNA and cyclophilin A (CypA) were used as loading and transfer controls of RNA samples from liver and testis, respectively. B, human SHBG transcripts were only detectable by Northern blotting in poly(A)+ RNA extracts of a mixed population of Sertoli and germ cells from mice containing a 4.3-kb human SHBG transgene lacking FP4. By contrast, human SHBG transcripts were undetectable in poly(A)+ RNA extracts of a pure population of testicular germs irrespective of the presence or absence of FP4 within the human SHBG proximal promoter. A mouse CypA cDNA was used to control for RNA loading and transfer to the Northern blots. C, in a reverse transcription-PCR of total RNA extracts from separated testicular cell types, a product was only obtained using human SHBG-specific primers in the extracts of the mixed population of Sertoli and germ cells from mice expressing the modified (FP4–) human SHBG transgene. By contrast, reverse transcription-PCR products were obtained in all extracts for mouse CypA, which was used as an internal control.

 
To determine which cell types in the testis express the modified human SHBG transgene, RNA samples from testicular germ cells and a mixed population of Sertoli cells and germ cells were examined by Northern blotting (Fig. 2B) and reverse transcription-PCR analysis (Fig. 2C). These experiments showed that human SHBG transcripts were present in mixed populations of Sertoli cells and germ cells from mice containing the FP4 deleted human SHBG transgene but were undetectable in pure populations of germ cells from these mice. The presence of human SHBG transcripts in Sertoli cells of transgenic mice that contain the FP4 deleted transgene suggested that they might produce and secrete SHBG. To confirm this, primary cultures of Sertoli cells from the different transgenic mouse lines were established, and culture medium was harvested for SHBG analysis using a highly sensitive and specific immunoassay. This demonstrated that Sertoli cells from mice expressing the FP4 deleted human SHBG transgene secrete SHBG and that it accumulates in the culture medium over a 7-day incubation, whereas immunoreactive human SHBG was undetectable in culture media of Sertoli cells derived from wild-type mice or mice containing the unmodified 4.3-kb human SHBG transgene (Fig. 3).



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  		FIG. 3.
Human SHBG is secreted only by Sertoli cells of mice expressing a 4.3-kb human SHBG transgene that lacks FP4 within the proximal promoter. Primary cultures of highly enriched populations of Sertoli cells (106 cells per well in 24-well plates) from wild-type (WT) mice or mice containing a 4.3-kb human SHBG transgene that either includes (FP4+) or lacks (FP4–) the FP4 region within the proximal promoter were maintained in culture over a period of 7 days. The concentrations of human SHBG in the culture medium were monitored using a time resolved immunofluorimetric assay at the times indicated. Mean + S.D. values are shown for samples from triplicate cultures. Human SHBG was not detectable (ND) in culture medium from WT or mice containing the unmodified 4.3-kb human SHBG transgene (FP4+).

 
Given the substantial amount of information about the hormonal regulation of rat androgen-binding protein production by Sertoli cells, we examined the effect of various hormone treatments on SHBG secretion by primary Sertoli cells from mice expressing the FP4 deleted human SHBG transgene (Fig. 4). In these experiments, both FSH and retinoic acid increased human SHBG secretion by these cells over an 8-day period (Fig. 4, A and B). Estradiol also increased SHBG secretion but only when added at a concentration 100 nM, whereas testosterone at this and lower concentrations had no effect (Fig. 4, C and D).



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  		FIG. 4.
Effects of FSH (A), retinoic acid (B), estradiol (C), and testosterone (D) on the production of human SHBG by Sertoli cells from mice containing a 4.3-kb human SHBG transgene that lacks FP4 within the proximal promoter. Primary cultures of highly enriched populations of Sertoli cells (106 cells per well in 24-well plates) from mice containing a 4.3-kb human SHBG transgene that lacks the FP4 region within the proximal promoter were maintained in culture over a period of 8 days. The concentrations of human SHBG in the culture medium were monitored using a time resolved immunofluorimetric assay at the times indicated. Mean + S.D. values are shown for samples from triplicate cultures. Differences (*, p < 0.05, **, p < 0.01) in SHBG concentrations after treatments with hormones at each time point were compared with respect to untreated controls.

 
Binding of USF-1 and USF-2 to FP4 within the Human SHBG Promoter in Sertoli Cells in Vitro and in Vivo—Similar results were obtained when two different mouse Sertoli cell lines (MSC-1 and TM4) were used as the source of nuclear protein for EMSAs, and the data for TM4 cells are presented (Fig. 5). The electrophoretic mobilities of the two band shifts obtained using the labeled FP4 oligonucleotide are similar to those obtained using a MLP oligonucleotide, which contains a well characterized USF binding site (25). However, the lower band shift (i.e. the one with the greatest mobility) does not represent a specific USF complex because it is not supershifted by antibodies against USF-1 or USF-2 (Fig. 5). Moreover, although the identity of the protein responsible for this complex is unknown, it does not appear bind to the core USF recognition sequence because the resulting band shift is eliminated using excess unlabeled oligonucleotide with a disruption in the USF-binding site (Fig. 5) or a totally unrelated oligonucleotide sequence (data not shown). By contrast, the upper band shift could be competed with excess (10x and 100x) unlabeled FP4 oligonucleotide as well as excess (100x) MLP oligonucleotide but not with excess (100x) FP4 oligonucleotide mutated to disrupt the USF-1 binding site (Fig. 5). Furthermore, addition of antibodies against either USF-1 or USF-2 to the EMSA reactions using both labeled FP4 and MLP oligonucleotides resulted in supershifts of only the upper band shifts observed in the EMSA assays, which was not seen when nonspecific rabbit IgG was used (Fig. 5).



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  		FIG. 5.
EMSA and supershift assays demonstrate that USF-1 and USF-2 interact with FP4 within the human SHBG proximal promoter. For EMSA, double stranded, end-labeled oligonucleotides spanning FP4 (lanes 1–9) and a consensus USF-binding site within the MLP of adenovirus (lanes 10–16) were incubated with 10 µg of nuclear proteins from the mouse TM4 Sertoli cell line in the absence (lanes 2 and 11) or presence (lanes 3–6 and 12–13) of excess unlabeled oligonucleotides as competitors. Free probe was separated from the protein-DNA complex by non-denaturing polyacrylamide gel electrophoresis. No nuclear protein was added to reaction mixtures applied to lanes 1 and 10. Antibody supershift assays were performed by incubating (15 min at room temperature) 1 µg of rabbit anti-USF-1 (lanes 7 and 14) or anti-USF-2 (lanes 8 and 15) antibodies or the same amount of rabbit IgG (lanes 9 and 16) with EMSA reaction mixtures.

 
To determine whether USF-1 and/or USF-2 bind the FP4 sequence in vivo we performed a ChIP assay using oligonucleotide primers to amplify the human SHBG promoter region containing binding sites for USF (FP4) as well as HNF4/COUP-TF (FP3). In these experiments, antibodies against USF-1, USF-2, COUP-TF II, and HNF4{gamma} were used for ChIP because these transcription factor isoforms are all present in the testis (28–30). As a positive control for the ChIP assay, oligonucleotide primers were designed to amplify the murine GAPDH proximal promoter, and TFIIB antibodies were used for ChIP, whereas normal mouse IgG was routinely used as a negative control (Fig. 6). The results indicate that anti-COUP-TF II and anti-HNF4{gamma} antibodies immunoprecipitate the human SHBG promoter from mice that harbor the 4.3-kb human SHBG transgene irrespective of the presence or absence of the FP4 sequence, whereas the USF-1 and USF-2 antibodies only precipitated the SHBG promoter fragment from mice containing the unmodified 4.3-kb human SHBG transgene (Fig. 6).



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  		FIG. 6.
ChIP assay demonstrating that USF-1 and USF-2 interact with FP4 within the human SHBG proximal promoter in vivo. Nuclear proteins bound to genomic DNA in testicular cells (Sertoli cells and germ cells) from mice containing 4.3-kb human SHBG transgenes that either include (FP4 +) or lack (FP4 –) the FP4 region within the proximal promoter were cross-linked and subjected to a ChIP assay using antibodies against USF-1, USF-2, COUP-TFII, and HNF4{gamma}, and oligonucleotide primers to PCR amplify the region containing FP3 and FP4 within the human SHBG proximal promoter. As a control for the ChIP protocol, anti-TFIIB antibodies provided in the ChIP assay kit were used together with mouse specific oligonucleotide primers to PCR amplify the GAPDH promoter. A nonspecific mouse IgG was used in all ChIP reactions as a control for nonspecific immunoprecipitation. Positive PCR controls of sheared genomic DNA templates indicated the integrity of the input DNA used in the ChIP reactions, whereas PCR reactions performed in the absence of template were used as negative controls.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have sought to define the molecular mechanisms that account for cell type-specific differences in SHBG expression in testis of humans when compared with other mammals in which Sertoli cells are known to secrete an androgen-binding protein into the seminiferous tubules (2). A phylogenetic analysis of SHBG proximal promoter sequences alerted our attention toward a sequence within the human sequence that is absent in the SHBG promoters of the other species. This sequence is highly conserved in the chimpanzee SHBG proximal promoter and contains a region that we have previously found contains a putative USF binding site by EMSA and supershift assays of mouse liver nuclear protein extracts.2 Because the SHBG genes in rabbits (31) and sheep2 are expressed (like the human SHBG gene) in the livers of adult animals, we assumed that the absence of this putative USF binding site in their SHBG promoters does not account for the lack of SHBG expression in the livers of adult rodents. This assumption was confirmed by the finding that removal of the USF binding site in the context of an otherwise identical 4.3-kb human SHBG transgene does not alter its expression in the liver when stably integrated within the genomes of two independent transgenic mouse lines.

Previously we have shown that the 4.3-kb human SHBG transgene is not transcribed in vivo in the mouse testes (14), and removal of the USF binding site from this transgene not only relieved this but resulted in the expression of the human SHBG transgene within mouse Sertoli cells. Moreover, this resulted in secretion of an immunoreactive human SHBG from primary cultures of mouse Sertoli cells that express the modified transgene. Even more remarkably, the way the modified human SHBG transgene responds to hormones resembles that of the rat SHBG gene in isolated Sertoli cells in that FSH and retinoic acid stimulates its expression whereas testosterone does not (6). There have been no previous reports to indicate that estradiol alters the expression of the rat SHBG gene in isolated Sertoli cells, and the stimulation of expression of the modified human SHBG transgene in primary cultures of mouse Sertoli cells by estradiol was unexpected. Although the response was obtained at high concentrations (100 nM) of estradiol, this could be relevant given the high intratesticular concentration of testosterone and the presence of P450 aromatase enzyme in Sertoli cells (32).

Our data show that the FP4 site within the human SHBG proximal promoter acts as a repressor of transcription specifically in Sertoli cells and that this is mediated by the binding of USF-1 and USF-2 as demonstrated by EMSA and ChIP experiments. The ubiquitous USF-1 and USF-2 transcription factors are present in Sertoli cells (33) and often function as heterodimers (34). In most instances they act as activators of transcription (29, 35), but there is evidence that they repress transcription of target genes as components of a repressive complex in some cell types (36, 37). There have been several reports that indicate that USF binding sites either overlap or are located in close proximity to a HNF4/COUP-TF binding site within the proximal promoters of several genes, including the FSH receptor (29) and L-Type pyruvate kinase (38) genes, and this also occurs in the human SHBG proximal promoter where FP3 is a known HNF4/COUP-TF binding site (17). In the case of the FSH receptor promoter, where USF and COUP-TF binding sites overlap, USF-1/USF-2 binding alone is insufficient to enhance cell-specific expression (33), and it is possible that interactions between USF transcription factors at FP4 and HNF4 or COUP-TF isoforms at FP3 co-operate to repress the transcriptional activity of the human SHBG promoter in Sertoli cells. However, various USF, HNF4, and COUP-TF transcription factor isoforms are also expressed in the liver, and human SHBG transcription in this tissue is not effected by the presence or absence of FP4. Therefore, the repression of human SHBG transcription in Sertoli cells via USF-1 and USF-2 binding to FP4, most likely as a heterodimer, must involve cell-specific differences in the relative abundance of these interacting transcription factors or the presence of some other cell-specific co-regulatory proteins that act cooperatively with USF.

The present data reinforce our previous conclusion that SHBG gene expression in the human testis is fundamentally different when compared with mammalian species in which an androgen-binding protein has been identified within the male reproductive tract (13) and provide a molecular basis for this. Because the chimpanzee SHBG proximal promoter is identical to the corresponding human sequence, it is likely that this difference in the way that the SHBG gene is expressed in the human testis is a relatively recent evolutionary development that may be shared by other primates. The chimpanzee testis, like the human testis, is characterized by multistage spermatogenesis throughout seminiferous tubules (39), and this morphological feature is shared by most other higher apes and new world monkeys but not old world monkeys (40). Therefore it remains to be seen whether the SHBG expression in the testes of different primate species resembles that in the human testis in which the predominant human SHBG transcript comprises an alternative exon 1 sequence (11, 13) and if this correlates with the occurrence of multistage spermatogenesis. Moreover, because the biological significance of the protein products of the alternative SHBG transcript in the human testis is unknown, studies in the testes of primates may represent the only appropriate model for the function of the SHBG gene in the human testis.


   FOOTNOTES
 
* This work was supported in part by an operating grant from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number AY838765 [GenBank] . Back

{ddagger} Recipient of a Canadian Institutes of Health Research graduate studentship. Back

§ A Canada Research Chair in reproductive health. To whom correspondence should be addressed: B. C. Research Institute for Children's and Women's Health, 950 West 28th Ave., Vancouver, BC V5Z 4H4, Canada. Tel.: 604-875-2435; Fax: 604-875-2496; E-mail: ghammond{at}cw.bc.ca.

1 The abbreviations used are: SHBG, sex hormone-binding globulin; FSH, follicle-stimulating hormone; FP4, footprint 4; EMSA, electrophoretic mobility shift assay; USF, upstream stimulatory factor; MLP, major late promoter; ChIP, chromatin immunoprecipitation; COUP-TF, chicken ovalbumin upstream promoter-transcription factor. Back

2 M. Talikka and G. L. Hammond, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Sui-pok Yee and Dr. Sara Gatchel (London, Ontario) for help generating the transgenic mice and Magid Fallahi for re-derivation and breeding of lines.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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