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Department of Obstetrics and Gynecology, University of British Columbia (C.K.C., C.M.Y., P.C.K.L.), Vancouver, Canada V6H 3V5; and Department of Zoology, University of Hong Kong (R.L.C.H., B.K.C.C.), Hong Kong
Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H30-4490 Oak Street, British Columbia Women’s Hospital, Vancouver, Canada V6H 3V5. E-mail: peleung{at}interchange ubc.ca.
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Abstract |
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T3-1 cells. Mutation of the AAAC residues of the octamer sequence completely removed its silencing activity. Interestingly, conversion of the octamer sequence into that of the rodent GnRHR promoter (5'AAGCAAAGT3') did not attenuate its silencing effect, indicating that the repressive role of the octamer sequence is evolutionarily conserved. EMSAs showed that common DNA-protein complexes of the same mobility were formed with nuclear extracts from the reproductive cells and gonadotropes, and a consensus octamer transcription factor-1 (Oct-1) binding sequence could dose dependently inhibit the complex formation. Antibody supershift and Southwestern blot assays confirmed that the protein binding to the octamer sequence was the ubiquitously expressed transcription factor Oct-1. Overexpression of Oct-1 augmented the silencing activity of the octamer sequence in T3-1 cells. Taken together, our results clearly indicate a role of Oct-1 in the transcriptional repression of the human GnRHR gene. |
Introduction |
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Apart from positive regulation by transcriptional activators, gene transcription may also be negatively controlled by silencer elements and their associated repressor proteins. Although an earlier study showed that there was an interplay between the pituitary adenylate cyclase-activating polypeptide (PACAP) and a silencer element located between nt -1676 and -1648 to regulate the activity of human GnRHR promoter in pituitary T3-1 cells (9), the transcriptional repression of the human GnRHR gene remains poorly understood. Interestingly, previous deletion analysis of the human GnRHR 5'-flanking region has revealed a very powerful negative regulatory element (NRE) located between nt -1017 and -771. This NRE can completely suppress the basal activities of the homologous promoter in pituitary T3-1 cells (10), ovarian carcinoma OVCAR-3 cells (10), placental JEG-3 cells (7), and immortalized granulosa-lutein cells (8), suggesting that the transcription factor(s) interacting with this element is ubiquitously expressed. However, the functional significance of this NRE remains unknown. In the present study, we demonstrated that this element could function as an orientation-dependent silencer in these cells and showed that Oct-1 was a transcriptional repressor of the GnRHR promoter and might play a very crucial role in silencing the GnRHR gene transcription.
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Materials and Methods |
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T3-1 cells were provided by Dr. P. L. Mellon (Department of Reproductive Medicine, University of California, San Diego, CA). The SVOG-4o, SVOG-4m, IOSE-29EC, and OVCAR-3 cells were maintained in M199/MCDB105 (1:1) supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, UT). The JEG-3, HEK-293, and T3-1 cells were maintained in DMEM (Life Technologies, Inc., Burlington, Ontario, Canada) containing 10% FBS. Cultures were maintained at 37 C in humidified atmosphere with 5% CO2 in air, and medium was renewed every 3 d. Cells were passaged using trypsin/EDTA solution (0.05% trypsin and 0.53 mM EDTA) or harvested for protein extraction when they reached about 80% confluence.
Plasmid construction and site-directed mutagenesis
The NRE (located between nt -1017 and -771 of human GnRHR 5'-flanking region) was amplified by PCR using primers containing a BamHI site (Table 1
). The fragment was cloned into the BamHI site of pTK-Luc (kindly provided by Dr. V. Giguere, Molecular Oncology Group, McGill University Health Center, Montréal, Québec, Canada) and p(-1300/-1018)-Luc in both orientations. The p(-1300/-1018)-Luc contains a minimal human GnRHR promoter (from nt -1300 to -1018) cloned upstream of a luciferase reporter gene in the promoterless pGL2-Basic vector (Promega Corp., Nepean, Canada). Deletion constructs for fine mapping of the NRE were generated by PCR amplification of the corresponding regions of the human GnRHR 5'-flanking region, followed by subsequent cloning into the pGL2-Basic vector. The PCR was carried out for 30 cycles with denaturation for 30 sec at 94 C, annealing for 1 min at 60 C, extension for 1 min at 72 C, and a final extension for 15 min at 72 C. Restriction site (KpnI or HindIII) was artificially introduced into the primers (Table 1). For site-directed mutagenesis, four octamer sequence variants were generated by PCR amplification using a forward primer p1300 and reverse primers containing the desired mutations (Table 1). Mutations were confirmed by DNA sequence analysis. Oct-1 expression plasmid (pcDNA3-HA-Oct-1) and control plasmid (pcDNA3-HA) were provided by Dr. H. Singh (Howard Hughes Medical Institute, University of Chicago, Chicago, IL). Plasmid DNA for transient transfection was prepared using plasmid midi kits (QIAGEN, Chatsworth, CA) following the manufacturer’s suggested procedures. The concentration and quality of DNA were determined by measuring absorbance at 260 nm and by agarose gel electrophoresis, respectively.
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EMSAs
Overlapping oligonucleotides (Table 1
) corresponding to the putative octamer sequence were synthesized by the Oligonucleotide Synthesis Laboratory (University of British Columbia) and annealed to form double-stranded DNA. Consensus Oct-1 (Oct-1-c), activating protein-2 (AP-2-c), nuclear factor-
B (NF-B-c), and transcription factor IID (TFIID-c) oligonucleotides were purchased from Promega Corp. Probe for EMSA was end radiolabeled with [32P]-ATP by T4 polynucleotide kinase (Life Technologies, Inc.) and separated from unincorporated radionucleotides by the Microspin G-25 columns (Amersham Pharmacia Biotech, Morgan, Canada). Nuclear extracts were prepared from OVCAR-3, JEG-3, and T3-1 cells according to the method described previously (8). Protein concentrations were determined by a modified Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). EMSAs were carried out in 20 µl containing 20 mM HEPES (pH 7.5), 50 mM NaCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 1 µg poly (dI:dC), 2–10 µg nuclear extracts, and 50 fmol radiolabeled probe (30,000 cpm). For the competitive assays, the competitor oligonucleotides were added simultaneously with the labeled probe. For antibody supershift assays, nuclear extracts were preincubated with either anti-Oct-1 (sc-232x) or anti-GATA-4 antibody (sc-1237x) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at room temperature for 30 min before the addition of the radiolabeled probe. The binding reaction was incubated at room temperature for 15 min and separated on a 6% polyacrylamide gel containing 1x 0.09 M Tris-borate and 2 mM EDTA (pH 8.0). Before loading of samples, the gel was prerun for 90 min at 100 V at 4 C. Electrophoresis was carried out at 30 mA at 4 C. The gel was then dried and exposed to x-ray film (Eastman Kodak Co., Rochester, NY) at -70 C.
Southwestern and Western blot analyses
Southwestern blot was performed essentially as previously described (13). Crude nuclear extracts from OVCAR-3, JEG-3, and T3-1 cells (100 µg and 20 µg proteins for Southwestern and Western blot analyses, respectively) were separated in duplicate lanes by sodium dodecyl sulfate-10% polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond-C, Amersham Pharmacia Biotech). For Southwestern blot analysis, proteins were allowed to renature in TNED buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 0.1 mM EDTA (pH 7.5), and 1 mM dithiothreitol] containing 5% milk overnight at room temperature. Afterward the membrane was rinsed three times in the TNED buffer, and incubated with 11 ml of the same buffer containing 75 pmol of the 32P-radiolabeled probe used in the EMSA studies at room temperature overnight. After hybridization, the membrane was washed three times (10 min each) with the TNED buffer, dried, and subjected to autoradiography. For Western blot analysis, the membrane was blocked with 5% (wt/vol) nonfat dried milk in Tris-buffered saline containing 20 mM Tris-Cl (pH 8.0), 140 mM NaCl, and 0.05% (vol/vol) Tween-20 for 3 h at room temperature before incubating with the anti-Oct-1 antibody (1:10,000) (sc-232x, Santa Cruz Biotechnology, Inc.) overnight at 4 C. Immunostained proteins were visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech).
Data analysis
For all transfection assays, data were shown as the mean ± SEM of triplicate assays in at least three independent experiments. For the Oct-1 overexpression study, data were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test using the computer software PRISM (GraphPad Software, Inc., San Diego, CA). Data were considered significantly different from each other at P < 0.05. For EMSA and Southwestern blot analysis, all experiments were performed at least three times.
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Results |
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T3-1 cells (Fig. 1A). The activities of the TK promoter were found to be completely abolished in these cell lines when the NRE was cloned in a sense orientation (sNRE-pTK-Luc). However, no significant changes of promoter activities (relative to the pTK-Luc) were observed when it was cloned in the opposite orientation (rNRE-pTK-Luc), indicating that the NRE can function as an orientation-dependent silencer. The construct sNRE-pTK-Luc was further analyzed in SVOG-4o, IOSE-29EC, and HEK-293 cells to demonstrate its silencing activity in other cell types. Similarly, the activities of the TK promoter were completely removed in these cells (Fig. 1B).
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T3-1 cells, whereas approximately 57% reduction of promoter activities were detected in OVCAR-3 and JEG-3 cells when the NRE was cloned in a sense orientation (sNRE-p(-1300/-1018)-Luc). In contrast, only about 28% reduction of promoter activities was observed when the NRE was cloned in the opposite orientation (rNRE-p(-1300/-1018)-Luc) (Fig. 2). These results indicate that the NRE can function in a position-independent manner, but the repressive effect is weaker when the NRE is located further apart from the homologous promoter.
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T3-1 cells and similar promoter activity profiles were observed in these cell lines (Fig. 3). 3'-Deletion from nt -771 to -954 did not eliminate the silencing activity of the NRE, indicating that the core-repressive activity of the NRE resides in the distal 64-bp region (i.e. from nt -1017 to -954). To delineate the nucleotide sequence required for the silencing activity of the NRE, a more detailed 3'-deletion mapping was performed. Deletion from nt -954 to -1009 did not significantly increase the promoter activities in the reproductive cells and gonadotropes, and a putative octamer regulatory sequence (5'AAGCAAACT3', nt -1017 to -1009) alone could reduce the activities of the homologous promoter by 76%, 78%, 90%, and 69% in SVOG-4m, OVCAR-3, JEG-3, and T3-1 cells, respectively (Fig. 4; the activities of p(-1300/-1009)-Luc were 4434 RLU, 65253 RLU, 5336 RLU, and 16110 RLU in SVOG-4m, OVCAR-3, JEG-3, and T3-1 cells, respectively). This observation clearly indicates that most of the silencing activity of the NRE retain in this octamer sequence.
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T3-1 cells. Mutation of the sequence AAGC to TGTG within the putative octamer motif restored the promoter activities by 64%, 48%, and 74% in OVCAR-3, JEG-3, and T3-1 cells, respectively. However, mutation of the sequence AAAC to TGTG completely resumed the promoter activities in OVCAR-3 and T3-1 cells, and 80% recovery of promoter activity was observed in the JEG-3 cells (Fig. 5). Alteration of the octamer sequence into the consensus Oct-1 binding motif (5'ATGCAAAT3') alleviated its silencing activity in all the three cell lines. Interestingly, a single-point mutation (AAAC to AAAG) converting the octamer sequence into that of the rodent GnRHR promoter did not attenuate its silencing activity, suggesting that the repressive role of the octamer sequence is evolutionarily conserved.
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T3-1 cells, we sought to examine whether the same transcription factor from these cell lines binds to this putative octamer sequence. To address this question, EMSA studies using various amounts of nuclear extracts from OVCAR-3, JEG-3, and T3-1 cells were performed. Common DNA-protein complexes of the same mobility (complex-A) were formed in a dose-dependent manner when nuclear extracts from these cells were used. However, the intensity of complex-A was much weaker, and an additional DNA-protein complex (complex-B) was observed with nuclear extracts from T3-1 cells (Fig. 6A). Competitive EMSA studies showed that the formation of these complexes could be inhibited by the unlabeled probe (100- to 500-fold excess) but not by other sequences including NF-B-c, AP-2-c and TFIID-c (500-fold excess; Fig. 6B). These observations indicate that the formation of these complexes is specific to the octamer sequence. Interestingly, an oligonucleotide containing the consensus Oct-1 binding motif was found to inhibit the formation of complex-A in a dose-dependent manner (Fig. 6C). Addition of anti-Oct-1 but not anti-GATA-4 antibody in the supershift assays prevented complex-A formation specifically (Fig. 6D). Western blot analysis of nuclear extracts from OVCAR-3, JEG-3, and T3-1 cells revealed that the endogenous expression levels of Oct-1 protein were much lower in the gonadotropes (Fig. 7A), and this observation was in agreement with the weaker Oct-1-binding signal detected in both Southwestern blot (Fig. 7B) and EMSA studies (Fig. 6A, complex-A formation). Taken together, these results clearly indicate that the POU domain transcription factor Oct-1 binds to the putative octamer sequence of the human GnRHR promoter.
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T3-1 cells (Fig. 8). Although overexpression of Oct-1 in the ovarian and placental cells did not augment the silencing activity of the octamer sequence, a further 31% reduction (P < 0.05 vs. control) of the GnRHR promoter activity was observed in the gonadotropes. The lack of response to Oct-1 overexpression in the reproductive cells may be due to their high endogenous levels of Oct-1 proteins (Fig. 7A). Nevertheless, these results confirm the role of Oct-1 as a repressor on the GnRHR promoter.
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Discussion |
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T3-1 cells, and the PACAP-responsive element was mapped to nt -1674/-1648 by scanning mutation analysis. However, none of these reports could identify the repressor proteins interacting with the corresponding silencer elements. Previous deletion analysis of the 5'-flanking region of the human GnRHR gene has revealed a very powerful NRE located between nt -1017 and -771. Nevertheless, the functional significance of this NRE is unknown. In the present study, four different cell lines, including the SVOG-4m, OVCAR-3, JEG-3, and T3-1 cells, which express the GnRHR gene, were used as model systems to identify the DNA motif(s) and its interacting transcription factor(s) that mediate the silencing activity of the NRE. The ability of the NRE to completely abolish the activity of a heterologous TK promoter and silence the homologous promoter in a position-independent manner suggests that the element can function as a transcriptional silencer. However, unlike other classical silencers, the action of the NRE appears to be orientation dependent because no significant change of TK promoter activity was observed when the NRE was cloned in the opposite direction. In addition, the NRE also demonstrated some degrees of promoter-dependence because there was about 25% reduction of homologous promoter activity even when the NRE was placed in the opposite direction. To date, only a small numbers of silencers have been described as orientation dependent (15, 16, 17), and their functional significance is still unclear. One possible mechanism for the functioning of this kind of silencers is that these elements present their specific binding factors in a particular position or direction relative to other regulatory sequences or factors. For instance, DNA bending as a result of silencer complex binding has been shown to repress gene transcription by physically hindering upstream elements (18). Therefore, it is possible that inversion of the silencer element can produce a bend in the opposite direction, thus eliminating the steric hindrance of the upstream enhancer elements. Because the NRE works ubiquitously and independent of its position, it is reasonable to believe that this element may functionally interact with different cell-specific GnRHR promoters (or enhancers) to tightly regulate the transcription of the GnRHR gene.
Progressive deletion analysis revealed that most of the silencing activity of the NRE resided in a 9-bp sequence (AAGCAAACT) located at the distal end of the NRE. This sequence shares high level of identity with the consensus octamer regulatory element (ATGCAAAT). Antibody supershift assay and Southwestern blot analysis confirmed that the repressor protein binding to this element was the widely expressed transcription factor Oct-1. More importantly, mutation of this motif into the octamer sequence of the rodent GnRHR promoter did not attenuate its silencing activity, indicating that the repressive role of this octamer sequence is evolutionarily conserved. In fact, the role of Oct-1 as a transcriptional repressor has been well studied in promoters of thyrotropin ß subunit gene (19), pituitary-specific transcription factor pit1/ghf1 gene (20), von Willebrand factor gene (21), and B cell-specific B29 (Igß) gene (22), and Kim et al. (19) showed that the silencing activity of Oct-1 resided in its alanine-rich C-terminal domain.
In our present study, the octamer binding sequence was found to repress the GnRHR promoter activity in a wide variety of cell types, a phenomenon that could be explained by the ubiquitous expression of the Oct-1 protein. However, Oct-1 has also been shown to participate in tissue-specific expression of the human GnRHR gene via cooperation with other transcription factors. For instance, a previous study from our laboratory revealed that Oct-1 bound to an AT-rich octamer sequence (5'ATTTGTAT3') located within the upstream placenta-specific human GnRHR promoter, and it functioned as a transcriptional activator because mutation of this AT-rich sequence reduced the promoter activity drastically (7). However, Oct-1 regulated this placenta-specific gene expression of the GnRHR via cooperation with other transcription factors including cAMP-responsive element-binding protein, GATA-2, GATA-3, and activating protein-1. On the other hand, we recently found that there was a possible cross-talk between Oct-1 and a putative C/EBP motif, which cooperated synergistically with a GATA and another C/EBP motif to specifically regulate GnRHR gene transcription in human granulosa-lutein cells (8). Another important information obtained from these studies is that Oct-1 possesses dual transcriptional functions (both activators and repressors) in regulating human GnRHR gene transcription. These observations may be explained by the fact that Oct-1 can adopt different conformations, depending on the precise nature of the binding and flanking sequences, which in turn determines its transcriptional functions (23). Indeed, our present mutational analysis showed that alteration of the octamer sequence into the consensus Oct-1 binding motif alleviated its repressive effect on the GnRHR promoter (Fig. 5), further supporting the notion that the exact identity of the Oct-1-binding sequence is a critical determinant of its transcriptional activity.
The mechanisms by which Oct-1 mediates its activator function have been well documented, and it has been proven that Oct-1 activates transcription by directly interacting with other transcription factors including TATA binding protein, transcription factor IIB, high mobility group protein 2, and Oct-binding factor 1 (24, 25, 26, 27, 28, 29). Although a recent study has shown that Oct-1 physically interacts with the silencing mediator of retinoid and thyroid receptors to mediate its repressor function (30), the exact mechanisms by which Oct-1 mediates its repressor function on the GnRHR promoter remain to be elucidated. However, the fact that Oct-1 has the ability to bend DNA through its POUS domain (31) may help explain the orientation-dependence for the proper functioning of the NRE. Therefore, mutation of the AAGC subsite of the octamer regulatory sequence may impair the DNA-bending ability of the POUs domain and thus account for the partial elimination of the silencing activity mediated by Oct-1 (Fig. 5).
Interestingly, a recent report from Belsham and Mellon (32) demonstrated that Oct-1 was one of the downstream transcriptional regulators of the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate signal transduction pathway because treatment with a nitric oxide donor increased the DNA-binding affinity of Oct-1 to its target site, and this enhanced binding affinity was likely due to increased phosphorylation of Oct-1 by cGMP-dependent protein kinase. This finding is in agreement with previous reports (33, 34) demonstrating that the DNA-binding specificity of Oct-1 could be regulated by different kinases in vitro. These studies implicate that Oct-1-regulated-gene expression can possibly be under the influence of various intracellular signal transduction pathways, which control the phosphorylation status and thus the binding activity of Oct-1. In addition, the transcriptional function of Oct-1 has also been shown to be regulated by nuclear receptors (35, 36, 37), and of particular interest, Kutoh et al. (37) showed that the glucocorticoid receptor could inhibit Oct-1 function by a mechanism involving direct protein-protein interaction in a hormone-dependent manner. This functional interference between Oct-1 and the glucocorticoid receptor thus represents a novel mechanism by which Oct-1 cross-couples with the nuclear receptor signal transduction pathway. These findings implicate that Oct-1-regulated GnRHR gene transcription may be influenced by a variety of extracellular stimuli and depend on the physiological status of the cell.
EMSA studies revealed that an additional DNA-protein complex (complex-B) was formed merely with nuclear extracts from the gonadotrope-derived T3-1 cells (Fig. 6A). Both competitive EMSA and antibody supershift assays indicated that Oct-1 was not likely the transcription factor present in this complex (Fig. 6, C and D). Because this complex was not observed from nuclear extracts of reproductive cell types, we speculated that the transcription factor(s) present in complex-B may be gonadotrope specific. However, we failed to detect additional bands in Southwestern blot analysis of nuclear extracts from T3-1 cells. The significance of these factors in the transcriptional repression of the GnRHR gene demands further investigation, and we cannot exclude the possible existence of a different (or additional) negative regulatory mechanism to control the GnRHR gene expression in the gonadotropes.
In summary, we have identified an octamer regulatory sequence as a core cis-acting silencing element within the human GnRHR 5'-flanking region and revealed for the first time that the POU domain transcription factor Oct-1, via binding to this octamer sequence, played a role in the transcriptional repression of the GnRHR gene.
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Acknowledgments |
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Footnotes |
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Abbreviations: AP-2, Activating protein-2; C/EBP, CCAAT/enhancer binding protein; FBS, fetal bovine serum; GnRHR, GnRH receptor; NF-B, nuclear factor-B; NRE, negative regulatory element; nt, nucleotide; PACAP, pituitary adenylate cyclase-activating polypeptide; RSV, Rous sarcoma virus; SV, simian virus; TFIID, transcription factor IID; TK, thymidine kinase.
Received June 3, 2002.
Accepted for publication August 30, 2002.
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