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Molecular Endocrinology, doi:10.1210/me.2004-0025
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Molecular Endocrinology 19 (1): 148-162
Copyright © 2005 by The Endocrine Society

Oct-1 and Nuclear Factor Y Bind to the SURG-1 Element to Direct Basal and Gonadotropin-Releasing Hormone (GnRH)-Stimulated Mouse GnRH Receptor Gene Transcription

Kyung-Yoon Kam, Kyeong-Hoon Jeong, Errol R. Norwitz, Elisa M. Jorgensen and Ursula B. Kaiser

Departments of Medicine (K.-Y.K., K.-H.J., E.M.J., U.B.K.) and Obstetrics, Gynecology and Reproductive Biology (E.R.N.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Ursula B. Kaiser, M.D., Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: UKaiser{at}partners.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The cis-regulatory element localized to position –292/–285 of the mouse GnRH receptor (mGnRHR) gene promoter, designated Sequence Underlying Responsiveness to GnRH 1 (SURG-1), has been shown previously to contribute to stimulation of mGnRHR gene expression by GnRH. We have identified three specific protein-DNA complexes on the SURG-1 element by EMSA using nuclear extracts from the gonadotrope-derived {alpha}T3–1 and LßT2 cell lines. Serial mutagenesis and supershift assays identified nuclear factor Y (NF-Y) binding to –288/–284 and Oct-1 binding to a TAAT sequence at –290/–287. Binding of these two transcription factors was confirmed in vivo by chromatin immunoprecipitation assay and increased in response to GnRH stimulation. To define the functional significance of these sequences in the regulation of mGnRHR gene transcription, transient transfection assays were performed in {alpha}T3–1 cells using a 1.2-kb mGnRHR (–1164/+62) gene promoter-luciferase reporter construct with selective mutations of the Oct-1, NF-Y, and/or the previously characterized activating protein 1 (AP-1) binding site (–274/–268). Individual mutations in the Oct-1, NF-Y, and AP-1 sites decreased both basal expression and stimulation by GnRH agonist, and the combined mutation of the Oct-1 and AP-1 binding sites further reduced basal transcriptional activity and abolished GnRH stimulation. Overexpression of NF-YA increased GnRHR promoter activity, whereas expression of a dominant negative NF-YA mutant decreased activity, further supporting a role of NF-Y in regulation of mGnRHR gene transcription. In addition, knockdown of Oct-1 by small interfering RNA confirmed that Oct-1 is important for mGnRHR gene expression. In conclusion, NF-Y and Oct-1 bind to the SURG-1 element to direct basal and GnRH-stimulated expression of the mGnRHR gene.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE REGULATION OF mammalian sexual maturation and reproductive function requires the integration and precise orchestration of hormonal action throughout the hypothalamo-pituitary-gonadal axis. GnRH, a hypothalamic decapeptide, plays a critical role in this axis (1). GnRH released from the hypothalamus binds to GnRH receptor (GnRHR) expressed on the plasma membrane of gonadotropes and stimulates gene expression and release of the gonadotropins, LH and FSH. LH and FSH, in turn, act on the gonads to stimulate steroidogenesis and gametogenesis (2). The gonadotropin secretion pattern shows distinct rhythmicity in response to the GnRH secretion pattern, and this feature is important for maintaining normal reproductive function. The response of pituitary gonadotropes to GnRH and secretion of LH and FSH correlate directly with the concentration of GnRHR on the cell surface (3, 4, 5). Therefore, the GnRHR is the input site of the hypothalamic stimulatory signal and serves as a regulatory point in control of gonadotropin secretion. Previous studies have demonstrated that the regulation of GnRHR concentration is mediated, at least in part, at the level of GnRHR gene expression (6, 7). There are a number of factors that affect GnRHR gene expression, including activin (8, 9), estrogen (10, 11), and glucocorticoids (12). There are, however, none more fundamental than GnRH (13, 14). Therefore, it is important to understand the molecular mechanisms controlling responsiveness to GnRH in terms of the regulation of GnRHR gene expression.

A 1.2-kb 5'-flanking region of the mouse GnRHR (mGnRHR) gene promoter has been cloned and characterized (7, 15). Although we have relatively little knowledge about the transcriptional mechanisms controlling the GnRHR gene, several cis-elements have been identified to be involved in mGnRHR gene expression. These include a steroidogenic factor 1 (SF-1) site at position –181/–173 relative to the major transcriptional start site (16), and a GnRHR-activating sequence at –329/–318 that binds activating protein 1 (AP-1), Smad proteins, and FoxL2 and mediates both activin and GnRH responsiveness (17, 18, 19). Previous studies by our group and others have identified two cis-elements, designated as SURG (Sequence Underlying Responsiveness to GnRH)-1 (at position –292/–285) and -2 (at position –276/–269), that are important for GnRH-stimulated transcription of the mGnRHR. SURG-2 overlaps with a conserved AP-1 consensus binding site, and mutation of SURG-2 results in markedly reduced transcription in GnRH-stimulated cells (7, 13). Mutation of SURG-1 also significantly diminished, but did not fully abolish, GnRH stimulation (7). Although it is established that SURG-2 is an AP-1-binding site, it is not known which protein(s) binds to SURG-1 and how it mediates the response to GnRH. The identification of the trans-acting factor(s) binding to the SURG-1 element, its role in regulation of transcription, and possible interaction with other transcription factors are essential for a better understanding of the control of GnRH-stimulated GnRHR gene expression.

Considering the dynamic regulation of gonadotropin secretion, we can expect that regulation of GnRHR gene expression might be complex. In spite of its significance, however, the molecular mechanisms by which GnRHR gene transcription is regulated have not been intensively investigated. In the present study, we have identified and characterized two overlapping cis-acting motifs within the SURG-1 element that bind to nuclear proteins and are involved in regulation of basal and GnRH-stimulated mGnRHR gene expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of SURG-1 Binding Proteins by EMSA
To determine whether any nuclear proteins bind specifically to the SURG-1 element, we performed EMSA using nuclear extracts from {alpha}T3–1, LßT2, and CV-1 cells, and a 32P-end-labeled oligonucleotide corresponding to –300/–277 of the mGnRHR gene promoter (S1) as probe (Fig. 1Go). Two closely migrating protein-DNA complexes, and a third less discrete complex with faster mobility, were identified and are referred to as complexes A, B, and C, respectively (Fig. 1AGo). Complexes A and B were present in common in nuclear extracts from the gonadotrope-derived cell lines, {alpha}T3–1 and LßT2, as well as in the nongonadotropic CV-1 cell line, derived from monkey kidney fibroblast cells. Complex C was less intense than complexes A and B, was variable in its presence, and was less easily detected in LßT2 cells than in {alpha}T3–1 cells. Nuclear extracts from CV-1 cells had a distinct third complex with slower mobility than the complex C of {alpha}T3–1 and LßT2 cells, suggesting the formation of a different protein-DNA complex.



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  		Fig. 1. Characterization of SURG-1 Binding Proteins by EMSA

A, {alpha}T3–1, LßT2, and CV-1 cells were grown to 50% confluence and treated with GnRH agonist (100 nM) for 0, 1, or 4 h before nuclear protein extraction. Using an oligonucleotide corresponding to –300/–277 of the mGnRHR gene promoter as probe, three specific protein-DNA complexes (designated by A, B, and C) were identified. Complex C from CV-1 cells is distinct from complex C found using nuclear extracts from {alpha}T3–1 and LßT2. The inset shows clearly the two distinct bands representing complexes A and B. B, Specificity of the complexes was assessed by competition with 500-fold excess of homologous (S1) and heterologous (SF-1) unlabeled oligonucleotides.

 
To determine whether GnRH treatment has any effects on the binding pattern of nuclear proteins to SURG-1, nuclear extracts were prepared from {alpha}T3–1, LßT2, and CV-1 cells treated with 100 nM GnRH agonist for 1 or 4 h and used in EMSA. The intensity of complexes A, B, and C was not affected by GnRH treatment, and no new complexes were formed (Fig. 1AGo).

To confirm the specificity of protein-DNA binding in these complexes, cold competition studies were performed with either {alpha}T3–1 or LßT2 nuclear extracts. A 500-fold excess of unlabeled (cold) competitor oligonucleotides was added to the EMSA reaction mixtures. S1 and an oligonucleotide corresponding to the SF-1-binding site in the rat LHß gene promoter (SF-1) (20) were used as homologous and heterologous competitors, respectively. Complexes A, B, and C were all effectively competed by S1 but not by SF-1, confirming the specificity of protein-DNA binding to the SURG-1 element (Fig. 1BGo). In summary, the results of these EMSA studies indicate that nuclear proteins bind specifically to –300/–277 of the mGnRHR gene promoter.

Complex B Binds to an Inverse CCAAT Sequence in the SURG-1 Element of the mGnRHR Gene Promoter and Includes Nuclear Factor Y (NF-Y)
To further characterize and localize the binding sites for the complexes, additional EMSA experiments were performed using the S1 oligonucleotide as probe and {alpha}T3–1 nuclear extracts. Oligonucleotides with serial 2-bp mutations between –294/–279 (designated M1–M8, Fig. 2AGo) were used as competitors. Oligonucleotides M3–M7 failed to effectively compete for complex B, with the greatest effect by mutations in oligonucleotides M4, M5, and M6 (Fig. 2BGo). The sequence defined by these mutations is ATTGGA, which contains the sequence CCAAT on the antisense strand. Although many DNA-binding proteins—in whose acronym the word CCAAT is present [e.g. CCAAT transcription factor/nuclear factor 1; C/EBP (CCAAT/enhancer binding protein); and CDP (CCAAT displacement protein)]—have been isolated and characterized based on their ability to bind to this consensus sequence, NF-Y is known to be a major protein recognizing the CCAAT box (21). To determine whether NF-Y binds to this element in {alpha}T3–1 cells, supershift EMSA was performed, using antibodies to the three subunits of NF-Y (A, B, and C). All three antibodies led to the formation of supershifted complexes, confirming the presence of NF-Y in complex B (Fig. 2CGo). In contrast, antibodies for C/EBPß and CDP had no effect. Taken together, these results indicate that complex B binds to –288/–284 in the SURG-1 element of the mGnRHR gene promoter and contains NF-Y.



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  		Fig. 2. Localization and Identification of NF-Y Binding to SURG-1

A, Serial 2-bp mutant oligonucleotides of region –300/–277 of the mGnRHR gene promoter were generated. The mutated bases are indicated in italics and bold. The SURG-1 element, as previously defined (7 ), is boxed. B, Competition EMSA using 32P-labeled –300/-277 oligonucleotide as probe with 500-fold excess unlabeled mutant oligonucleotides as competitors (designated M1–M8). C, Supershift EMSA with antibodies for NF-Y subunits (NF-YA, NF-YB, and NF-YC) and putative CCAAT-binding proteins (C/EBPß and CDP). Antirabbit and antigoat IgGs were used as negative controls. Complexes A, B, and C are indicated with arrows, and the supershifted bands for the NF-Y subunits are indicated by open arrowheads.

 
Complexes A and C Bind to –290/–287 of the mGnRHR Gene Promoter and May Represent a Homeodomain or Related Protein(s)
It was difficult to localize the binding site of complex A due to interference from complex B, but complex A did not appear to be affected by the anti-NF-Y antibodies (Fig. 2CGo). To more carefully delineate the sequences bound by complex A, we performed EMSA using {alpha}T3–1 nuclear extracts in the presence of anti-NF-YA antibody, using the S1 oligonucleotide as probe and the serial 2-bp mutant oligonucleotides of the region –294/–279 as competitors. Complex B was supershifted by the anti-NF-YA antibody, allowing complex A to be seen more clearly (Fig. 3AGo). Oligonucleotides M3 and M4 failed to effectively compete for both complexes A and C, thus localizing the binding of complexes A and C to –290/–287 of the mGnRHR gene promoter, corresponding to the DNA sequence, TAAT. This TAAT sequence is known to be the core motif of the homeodomain protein-binding consensus sequence (22). Several homeodomain proteins have been shown to be expressed in pituitary gonadotropes and to play a role in the expression of other gonadotrope-specific genes, including LHß and FSHß. These include Ptx-1, Ptx-2, and Otx1/2 (23, 24, 25). Antibodies for these homeodomain proteins were used in supershift EMSA to determine whether complexes A and C contain any of these gonadotrope-expressed homeodomain proteins (Fig. 3BGo). For this study, the M6 oligonucleotide (see Fig. 2AGo) was used as probe, to eliminate NF-Y binding and allow complex A to be seen more clearly. Complexes A and C were not supershifted by antibodies for Ptx-1, Ptx-2, or Otx-1/2. Lhx2 and 3, LIM-homeodomain factors, and Msx-1 are known to play roles in expression of the glycoprotein hormone {alpha}-subunit gene in gonadotropes (Lhx2 and 3) and thyrotropes (Msx-1). Supershift EMSA using antibodies to these homeodomain proteins also failed to supershift complexes A and C (Fig. 3BGo).



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  		Fig. 3. Localization and Identification of a Putative Homeodomain Protein Binding Site in SURG-1

A, Competition EMSA using 32P-labeled S1 (–300/–277) oligonucleotide as probe with 500-fold excess unlabeled mutant oligonucleotides as indicated. The NF-Y complex (complex B) was supershifted with anti-NF-YA antibody. B, Supershift EMSA with antibodies for Ptx-1, Ptx-2, Otx1/2, Lhx2, Lhx3, and Msx-1 using 32P-labeled M6 oligonucleotide. NF-Y complex cannot bind to M6 oligonucleotide, allowing complex A to be seen clearly. Antirabbit and antigoat IgGs were used as negative controls. The NF-Y supershifted complexes are indicated by open arrowheads.

 
Complex A Includes Oct-1, and the Octamer Sequence (–294/–287) Is Necessary for Oct-1 Binding
We hypothesized that Oct-1, which belongs to the POU domain transcription factor family, may represent a component of complex A and/or complex C. Recently, Oct-1 was reported to be involved in transcriptional repression of the human GnRHR gene through an upstream negative regulatory element in the {alpha}T3–1 cell line as well as in ovarian and placental cell lines (26). Oct-1 is known to show affinity toward TAAT core sites (27), although its canonical binding site is ATGCAAAT. Therefore, competition EMSA with a 200- or 1000-fold excess of an oligonucleotide containing the Oct-1 consensus binding sequence (5'-TGTCGAATGCAAATCACTAGAA-3') as cold competitor was performed (Fig. 4AGo). 32P- labeled oligonucleotides corresponding to wild-type sequence (S1) and mutants M3 and M6 (see Fig. 2AGo) were used as probes. M3 eliminates binding of complexes A and C, whereas M6 eliminates NF-Y binding, thereby allowing complex A to be seen more clearly. In these studies, complex A was effectively competed by even a 200-fold excess of the Oct-1 consensus oligonucleotide, whereas complex C was partially competed by a 1000-fold excess (Fig. 4AGo). Complex B was not affected by the presence of the Oct-1 consensus oligonucleotide. These results indicate that the protein(s) present in complex A can recognize the Oct-1 consensus sequence and suggest that Oct-1 and Oct-2 are candidate components of complex A.



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  		Fig. 4. Identification of Oct-1 Binding to SURG-1

A, Competition EMSA using 32P-labeled S1 (–300/–277) and M3 and M6 mutant oligonucleotides as probes with a 200- or 1000-fold excess of Oct-1 consensus oligonucleotide as cold competitor. B, Supershift EMSA with antibodies for Oct-1 and Oct-2 using 32P-labeled S1, M3, M6, and Oct-1 consensus oligonucleotides as probes. Antirabbit IgG was used as negative control. The Oct-1 supershifted complexes are indicated by the open arrowhead. C, Wild-type, M6, and mutant oligonucleotides of the Oct-1-binding site and flanking sequences of the SURG-1 element. Mutated bases are in italics. The octamer site is in bold. The SURG-1 element, as previously defined (7 ), is boxed. D, EMSA performed using probes described in panel C. Complexes A, B, and C are indicated with arrows.

 
To further identify the protein(s) present in complex A and test the hypothesis that it is related to Oct-1, a supershift EMSA was performed using anti-Oct-1 and Oct-2 antibodies, again using S1, M3, and M6 as probes (Fig. 4BGo). A supershifted complex was clearly identified in the presence of Oct-1 antibody, but not with Oct-2 antibody. This supershifted complex was not present when M3 was used as probe, which eliminated protein binding to the TAAT sequence. Using M6 as probe, it was clear that the Oct-1 antibody, but not the Oct-2 antibody, disrupted complex A formation. Using the Oct-1 consensus oligonucleotide as probe, it was demonstrated that the Oct-1 binding complex formed on this probe comigrated with complex A. Taken together, these data strongly suggest that complex A contains Oct-1.

Closer examination of the sequences flanking the TAAT core sequence in the SURG-1 element reveals a sequence (5'-AGGCTAAT-3') with homology to the Oct-1 consensus octamer sequence (5'-ATGCAAAT-3'), differing by two of eight nucleotides. To further characterize the sequence requirements for Oct-1 and NF-Y binding to overlapping sequences within the SURG-1 element, the nucleotides of the Oct-1 consensus sequence were substituted for the two differing nucleotides in S1 as well as in the M6 mutant oligonucleotide. These oligonucleotides were designated S1-Oct-1 and M6-Oct-1, respectively. The oligonucleotide sequences used are summarized in Fig. 4CGo. When these mutant oligonucleotides were used as probes in EMSA with {alpha}T3–1 nuclear extracts, Oct-1 binding was increased (Fig. 4DGo). Interestingly, this was associated with elimination of NF-Y binding. In several gene promoters, it has been reported that the nucleotide sequence 3' to the TAAT sequence is also important for Oct-1 binding (27, 28). Therefore, it is worth determining the significance of sequences distal and proximal to TAAT in Oct-1 binding to the SURG-1 element. For this purpose, two additional mutants were prepared (S1-distal mut and S1-proximal mut, see Fig. 4CGo). The mutations both distal and proximal to TAAT also markedly diminished Oct-1 binding to SURG-1 (Fig. 4DGo). Taken together, these results illustrate the importance of the SURG-1 sequence in maintaining and integrating Oct-1 and NF-Y binding. Mutations in the TAAT sequence as well as in both distal and proximal flanking sequences eliminate Oct-1 binding, whereas nucleotide changes that increase Oct-1 binding lead to loss of NF-Y binding.

Oct-1 and NF-Y Bind to the SURG-1 Element in Vivo
The EMSA studies indicated that NF-Y and Oct-1 present in {alpha}T3–1 and LßT2 cells were able to bind to the SURG-1 element in the mGnRHR gene promoter in vitro. To confirm the interaction of NF-Y and Oct-1 with the mGnRHR gene promoter in vivo in the context of the chromatin structure of the endogenous gene, ChIP assays were performed, using {alpha}T3–1 cells and anti-Oct-1 and anti-NF-YA antibodies (Fig. 5Go). Anti-c-Jun antibody was used as a positive control [to identify AP-1 binding to the previously characterized AP-1 binding site in the mGnRHR (7, 13, 29)], and preimmune rabbit IgG was used for a negative control. PCR amplification with a primer pair which amplified the region –337/–170, encompassing the GnRHR-activating sequence, SURG-1 and AP-1/SURG-2 elements, was used to detect protein-DNA binding. A PCR product was detected with the anti-c-Jun antibody and was increased by 1 and 4 h of GnRH treatment, suggesting a GnRH-stimulated increase in AP-1 binding, in agreement with previous reports that AP-1 binding to –327/–322 and to –274/–268 is important for GnRH regulation of GnRHR gene transcription (7, 13, 18). Weak bands for Oct-1 also appeared after 1 and 4 h of GnRH treatment. The weak signal obtained for Oct-1 may be due to reduced interaction of the antibody, raised against an epitope in human Oct-1, for mouse Oct-1. Western blot analysis supported this reduced cross-species affinity (data not shown). Interestingly, NF-Y binding also appeared to be increased by GnRH treatment, with a clear band for NF-Y binding seen after 4 h of GnRH treatment. These results indicate that Oct-1 and NF-Y bind to the SURG-1 element in vivo and suggest that binding in vivo is regulated by GnRH.



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  		Fig. 5. ChIP Assay of Oct-1, NF-Y, and AP-1 Binding to the mGnRHR Gene Promoter

Promoter sequences spanning the SURG-1 element (–337/–170) were analyzed by PCR amplification of the immunoprecipitated chromatin of {alpha}T3–1 cells treated with 100 nM GnRH agonist for 0, 1, and 4 h and immunoprecipitated with Oct-1, NF-YA, or c-Jun antibodies as indicated, or with preimmune serum (rabbit IgG) as a negative control. Input samples (10-fold diluted) were subjected to PCR as positive controls.

 
The NF-Y and Oct-1 Binding Sites in the SURG-1 Element Contribute to Basal and GnRH-stimulated mGnRHR Gene Transcription
We previously isolated 1.2 kb of the 5'-flanking region of the mGnRHR gene (15). Functional studies of this promoter region were performed by fusing this sequence upstream of a luciferase reporter, followed by transient transfections in the gonadotrope-derived {alpha}T3–1 cell line. The AP-1 binding site at position –274/–268 was previously identified as necessary for GnRH stimulation of mGnRHR gene expression (7, 13). In addition, sequences between –292/–285, referred to as SURG-1, were also shown to be necessary for full GnRH responsiveness (7). We have now identified two cis-elements, one for Oct-1 and the other for NF-Y, within this SURG-1 element. This localization suggests that either or both of these cis-elements may contribute to GnRH responsiveness, either alone or in combination with the AP-1-binding site.

To test the functional importance of these newly defined cis-elements in mediating GnRH stimulation of mGnRHR gene transcription, we introduced mutations in the Oct-1, NF-Y, and AP-1 binding sites, individually and in combination, into –1164/+62 mGnRHR-Luc (Fig. 6AGo). To determine the effects of these mutations on GnRH responsiveness, these constructs were transfected into {alpha}T3–1 cells by calcium phosphate coprecipitation for 4 h, followed by treatment with 100 nM GnRH agonist for 4 h. This experimental paradigm was selected to optimize the GnRH response. Using this transfection paradigm, luciferase activity was low in the absence of GnRH. Wild-type –1164/+62 mGnRHR-Luc activity increased by 7.4 ± 0.6-fold in response to 100 nM GnRH agonist (Fig. 6BGo). Point mutation of the Oct-1, NF-Y, or AP-1 binding site resulted in a significant reduction in GnRH stimulation compared with wild type, to 3.1 ± 0.6, 4.6 ± 0.6, or 3.6 ± 0.6-fold, respectively. The combined mutation of the Oct-1 and NF-Y binding sites did not lead to any further reduction in GnRH stimulation, compared with mutation of the Oct-1 or NF-Y binding site alone. Interestingly, the combined mutation of the Oct-1 and AP-1 elements, or of all three elements, was sufficient to completely abolish GnRH responsiveness of the mGnRHR gene promoter (Fig. 6BGo).



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  		Fig. 6. Effect of Mutations in the Oct-1-, NF-Y-, and AP-1-Binding Sites of the mGnRHR Gene Promoter on Basal and GnRH-Stimulated Luciferase Activity

A, Wild-type and mutant constructs of –1164/+62 mGnRHR gene promoter fused to a luciferase vector. Two base pairs were mutated in the Oct-1 or NF-Y binding sites. A point mutation was introduced at position –269 by substituting thymidine for cytosine in the AP-1 binding site. Combined mutants were also prepared. B, {alpha}T3–1 cells were transfected for 4 h with wild-type –1164/+62 mGnRHR-Luc, the corresponding Oct-1, NF-Y, and/or AP-1 binding site mutants as indicated, or pXP2 followed by GnRH agonist stimulation (100 nM for 4 h). C, {alpha}T3–1 cells were transfected for 20 h and harvested for measurement of luciferase activity and ß-galactosidase assay. Measurements were normalized to pXP2 (not treated with GnRH agonist) for each experiment. Data represent mean ± SE from five (B) and eight (C) independent experiments, each performed in triplicate. *, P < 0.05 vs. pXP2; #, P < 0.05 vs. wild-type –1164/+62 mGnRHR-Luc.

 
To determine whether the Oct-1, NF-Y, and AP-1 sites also contribute to basal mGnRHR gene expression, the same constructs were again transfected into {alpha}T3–1 cells by calcium phosphate coprecipitation, this time for 20 h to measure effects on basal activity of the mGnRHR gene promoter (Fig. 6CGo). Wild-type –1164/+62 mGnRHR-Luc resulted in luciferase activity 57.2 ± 4.4 times higher than the promoterless pXP2 vector. Individual mutations in the Oct-1, NF-Y, or AP-1 binding site each resulted in a reduction in basal expression of the mGnRHR gene compared with wild type, with luciferase activity 14.7 ± 1.9-, 36.9 ± 5.6-, and 25.2 ± 4.7-fold higher than pXP2, respectively. The combined mutation of the Oct-1 and AP-1 elements, or of all three elements, further reduced basal transcriptional activity. Taken together, these results indicate that the Oct-1, NF-Y, and AP-1 binding sites play important roles in transcriptional regulation of both basal and GnRH-stimulated mGnRHR gene expression.

NF-YA Increases Mouse GnRHR Gene Transcription through the SURG-1 Element
Our results have demonstrated that NF-Y and Oct-1 bind to cis-elements within SURG-1 in the mGnRHR gene promoter, and that these cis-elements are involved in regulation of basal and GnRH-stimulated mGnRHR gene transcription. However, these results do not directly demonstrate a role of NF-Y and Oct-1 in regulation of mGnRHR gene transcription. To confirm this role for NF-Y, the effects of overexpression of NF-YA, and of interfering with the function of endogenous NF-Y by expressing a dominant negative (DN) mutant NF-YA, were determined (Fig. 7Go). Wild-type –1164/+62 mGnRHR-Luc was cotransfected into {alpha}T3–1 cells together with an expression vector encoding wild-type NF-YA. After 24 h, cells were harvested and luciferase activity was measured and normalized to ß-galactosidase activity. NF-YA overexpression increased GnRHR gene transcription by 1.7 ± 0.7-fold. Western blot analysis confirmed increased NF-YA expression in transfected cells (data not shown). Similar transfections were performed using an expression vector encoding a DN NF-YA mutant, which can bind to NF-Y subunits B and C to form a trimer but lacks DNA binding activity (30). Unlike the wild-type NF-YA, the DN NF-YA did not increase mGnRHR gene transcription, but rather reduced transcriptional activity. Parallel transfections were performed using –1164/+62 GnRHR-Luc containing a mutation in the NF-Y-binding site (µNF-Y), in which overexpression of either wild-type or DN NF-YA had no effect on luciferase activity. These results demonstrate directly a role for NF-Y in the regulation of mGnRHR gene transcription via the SURG-1 element.



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  		Fig. 7. Effects of Overexpression of Wild-Type and DN NF-YA on mGnRHR Gene Transcription

Expression vectors (0.5 µg/well) encoding either wild-type or a DN mutant mouse NF-YA, or the pSG5 empty vector control, were cotransfected into {alpha}T3–1 cells together with wild-type or µNF-Y –1164/+62 mGnRHR-Luc as indicated. Cells were harvested 24 h after transfection, and luciferase activity was measured and normalized to ß-galactosidase activity. ***, P < 0.001 vs. pSG5; ###, P < 0.001 vs. NF-YA.

 
Oct-1 Small Interfering RNA (siRNA) Results in a Decrease in GnRHR Gene Transcription
To determine the direct effects of Oct-1 on mGnRHR gene transcription, several approaches were attempted. Cotransfection of an Oct-1 expression vector (31) with –1164/+62 mGnRHR-Luc had no effect on luciferase activity (data not shown). Although Western blot analysis confirmed overexpression of Oct-1 protein in these transfection studies, binding to the SURG-1 element examined by EMSA was not increased (data not shown). Moreover, no DN Oct-1 mutants were available to interfere with endogenous Oct-1 protein activity. Therefore, RNA interference was used to reduce cellular Oct-1 levels (Fig. 8Go). Oct-1 siRNA (50 nM), or a scrambled siRNA used as a control, was transfected into {alpha}T3–1 cells, and after 24 h cells were harvested and total RNA was extracted. RNA was reverse transcribed and used as template for real-time quantitative PCR, with normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cells transfected with Oct-1 siRNA had a 53% decrease in Oct-1 mRNA levels compared with untransfected controls (Fig. 8AGo). Furthermore, this decrease in Oct-1 mRNA levels was associated with a significant decrease in GnRHR mRNA levels (40% of control) (Fig. 8BGo). These changes did not occur in cells transfected with scrambled siRNA, confirming that the observed decreases were due to target-specific knockdown of Oct-1. These results strongly support a significant role of Oct-1 in GnRHR gene transcription.



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  		Fig. 8. Effect of Oct-1 siRNA Transfection on Oct-1 and GnRHR mRNA Levels

Oct-1 siRNA (50 nM) or scrambled siRNA was transfected into {alpha}T3–1 cells for 4 h. After 24 h, total RNA was extracted and 1 µg of total RNA was subjected to reverse transcription. Reverse transcription product (1µl) was used for real-time PCR with primer sets for mouse Oct-1, GnRHR, or GAPDH. A, Representative electrophoresis results of Oct-1, GAPDH, and GnRHR mRNA amplified by conventional RT-PCR after 28 cycles. B, Quantitative plots of Oct-1 and GnRHR mRNA levels calculated after real-time PCR. Both were normalized by GAPDH mRNA levels. *, P < 0.05 vs. control; #, P < 0.05 vs. Oct-1. CTL, Untransfected control; SCR, scrambled siRNA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The gonadotrope-specific and GnRH-stimulated expression of the GnRHR gene is dependent on multiple transcription factors that interact with specific cis- elements. The present study identifies binding sites for the transcription factors NF-Y and Oct-1 within the SURG-1 element of the mGnRHR gene promoter, an element reported previously to be important for mediating GnRH responsiveness (7), and demonstrates that these transcription factors are important for GnRHR gene regulation.

A CCAAT box, which binds NF-Y, was identified at position –288/–284 within the SURG-1 element of the mGnRHR gene promoter in the reverse orientation. NF-Y is a ubiquitous transcription factor and is known to be involved in basal or induced expression of various genes (32). It consists of three subunits (A, B, and C) that form a heterotrimer for binding to its target DNA element (33). Recently, it has been reported that NF-Y contributes to cell-specific expression of the mouse FSHß gene by physically and functionally interacting with SF-1 in the LßT2 cell line (34). Furthermore, an interaction between NF-Y and AP-1 at overlapping binding sites was suggested to be important for maximal induction of the FSHß gene by GnRH (35). In addition, an NF-Y binding site has been shown to be important for basal, but not GnRH-stimulated, expression of the bovine LHß subunit gene in gonadotropes in vivo and in vitro (36). In the case of the GnRHR gene, the NF-Y-binding site in SURG-1 mediates both basal expression and responsiveness to GnRH (Fig. 6Go, B and C). There is mounting evidence that NF-YA is a regulatory subunit of the trimeric complex (37, 38) and that regulation is achieved at a posttranscriptional level (39). Overexpression of NF-YA and interference with NF-Y function using a DN NF-YA mutant confirmed the role of NF-Y in GnRHR gene transcription. These effects were prevented by mutation of the NF-Y-binding site, further confirming that NF-Y binding to the SURG-1 element is critical for GnRHR gene expression (Fig. 7Go). Whereas GnRH had no effect on NF-Y binding to the mGnRHR gene promoter in vitro in EMSA studies (Fig. 1AGo), ChIP assay demonstrated an increase in NF-Y binding after GnRH agonist stimulation. This discrepancy suggests that in vivo binding is more finely regulated, possibly by other factors in a physiological context. Indeed, NF-Y is known to interact with components of the basal transcriptional machinery as well as with coactivators (21); such interactions may be regulated by GnRH.

Oct-1, a POU domain transcription factor, has been shown to play a role in the regulation of both GnRH and GnRHR gene expression. There has been increasing evidence for a dual role of Oct-1 in the regulation of the GnRH gene. Oct-1 is essential for activity of the proximal conserved region and distal neuron-specific enhancer of the GnRH gene promoter (40, 41). In addition, immunoneutralization of Oct-1 as well as mutation of an octamer-binding site in the rat GnRH promoter blocked the pulsatile nature of GnRH promoter activity in GT1–7 neuronal cells (42). On the other hand, Oct-1 is involved in tethering of the glucocorticoid receptor to a negative glucocorticoid response element, playing a role in glucocorticoid repression of the mouse GnRH gene. Moreover, Oct-1, together with C/EBPß, is a downstream transcriptional regulator involved in the repression of human GnRH gene expression by the glutamate/NO/cGMP signal transduction pathway (43). Interestingly, Oct-1 also appears to play opposing roles in the regulation of the GnRHR gene. Whereas Oct-1 has a role in the activity of a placenta-specific upstream promoter in the human GnRHR gene (44), the same factor has been reported to be involved in the transcriptional repression of the GnRHR gene through an element at –1017/–1009 (relative to the translational start site) in ovarian granulosa-luteal cells. The present study defines additional roles for Oct-1 in the regulation of the GnRHR gene. Oct-1 binds to the SURG-1 element in the mGnRHR gene promoter, and mutation of this Oct-1-binding site interfered with basal and GnRH-stimulated mGnRHR gene transcription. ChIP assay confirmed Oct-1 binding in vivo, and RNA interference studies further confirmed the role of Oct-1 in GnRHR gene expression. These results suggest that the role of Oct-1 in GnRHR gene regulation can vary in a tissue-specific fashion or according to the physiological context.

The octamer sequence ATGCAAAT is a classical Oct-1 and Oct-2 binding site. Some regulatory elements recognized by Oct-1 have a modified octamer sequence such as ATGATAATGAG and TAATGA(A/G)AT (45, 46). In vitro experiments have shown that Oct-1 and Oct-2 proteins can bind to TAAT-core- containing homeodomain binding sequences (27). The present study demonstrated that Oct-1 binds to the SURG-1 element. Binding of Oct-1 to the SURG-1 element requires the octamer sequence at –294/–287. When the four bases 5' to TAAT are mutated, Oct-1 binding is eliminated. Oct-1 has two POU domains. The N-terminal POUS domain makes its primary contacts with the 5'-half of the octamer site (ATGC), whereas the C-terminal POUH domain makes its primary contacts with the 3'-part of the octamer site (AAAT) on the opposite side of the double helix (47). The requirement of the full octamer sequence suggests that both POUS and POUH are involved in Oct-1 binding to SURG-1 in the mGnRHR gene promoter. In Fig. 3Go, each of the 2-bp mutations of the four bases 5' to TAAT was not sufficient to reduce Oct-1 binding, likely due to the less stringent sequence specificity for POUS. The region 3' to TAAT also has some effect on Oct-1 binding (Fig. 4CGo). Because, in addition to the core octamer sequence, the flanking bases make a significant contribution to the binding affinity of the POU domain (48), this region appears to play a supporting role for the binding affinity of Oct-1 to SURG-1.

The overlap of the binding sites for NF-Y and Oct-1 in the SURG-1 element may be related to interaction between the two factors, such as agonism or antagonism in their function. DNA binding by NF-Y and Oct-1 appear to be independent, because mutation of the NF-Y binding site (M6) retains intact binding of Oct-1, and, similarly, mutation in the Oct-1 binding site (M3) does not affect NF-Y binding (Figs. 3Go and 4BGo). However, the introduction of sequence modification that increased Oct-1 binding resulted in a decrease in NF-Y binding (Fig. 4DGo), suggesting the possibility of competition between these two transcription factors. The balance between the binding of these two factors in vivo may be dependent on the integrated effects of other factors, which can modify the abundance or binding affinity of each transcription factor.

Transient transfection experiments indicate that the Oct-1, NF-Y, and AP-1 binding elements all have roles in the regulation of mGnRHR gene expression. Although individual mutations of the Oct-1 and NF-Y binding sites decreased both basal expression of the mGnRHR gene and stimulation by GnRH, the combined mutation of both sites did not show any further reduction. This result suggests that both binding sites must be intact for Oct-1 and NF-Y to affect mGnRHR gene expression. Similarly, both Oct-1- and NF-Y-binding sites have been shown to be necessary for the histone deacetylase inhibitor-induced activation of the Gadd45 gene promoter, despite the absence of demonstrable protein-protein interactions between these two factors (49).

Mutations in the consensus AP-1 binding site at –274/–268 of the mGnRHR gene promoter have been reported previously to decrease GnRH responsiveness in {alpha}T3–1 cells (7, 13). Consistent with these reports, in the present study, a single base pair mutation of the AP-1 consensus sequence reduced the response to GnRH. This effect is consistent with previous observations that GnRH regulation of the mGnRHR gene is mediated, at least in part, by activation of PKC, leading to increased protein binding to the AP-1 element (7, 13). The AP-1 element is important for basal expression of the mGnRHR gene as well as for GnRH responsiveness (17). Interestingly, the combined mutation of the Oct-1 and AP-1 binding sites abolished both basal and GnRH-stimulated transcriptional activity of the mGnRHR gene promoter (Fig. 6Go). POU proteins cooperate with many transcription factors including AP-1 (50, 51, 52). Recently, it was reported that Oct-1 activates the profilaggrin promoter in cooperation with c-Jun, and that binding of both factors at their respective recognition sites is essential for the cooperation (53). Therefore, together with the present data, evidence supports the possibility of cooperative interaction between Oct-1 and AP-1 in the regulation of mGnRHR gene expression. Whereas the coupling of PKC signaling induced by GnRH to AP-1 transactivation of gene expression has been well described (29), the relationship of GnRH signaling to Oct-1 transcriptional activity is not yet known. Further studies will be necessary to detail the function of Oct-1 and its interaction with NF-Y and AP-1 in the regulation of mGnRHR gene transcription.

Complex C is not yet identified in the present study. This complex was weak in intensity, suggesting either lower protein abundance or reduced DNA affinity. The TAAT sequence is necessary for its binding, suggesting that it may also be a homeodomain protein and/or Oct-1-related protein. Complex C was competed partially by the Oct-1 consensus oligonucleotide but not recognized by anti-Oct-1 or -Oct-2 antibody. Interestingly, when the Oct-1 consensus oligonucleotide or the S1 oligonucleotide modified to an Oct-1 consensus sequence was used as probe, complex A was increased whereas complex C decreased, suggesting competition with Oct-1 for binding to the same site. Studies to identify the protein(s) in complex C are ongoing.

In summary, we have identified Oct-1 and NF-Y binding sites in the SURG-1 element of the mGnRHR gene promoter. These transcription factors are functionally involved in both basal expression and GnRH regulation of the mGnRHR gene, both independently and through possible interactions with the canonical AP-1 element.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Des-Gly10[D-Ala6]-GnRH-ethylamide (GnRH agonist) was obtained from Sigma Chemical Co. (St. Louis, MO). Antibodies for NF-YA (sc-10779X), NF-YB (sc-7711X), NF-YC (sc-7715X), C/EBPß (sc-150X), CDP (sc-6327X), Ptx-1 (sc-18922X), Ptx-2 (sc-8747X), Otx1/2 (sc-11026X), LHX2 (sc-19344X), LHX3 (sc-13263), MSX-1 (sc-15395X), Oct-1 (sc-232X), and Oct-2 (sc-233X) and antirabbit (sc-2027) and antigoat (sc-2028) IgGs were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All oligonucleotides were prepared by Life Technologies, Inc. (Carlsbad, CA). Sequences of oligonucleotides used are detailed in Results. {alpha}T3–1 and LßT2 cells were generously provided by Dr. Pamela Mellon (University of California, San Diego).

Plasmids
A 1.2-kb fragment of the 5'-flanking region of the mGnRHR gene was ligated into the luciferase reporter vector, pXP2, as described previously (designated –1164/+62 mGnRHR-Luc) (15). The nucleotide sequence of the mGnRHR gene promoter used in this study is based on previous work in this laboratory (15), with –1 assigned to the nucleotide immediately 5' of the major transcription start site. Mutations were introduced into –1164/+62 mGnRHR-Luc by site-directed mutagenesis, using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) with selected sense and antisense oligonucleotides, following the manufacturer’s instructions. Specific nucleotide changes introduced are detailed in Results. An expression vector expressing ß-galactosidase driven by the simian virus 40 (SV40) promoter (SV40-ß-gal) was used as an internal standard and control. The mouse wild-type and DN NF-YA expression vectors were kindly provided by Dr. Roberto Mantovani (30).

Cell Culture and Transfection Studies
{alpha}T3–1 and LßT2, mouse gonadotrope-derived cell lines (54, 55), were maintained in monolayer culture in high-glucose DMEM. CV-1 (African green monkey kidney fibroblast) cells were maintained in low-glucose DMEM. All media were supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. All cells were maintained at 37 C in humidified 5% CO2-95% air.

For transient transfection studies, cells were divided into six-well tissue culture plates and cultured overnight in DMEM. When cells reached 40–50% confluence, cells were transfected by calcium phosphate coprecipitation as described previously but with minor modifications (7). Briefly, in experiments to study effects on GnRH responsiveness, cells were incubated with calcium phosphate-DNA precipitates for 4 h in media containing 10% (vol/vol) fetal bovine serum. In each experiment, a luciferase reporter plasmid (2 µg/well) was added along with SV40-ß-gal (1 µg/well), used as an internal standard. After the 4-h transfection, cells were washed once at room temperature with PBS (pH 7.4). Thereafter, cells were treated with 100 nM GnRH agonist or vehicle in 10% serum-containing DMEM for 4 h, followed by harvest. These conditions were selected after optimization analysis to give maximal GnRH responsiveness. For studies of basal expression, cells were transfected under the same conditions described above, except a 20-h transfection and only 0.5 µg/well SV40-ß-gal was used. After the incubation, medium was aspirated, and cells were washed once with ice-cold PBS. Cells were lysed in the wells by addition of 200 µl lysis buffer [125 mM Tris (pH 7.6), 0.5% (vol/vol) Triton X-100]. Cellular debris was removed from the lysate by centrifugation at 14,000 x g for 10 min at 4 C. Supernatants were assayed immediately for luciferase and ß-galactosidase activity by standard protocols. Briefly, luciferase activity was determined by adding 100 µl of cell lysate to 200 µl of luciferin substrate (Pharmingen, San Diego, CA) and measuring luminescence with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) set for a 20-sec integration with no delay. ß-Galactosidase activity was determined by adding 80 µl of cell lysate to 300 µl of substrate [0.1M Na2HPO4 (pH 7.3), 0.013 M 2-nitrophenyl-ß-D-galactopyranoside, 0.1% (vol/vol) 0.1M MgCl2, 0.35% (vol/vol) ß-mercaptoethanol], incubating overnight at 37 C, and measuring colorimetrically at 410 nm in a Beckman DU640 spectrophotometer (Beckman, Fullerton, CA) after the addition of 100 µl of 0.1 M sodium carbonate. Luciferase activity was normalized to expression of SV40-ß-galactosidase.

For overexpression of NF-YA, the NF-YA, DN NF-YA, or corresponding pSG5 control expression vectors (0.5 µg/well) were cotransfected in {alpha}T3–1 cells along with a luciferase reporter plasmid (0.5 µg/well) and SV40-ß-gal (0.5 µg/well) using GenePORTER transfection reagent (Gene Therapy Systems Inc., San Diego, CA) with Opti-MEM I (Invitrogen Co., Carlsbad, CA) as serum-free medium according to the manufacturer’s protocol. After transfection, cells were incubated for 4 h in serum-free conditions, after which an equal volume of 20% fetal bovine serum-DMEM was added and cells were further incubated for an additional 20 h until harvest.

Preparation of Nuclear Extracts
{alpha}T3–1, LßT2, and CV-1 cells were grown to approximately 50–60% confluence and treated with 100 nM GnRH agonist or vehicle for 1 or 4 h. Thereafter, cells were harvested, and nuclear extracts were prepared by the method of Andrews and Faller (56).

EMSA
Two complementary strands of synthetic oligonucleotides were annealed in annealing buffer (100 mM NaCl; 10 mM Tris·Cl, pH 8.0; and 1 mM EDTA), and the annealed oligonucleotides were purified by electrophoresis on a 10% polyacrylamide gel using the QIAEX II gel extraction kit (QIAGEN, Valencia, CA). The oligonucleotides were 5'-end labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Labeled oligonucleotides were purified using a nick column nucleotide removal kit (Amersham Life-Science, Piscataway, NJ). The binding reaction for EMSA was performed by incubating 200,000 cpm of DNA probe with 5 µg of nuclear extract and 1 µg of salmon sperm DNA in reaction buffer [20 mM HEPES (pH 7.9), 60 mM KCl, 5 mM MgCl2, 2.5 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, 1 mg/ml BSA, and 5% (vol/vol) glycerol] for 30 min at 4 C. For competition studies, excess unlabeled oligonucleotide (500x) was added 5 min before the addition of probe. Supershift experiments were carried out by adding the indicated antibodies (0.5 µg) to the reaction mixtures 1 h before probe was added. Protein-DNA complexes were resolved by 5% low-ionic strength nondenaturing polyacrylamide gel electrophoresis in 0.5x Tris borate/EDTA buffer (45 mM Tris-HCl, pH 8.0; 45 mM boric acid; 1 mM EDTA). Gels were dried for 1 h and subjected to autoradiography for 24–48 h.

ChIP Assay
{alpha}T3–1 cells were treated with 100 nM GnRH agonist for 0, 1, or 4 h, after which cells were fixed with 1% formaldehyde at room temperature for 10 min. Cross-linked DNA was sonicated to fragments ranging from 200–500 bp in length. After cell lysis and sonication, the supernatant was diluted 5-fold in ChIP dilution buffer (0.01% sodium dodecyl sulfate; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris, pH 8.1; 167 mM NaCl; 1 µg/ml leupeptin; 1 µg/ml aprotinin; and 1 mM phenylmethylsulfonylfluoride) before incubation with antibodies. ChIP was carried out by the addition of 10 µg of Oct-1, NF-YA, or c-Jun antibody or normal rabbit serum as a negative control. Ten percent of the sample volume was reserved for use as input control. Cross-linking was reversed by incubation at 65 C for 6 h. DNA was subsequently purified using Qiaquick columns (QIAGEN). PCR was performed using 3 µl of DNA as template for 35 cycles, using primers spanning the SURG-1 element (–337/–170; sense: 5'-GTATCTGTCTAGTCACAACAG-3'; antisense: 5'-TCCTGAAGGCCAAGTGTAACC-3').

RNA Interference
Oct-1 siRNA was purchased from Santa Cruz Biotechnology, Inc. A scrambled siRNA for use as a negative control with a randomly generated 21-nucleotide sequence was prepared using the siRNA Construction Kit (Ambion, Inc., Austin, TX). {alpha}T3–1 cells were seeded into six-well culture plates 1 d before transfection. On d 1, the cells (30–40% confluence) were washed with PBS and transfected with 50 nM Oct-1 or scrambled siRNA using transfection reagent (Santa Cruz Biotechnology). The cells were incubated with siRNAs for 24 h. Total RNA from siRNA-transfected cells was then extracted using Tri reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s guidelines. Total RNA (1 µg) was subjected to reverse transcription using random hexamer and Maloney murine leukemia virus reverse-transcriptase (Ambion). The reverse transcription product (1 µl) was subsequently used for quantitative real-time PCR using the ABI Prism 7000 (Applied Biosystems, Foster City, CA). Oct-1 and GnRHR mRNA levels were normalized to GAPDH mRNA levels. The PCR primer sets used are as follows: Oct-1 sense, 5'-TTCAGTGCAGTCAGCCATTC-3'; antisense, GGCTTTGCTGAGGTAGTTGC-3'; GnRHR sense, 5'-CAGCTTTCATGATGGTGGTG-3'; antisense, 5'-TAGCGAATGCGACTGTCATC-3'; GAPDH sense, 5'-CGTCCCGTAGACAAAATGGT-3'; antisense, 5'-TCTCCATGGTGGTGAAGACA-3'.

Statistical Analysis
Transfections were performed in triplicate and repeated multiple times. Data in each experiment were expressed as luciferase/ß-galactosidase activity. Data were combined across experiments, and the results were expressed as mean ± SE for basal and GnRH agonist-stimulated activities for each construct. ANOVA followed by post hoc comparisons with Fisher’s protected least significant difference test was used to determine whether changes in basal activity or in GnRH agonist responsiveness among different GnRHR promoter-luciferase reporter constructs were significant and with Tukey test for overexpression and siRNA experiments. Significant differences were designated as P < 0.05.


   ACKNOWLEDGMENTS
 
We thank Dr. Pamela Mellon for the {alpha}T3–1 and LßT2 cells and Dr. Roberto Mantovani for the wild-type and DN NF-YA expression vectors.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant HD19938 (to U.B.K.).

First Published Online September 23, 2004

Abbreviations: AP-1, Activating protein-1; CDP, CCAAT displacement protein; C/EBP, CCAAT/enhancer binding protein; ChIP, chromatin immunoprecipitation; DN, dominant negative; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GnRHR, GnRH receptor; NF-Y, nuclear factor Y; SF-1, steroidogenic factor 1; siRNA, small interfering RNA; SURG, Sequence Underlying Responsiveness to GnRH; SV40, simian virus 40.

Received for publication January 22, 2004. Accepted for publication September 16, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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