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Molecular Endocrinology, doi:10.1210/me.2007-0455
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Molecular Endocrinology 22 (8): 1908-1923
Copyright © 2008 by The Endocrine Society

A Composite Element that Binds Basic Helix Loop Helix and Basic Leucine Zipper Transcription Factors Is Important for Gonadotropin-Releasing Hormone Regulation of the Follicle-Stimulating Hormone β Gene

Nick A. Ciccone, Charlemagne T. Lacza, Melody Y. Hou, Susan J. Gregory, Kyung-Yoon Kam, Shuyun Xu and Ursula B. Kaiser

Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Ursula B. Kaiser M.D., Brigham and Women’s Hospital, Division of Endocrinology, Diabetes and Hypertension, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: ukaiser{at}partners.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although FSH plays an essential role in controlling gametogenesis, the biology of FSHβ transcription remains poorly understood, but is known to involve the complex interplay of multiple endocrine factors including GnRH. We have identified a GnRH-responsive element within the rat FSHβ promoter containing an E-box and partial cAMP response element site that are bound by the basic helix loop helix transcription factor family members, upstream stimulating factor (USF)-1/USF-2, and the basic leucine zipper member, cAMP response element-binding protein (CREB), respectively. Expression studies with CREB, USF-1/USF-2, and activating protein-1 demonstrated that the USF transcription factors increased basal transcription, an effect not observed if the cognate binding site was mutated. Conversely, expression of a dominant negative CREB mutant or CREB knockdown attenuated induction by GnRH, whereas dominant negative Fos or USF had no effect on the GnRH response. GnRH stimulation specifically induced an increase in phosphorylated CREB occupation of the FSHβ promoter, leading to the recruitment of CREB-binding protein to enhance gene transcription. In conclusion, a composite element bound by both USF and CREB serves to integrate signals for basal and GnRH-stimulated transcription of the rat FSHβ gene.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HYPOTHALAMIC DECAPEPTIDE, GnRH, is released in a pulsatile manner to bind specific high-affinity cell surface receptors (GnRHR) on pituitary gonadotropes to activate signal transduction cascades that modulate the biosynthesis and secretion of LH and FSH. Collectively known as the gonadotropins, LH and FSH act on the ovary and testis to direct steroidogenesis and gametogenesis (1). Because these hormones are responsible for sexual maturation and normal reproductive function, the regulation of their synthesis and secretion is essential for the preservation of a species.

All glycoprotein hormones, which include LH, FSH, TSH, and chorionic gonadotropin, share a common {alpha}-glycoprotein subunit ({alpha}-GSU). The {alpha}-GSU is combined with the respective β-subunit, which confers biological specificity and activity (1, 2). Befitting their important roles in endocrine physiology, the synthesis and secretion of LH and FSH are under complex regulation by multiple interacting factors, both stimulatory and inhibitory, affecting subunit gene expression, protein synthesis, and gonadotropin secretion. Although synthesized in the same cell type, LH and FSH are differentially released; this differential regulation is achieved, at least in part, at the level of transcription under the control of GnRH pulsatility (3, 4). This differential regulation of gonadotropin subunits by GnRH pulse frequencies suggests that distinct molecular mechanisms exist within the gonadotrope to mediate gonadotropin gene transcription, synthesis, and secretion, either by activation of distinct signal transduction cascades or by activation/repression of different transcription factors.

The synthesis of FSHβ is the rate-limiting step in FSH production (5, 6), yet the molecular mechanisms dictating the transcriptional activity of this gene are not fully understood. Characterization of the FSHβ gene promoter has lagged behind that of the {alpha}-GSU and LHβ subunit genes, largely due to the lack of appropriate cellular models for study. The creation of the gonadotrope-derived pituitary cell line, LβT2 cells, has provided a model for further characterization of the FSHβ gene (7). Recent studies have identified several putative cis-elements and their cognate trans-factors important in maintaining transcription of the FSHβ gene. cis-Elements within the human and porcine FSHβ promoter include binding sites for the LIM homeodomain transcription factor LHX3 (8). Nuclear factor Y (NF-Y) and steroidogenic factor 1 (SF-1) have been shown to bind to the mouse (m)FSHβ gene promoter, underscoring the importance of these transcription factors in gonadotrope-specific basal expression (9), whereas pituitary homeobox 1 contributes to tissue-specific expression of the rat (r)FSHβ gene (10). Activin stimulates FSHβ transcription predominantly via binding of Smad- and Mad-related protein transcription factors together with cofactors (11, 12, 13). Steroid hormones of gonadal origin can affect FSHβ gene expression through classical feedback mechanisms that act directly at the level of the pituitary gonadotrope through steroid hormone response elements that have been characterized within the FSHβ promoter (14, 15).

The hypothalamic decapeptide GnRH is also an important modulator of FSHβ gene expression, but the transcriptional mechanisms that mediate GnRH responsiveness remain incompletely characterized. Possible GnRH response elements include two AP-1 sites at positions –120 and –83 of the ovine (o)FSHβ promoter, which have been shown to bind c-Fos in a heterologous nonpituitary cell line (16). However, the functional significance of this interaction remains unclear, because investigations in transgenic mice as well as in LβT2 cells did not support the necessity of these sites for GnRH stimulation (17, 18). Furthermore, these AP-1 sites are not completely conserved in the rat, mouse, or human promoters. A partial AP-1 site, overlapping with the previously reported NF-Y binding site, has been suggested to mediate GnRH-stimulated transcription of mFSHβ in LβT2 cells (19).

To better delineate the mechanisms underlying GnRH-mediated induction of FSHβ gene transcription, we have undertaken experiments to further identify important cis-elements and their cognate trans-acting factors. Herein we describe the identification of a composite cis-element in the rFSHβ gene, which binds members of the basic leucine zipper (bZip) CREB and basic helix loop helix (bHLH) USF families of transcription factors. The binding of USF transcription factors contributes to basal rFSHβ transcriptional activity in LβT2 cells, whereas GnRH stimulates CREB phosphorylation, leading to recruitment of the histone acetyl transferase coactivator CREB-binding protein (CBP) to the rFSHβ promoter, in a previously unrecognized mechanism by which GnRH can directly stimulate the FSHβ gene.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequences between –140 and –50 of the rFSHβ Gene Promoter Mediate Stimulation of Transcriptional Activity by GnRH
To first identify GnRH-responsive regions within the rFSHβ gene, we performed transient transfection analysis with progressive 5'- and 3'-deletion constructs of –2000/+698 rFSHβLuc together with simian virus (SV) 40βGal as an internal control. Forty-eight hours after transfection, cells were treated with vehicle or 100 nM GnRH agonist (GnRHAg) for 4 h, after which the cells were lysed and assayed for luciferase and β-galactosidase activity. Luciferase values were corrected for β-galactosidase activity and expressed as fold response to GnRH for each construct. Using this experimental paradigm, with several FSHβ promoter constructs, a 5- to 8-fold increase in luciferase activity in response to GnRH stimulation was observed (Fig. 1Go). This GnRH response was significantly reduced, from 6.69 ± 0.98-fold to 2.49 ± 0.24-fold, by 5'-deletion from –256/+698 rFSHβLuc to –50/+698 rFSHβLuc (P < 0.05). Additional constructs tested further localized the GnRH-responsive region to –140/–50, with an observed reduction in the GnRH response from 6.24 ± 0.29-fold for –140/+15 rFSHβLuc to 2.85 ± 0.82-fold for –50/+15 rFSHβLuc (P < 0.05) (Fig. 1Go). These studies suggest that the region between –140/–50 may contain a GnRH-responsive cis-element(s). No significant effect of the deletions on basal luciferase activity was observed (data not shown).


Figure 1
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  		Fig. 1. Identification of a GnRH-Responsive Region in the rFSHβ Gene Promoter

Serial 5'- and 3'-deletion constructs of rFSHβ fused to a luciferase reporter in pXP2 were transiently transfected in LβT2 cells. Results are expressed as fold response to GnRHAg for each construct and are shown as mean ± SEM of four independent experiments, each performed in triplicate. Statistically significant differences are denoted by different letters (P < 0.05).

 
Protein-DNA Complexes Form on –80/–51 of the rFSHβ Gene Promoter
Based on the transient transfection results suggesting the importance of sequences between –140/–50 of the rFSHβ gene promoter in mediating GnRH responsiveness, we proceeded to identify sequences within this region that bind nuclear proteins by EMSA. The 5'-flanking sequence of the rFSHβ gene from –140 to –31 was divided into five 30-bp oligonucleotides (I–V), each overlapping by 10 bp (supplemental Table 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend. endojournals.org). These oligonucleotides were radioactively labeled for use as probes in EMSA studies using nuclear extracts from LβT2 cells treated with either 100 nM GnRHAg or vehicle for 1 h. Three specific protein-DNA complexes, referred to as complexes A, B, and C, formed on probe IV (–80/–51) (Fig. 2AGo). All three complexes increased in intensity after GnRHAg treatment. All three complexes were effectively competed with 100-, 250-, and 500-fold excess oligonucleotide IV in both vehicle- and GnRHAg-treated LβT2 nuclear extracts, but not with excess oligonucleotide I, confirming the specificity of the protein-DNA complexes formed (Fig. 2BGo). None of the other probes formed any specific protein-DNA complexes with either vehicle- or GnRH-treated nuclear extracts. These results indicate that nuclear proteins from LβT2 cells bind specifically and in a GnRH-regulated manner to sequences within –80/–51 rFSHβ, within the region which conferred GnRH responsiveness in functional transfection assays.


Figure 2
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  		Fig. 2. Three Specific Protein-DNA Complexes Form on –80/–51 of the rFSHβ Gene Promoter

A, Five overlapping oligonucleotides spanning –140/–31 of the rFSHβ gene promoter were used as probes for EMSA. Three protein-DNA complexes were identified on probe IV (–80/–51), labeled as complexes A, B, and C. B, Increasing amounts of unlabeled excess oligonucleotide I or IV (100-, 250-, or 500-fold) were added to nuclear extracts from vehicle- or 100 nM GnRHAg-treated LβT2 cells, followed by the addition of probe IV. Complexes A, B, and C are labeled. N.E., Nuclear extract.

 
Localization of Complexes A and C to –72/–65 of the rFSHβ Gene Promoter
To more clearly delineate the cis-elements within –80/–51 rFSHβ and identify their cognate binding proteins, a series of sequential 2-bp mutations in oligonucleotide IV, designated as mIV1–IV13, were generated (Fig. 3AGo). These mutant oligonucleotides were used as competitors in EMSA studies. Oligonucleotide IV was radiolabeled and combined with nuclear extracts from LβT2 cells together with a 500-fold excess of unlabeled mIV1–mIV13 (Fig. 3BGo). Oligonucleotides mIV6 (mutation at positions –70/–69) and mIV7 (–68/–67) failed to effectively compete for formation of complexes A and C, and mIV5 (–72/–71) and mIV8 (–66/–65) were only partially able to compete. These results indicate that the proteins in complexes A and C bind to –72/–65 of the rFSHβ gene promoter.


Figure 3
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  		Fig. 3. Serial 2-bp Mutations in –80/–51 rFSHβ Localize the cis-Elements Necessary for Protein-DNA Complex Formation

A, Oligonucleotides harboring sequential 2-bp mutations between –80/–54 rFSHβ were generated. Nucleotide mutations C {leftrightarrow} T and A {leftrightarrow} G were used. The mutants are labeled mIV1 to mIV13, and the mutated nucleotides are italicized and printed in small caps. B, Competition EMSA studies were performed with a 500-fold excess of oligonucleotides I, IV, or mIV1 to mIV13, nuclear extracts, and radioactively labeled probe IV. Complexes A, B, and C are marked by arrows. N.E., Nuclear extract.

 
Identification of CREB, USF-1, and USF-2 as Transcription Factors that Bind to –80/–51 of the rFSHβ Gene Promoter
To identify putative transcription factors in the protein-DNA complexes formed on probe IV, we performed sequence homology analyses using MatInspector (32). Search results identified putative NF-Y, E-Box, CREB, and AP-1 binding sites (Fig. 4AGo). The putative NF-Y, CREB, and AP-1 binding sites are fully conserved across human, rat, and mouse species, whereas the E-box site (CANNTG) is also highly but not perfectly conserved (33). The ovine FSHβ gene promoter has little recognizable homology in this region.


Figure 4
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  		Fig. 4. Identification of Transcription Factors that Bind to –80/–51 of the rFSHβ Gene Promoter

A, The sequences of human, mouse, and ovine FSHβ were aligned and compared with –80/–51 of the rat FSHβ gene promoter. Putative NF-Y, CREB/AP-1, and E-box elements are boxed with solid, broken, and heavy lines, respectively, in the rat FSHβ gene promoter. For mouse FSHβ, the previously reported NF-Y and AP-1 binding sites are boxed with solid and broken lines, respectively. Similarly, the previously reported AP-1 site in the ovine FSHβ is boxed with a broken line. B and C, Competition EMSA was performed using a 500-fold excess of the following unlabeled oligonucleotides as indicated: I, IV, AP-1, mutated (mut) AP-1, CREB, mutCREB, USF, mutUSF, MycMax, mutMycMax, NF-Y, and mutNF-Y. Nuclear extracts from LβT2 cells were used together with radioactively labeled probe IV. Complexes A, B, and C are indicated. N.E., Nuclear extract.

 
In light of the identified putative transcription factors, we designed oligonucleotides corresponding to consensus and mutated AP-1, CREB, NF-Y, USF, and MycMax binding sequences (supplemental Table S1). These were used in competition EMSA studies, with addition of excess unlabeled oligonucleotide, nuclear extracts from LβT2 cells, and radiolabeled probe IV (Fig. 4Go, B and C). Complexes A and C were entirely competed by consensus USF and MycMax oligonucleotides, but not with the corresponding mutant USF and MycMax oligonucleotides (Fig. 4BGo, lanes 6–7 and 10–11). Complex B was competed by consensus AP-1 and CREB oligonucleotides, and not with the mutated AP-1 and CREB oligonucleotides (Fig. 4BGo, lanes 4–5 and 8–9). The consensus NF-Y oligonucleotide failed to compete for formation of complex A, B, or C (Fig. 4CGo, lanes 5 and 7). These results suggest that a member of the basic helix-loop-helix transcription factor family (such as USF, c-Myc, or Max) is responsible for the formation of complexes A and C, and that a member of the basic leucine zipper transcription factor family [such as AP-1, CREB, or CCAAT enhancer binding protein (C/EBP)] is responsible for the formation of complex B, whereas NF-Y does not appear to bind to this region of the rFSHβ gene.

To further confirm the identity of the binding proteins, we performed EMSA using specific antibodies for these transcription factors. Specific reactions contained antibodies against Jun family members, Fos family members, CREB and CREB family members, USF-1, USF-2, c-Myc, C/EBP-β, or NF-YA, combined with vehicle- or GnRHAg-treated LβT2 nuclear extracts and radiolabeled probe IV (Fig. 5Go). Using an AP-1 antibody that recognizes all isoforms of Fos, a weak supershifted complex was observed with both vehicle- and GnRHAg-treated nuclear extracts (Fig. 5AGo, lanes 5–6). The origin of this complex may have been from complex B, but this could not be definitively determined. The Jun antibody may have partially blocked the formation of complex B in vehicle-treated nuclear extract, but again no definitive effect could be observed (Fig. 5AGo, lanes 7–8). However, using either of two CREB antibodies, which recognize all CREB family members or the CREB isoform only, respectively, complex B was clearly and completely supershifted. The intensity of the supershifted CREB complex increased with the use of GnRHAg-treated LβT2 nuclear extracts (Fig. 5AGo, lanes 9–12). In contrast, the C/EBP-β antibody had no detectable effect (Fig. 5AGo, lanes 19 and 20). Similarly, antibodies to activating transcription factor (ATF)-1, -2, and -3 also failed to generate a supershifted complex (data not shown). These results suggest that CREB is primarily responsible for complex B.


Figure 5
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  		Fig. 5. Characterization of the Transcription Factors that Bind to –80/–51 of the rFSHβ Gene Promoter

A, EMSA supershift assays were performed with the following antibodies: IgG, c-Fos (recognizing Fos family members), c-Jun (recognizing Jun family members), CREB family, CREB, USF-1, USF-2, c-Myc, C/EBP-β, and NF-YA. Complexes A, B, and C are indicated, as are the supershifted complexes. B, EMSA supershift assays were performed with NF-YA antibody or IgG, with oligonucleotide IV or mouse GnRH receptor SURG-1, used as a positive control for NF-Y binding and supershift, as probe. Complexes A, B, and C and supershifted complexes are indicated. N.E., Nuclear extract; SURG-1, sequence underlying responsiveness to GnRH-1.

 
To identify complexes A and C, which were effectively competed with consensus oligonucleotides for USF and Myc/Max, we used antibodies specific to USF-1, USF-2, and c-Myc. Complexes A and C were completely supershifted by the USF-1 antibody in both vehicle- and GnRHAg-treated LβT2 nuclear extracts, and the supershifted complexes increased in intensity with GnRH treatment (Fig. 5AGo, lanes 13–14). With the USF-2 antibody, a similar pattern was observed, although complexes A and C were only partially supershifted (Fig. 5AGo, lanes 15–16). The c-Myc antibody had no effect on any of the protein-DNA complexes (Fig. 5AGo, lanes 17–18). In agreement with our cold competition EMSA studies, the NF-YA antibody failed to affect any of the complexes (Fig. 5AGo, lanes 21–22). To further confirm that NF-Y does not bind to the putative NF-Y site in rFSHβ, we used an oligonucleotide encoding SURG-1, an element in the mouse GnRH receptor gene that has been shown to bind NF-Y, as a positive control (Fig. 5BGo and Refs. 34 and 35). NF-Y binding to rFSHβ was again not detected despite a clear supershift of the NF-Y complex bound to the SURG-1 element. Taken together, these results provide strong indication that the transcription factors CREB, USF-1 and USF-2, and, to a lesser extent, AP-1 bind to probe IV and may be important in mediating GnRH-stimulated rFSHβ gene transcription.

Additional Mutational Analyses Localize All Three Protein-DNA Complexes (A–C) to –70/–67 rFSHβ
rFSHβ (–72 to –65) was shown by cold competition EMSA to be the binding site for both complexes A and C (Fig. 3BGo), and both were supershifted by USF-1 and USF-2 antibodies. However, whereas supershift studies indicated that CREB and possibly AP-1 contributed to complex B formation, the precise nucleotide sequence in –80/–51 rFSHβ responsible for this complex was not yet definitively determined. In search of a more appropriate mutation that might allow us to better define the cis-element(s) important for the formation of complex B, we designed different mutations and again performed competition EMSA (Fig. 6Go). In contrast to the original mIV6 mutation (CA->tg), the nucleotide substitution mIV6 (CA->ac) failed to compete for complex B, as well as complexes A and C (Fig. 6Go, lanes 4–5). Combined mutations in mIV567 also resulted in loss of competition for all three complexes (Fig. 6Go, lane 6). To further confirm that these new mutations eliminate complexes A, B, and C, we radiolabeled these new mutant oligonucleotides and used them as probes for EMSA. In accordance with our observations in the competition EMSA studies, these new mutations in mIV6 (CA->ac) completely eliminated all three protein-DNA complexes, as did the combined mutations in mIV567 (data not shown).


Figure 6
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  		Fig. 6. Further Localization of All Three Protein-DNA Complexes A, B, and C to –70/–67 rFSHβ

Competition EMSA studies were performed using the following oligonucleotides as competitors in 500-fold excess: I, IV, mIV6 (CA->tg), mIV6 (CA->gt), mIV6 (CA->ac), and mIV567 (GTCACG->cggccg). The excess oligonucleotides were added to LβT2 nuclear extracts together with radioactively labeled probe IV. Complexes A, B, and C are indicated. N.E., Nuclear extract.

 
USF-1/2 and CREB Bind the rFSHβ Promoter in Vivo under Basal and GnRH-Stimulated Conditions
We have demonstrated by our EMSA studies that USF-1/2 and CREB bind to cis-regulatory elements within the rFSHβ promoter. To investigate whether these protein-DNA interactions occur in vivo, and to further characterize the effects of GnRH stimulation on the binding of these transcription factors to their respective DNA binding sites within the rFSHβ promoter in vivo, chromatin immunoprecipitation (ChIP) assays were performed. LβT2 cells were transfected with –140/+15 rFSHβLuc and 48 h later were treated with vehicle or GnRHAg for 1 h before harvest. USF-1, USF-2, and CREB were all bound to the rFSHβ gene promoter under both basal and GnRH-stimulated conditions (Fig. 7Go). No PCR products were seen when the LβT2 cells were transfected with the empty pXP2 vector, and preimmune IgG likewise did not give rise to a detectable PCR product. The use of cyclophilin primers yielded a PCR product only when the input DNA was used as template, but not after immunoprecipitation with CREB or USF-1/2 antibodies. These controls confirm the specificity of the ChIP analysis results, which indicate that USF-1/2 and CREB bind to the rFSHβ gene promoter region containing the E-box and partial CRE sites in vivo as well as in vitro. No change in binding of either transcription factor was observed after 1 h of GnRH stimulation.


Figure 7
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  		Fig. 7. USF-1/2 and CREB Bind to the rFSHβ Promoter under Basal and GnRH-Stimulated Conditions

ChIP analysis was performed by specific PCR amplification of rFSHβ promoter in immunoprecipitated DNA from LβT2 cells treated with 100 nM GnRH or vehicle for 1 h and using CREB and USF-1/2 antibodies as indicated, or preimmune IgG as a negative control. Input samples (10-fold diluted) were subjected to PCR as positive controls.

 
The Role of the CREB and USF Binding Sites in Mediating GnRH Stimulation of rFSHβ Gene Transcription
To determine the roles of the CREB and USF binding sites in mediating GnRH stimulation of the rFSHβ gene promoter, we introduced the mIV6 (CA>tg), mIV6 (CA->ac), and mIV567 (GTCACG->cggccg) mutations, which were previously used for our EMSA studies (Fig. 6Go), into –140/+15 rFSHβLuc and performed transient transfection studies using LβT2 cells as before, treating the transfected cells with vehicle or GnRHAg for 4 h before harvesting (Fig. 8Go). Mutation of the USF binding site (–140/+15 rFSHβ mIV6 (CA->tg) did not significantly reduce the fold response to GnRH compared with wild type (WT) –140/+15 rFSHβ (WT 8.8 ± 1.4-fold; mIV6 (CA->tg) 6.6 ± 1.0-fold; P = N.S.), whereas both –140/+15 rFSHβ mIV6 (CA->ac) and –140/+15 rFSHβ mIV567 (GTCACG->cggccg), both of which eliminate both CREB and USF binding, resulted in a significant reduction in the GnRH response [mIV6 (CA->ac) 4.9 ± 0.3-fold; mIV567 (CTCACG->cggccg) 4.4 ± 0.4-fold; P < 0.05 compared with WT], similar in magnitude to the reduction observed by 5'-deletion of this region. None of the mutations had a significant effect on basal luciferase activity. The mIV567 construct will be referred to as –140/+15 {Delta}CREB{Delta}USF throughout the remainder of this manuscript. These results suggest that the CREB-binding site, with possible lesser contributions from the USF-binding site, is important for mediating GnRH stimulation of the rFSHβ gene transcription. We were not able to generate a mutant that selectively eliminated CREB binding but retained USF binding to fully evaluate the independent contribution of the CREB binding site to the GnRH response.


Figure 8
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  		Fig. 8. Mutation of the CREB- and USF-Binding Sites Reduces GnRH-Stimulated Transcriptional Activation of the rFSHβ Gene Promoter

LβT2 cells were transiently transfected with either wild type (WT) or mutant –140/+15 rFSHβLuc reporter constructs as indicated, and basal and GnRH-stimulated luciferase activities were measured. Results are shown as mean ± SEM of three independent experiments, each performed in triplicate. Fold stimulation by GnRH in comparison to vehicle treatment is shown for each construct. Statistically significant differences between the fold response to GnRH for each construct are denoted by different letters (P < 0.001). RLU, Relative light units.

 
USF-1 and USF-2 Increase Activity of the rFSHβ Gene Promoter
Our observations with mutagenesis, EMSA, and ChIP assays have given us insight into important cis-elements in rFSHβ and their cognate trans-acting factors that may mediate transcriptional regulation. To further characterize the role of these trans-acting factors in rFSHβ transcription, we next evaluated the effects of their expression on rFSHβ gene promoter activity. We performed transient transfection studies, using –140/+15 rFSHβLuc, –140/+15 {Delta}CREB{Delta}USF, or pXP2, cotransfected with expression vectors encoding CREB, USF-1, or USF-2, or the empty expression vector as a control. Overexpression of either USF-1 or USF-2 resulted in a marked increase in activity of –140/+15 rFSHβLuc (Fig. 9AGo). Mutation of the CREB- and USF-binding sites eliminated the stimulatory effects of USF-1 and USF-2. Similarly, USF-1 and -2 had no effect on pXP2, used as a negative control. Expression of CREB caused a modest increase in –140/+15 rFSHβLuc activity that did not reach statistical significance.


Figure 9
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  		Fig. 9. Overexpression of USF-1 and USF-2 Increases Transcriptional Activity of the rFSHβ Gene Promoter

A, LβT2 cells were transiently cotransfected with –140/+15 rFSHβLuc, –140/+15 {Delta}CREB{Delta}USF rFSHβLuc, or pXP2 together with an expression vector encoding USF-1, USF-2, CREB, or the empty expression vector as a control, and SV40βGal. Basal luciferase activity is shown as mean ± SEM of three independent experiments, each done in triplicate. Statistically significant difference between luciferase values are denoted by different letters (P < 0.05). B, The results from panel A are represented as fold stimulation in response to GnRH, calculated for each luciferase reporter. Statistically significant differences in the fold response to GnRH are denoted by different letters (P < 0.05).

 
The effects of CREB, USF-1, and USF-2 expression on GnRH responsiveness of rFSHβ were also evaluated. Fold induction of –140/+15 rFSHβLuc, –140/+15 {Delta}CREB{Delta}USF rFSHβLuc, or pXP2 activity by GnRH in the presence of overexpressed CREB, USF-1, and USF-2 was measured (Fig. 9BGo). Expression of CREB had no effect on induction of rFSHβ gene promoter activity by GnRH. Expression of USF-1 or -2 did reduce the response to GnRH, to the levels of –140/+15 {Delta}CREB{Delta}USF rFSHβLuc. These results may potentially indicate a role of USF in mediating the response to GnRH. Alternatively, the decrease in response to GnRH may result from the high basal activity levels of the rFSHβ gene promoter in the presence of USF-1 and USF-2, which may limit the extent to which activity can be further induced by GnRH. These results led us to explore the roles of CREB and USF in mediating the GnRH response further by using dominant-negative mutants of these transcription factors.

Expression of a Dominant-Negative CREB Attenuates GnRH Responsiveness of the rFSHβ Gene Promoter
To further understand the functional roles of USF-1, USF-2, and CREB in regulating the transcriptional activity of the rFSHβ gene, we used dominant-negative forms of USF and CREB, A-USF and A-CREB, in transient transfection studies. The basic region of CREB is highly conserved and is critical for sequence-specific DNA binding. In A-CREB, this region has been replaced by an acidic amphipathic motif (25). Similarly, the bHLH region of USF-1, which is important for both DNA binding and dimerization, has been replaced by an acidic sequence in A-USF (27). A-CREB and A-USF bind to and sequester their respective native transcription factor as well as related family members. Neither A-CREB nor A-USF had any effect on basal luciferase activity (Fig. 10AGo). However, in LβT2 cells treated with 100 nM GnRHAg for 4 h, the fold response of –140/+15 rFSHβLuc to GnRH was attenuated in the presence of A-CREB [6.7 ± 0.6-fold to 2.4 ± 0.3-fold (P < 0.05; Fig. 10BGo)]. In contrast, A-USF had no significant effect on GnRH stimulation of –140/+15 rFSHβLuc, and overexpression of A-CREB and A-USF together did not further attenuate the GnRH response compared with A-CREB alone. Overexpression of A-CREB reduced the GnRH response of –140/+15 rFSHβLuc to the levels of –140/+15 {Delta}CREB{Delta}USF rFSHβLuc, and neither A-CREB nor A-USF significantly reduced the GnRH response of –140/+15 {Delta}CREB{Delta}USF rFSHβLuc further. A-CREB and A-USF had no effect on pXP2. These results suggest a role for CREB in mediating GnRH stimulation of rFSHβ gene transcription.


Figure 10
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  		Fig. 10. Overexpression of Dominant-Negative CREB (A-CREB) Attenuates GnRH Responsiveness of the rFSHβ Gene Promoter

A, LβT2 cells were transiently cotransfected with –140/+15 rFSHβLuc, –140/+15 {Delta}CREB{Delta}USF rFSHβLuc, or pXP2, together with an expression vector encoding dominant negative A-CREB or A-USF or the empty expression vector as a control, and SV40βGal. Basal luciferase activity is shown as mean ± SEM of three independent experiments, each done in triplicate. No statistically significant differences between mean luciferase values were found. B, The results from panel A are represented as fold stimulation in response to GnRH, calculated for each luciferase reporter. Statistically significant differences in the fold response to GnRH are denoted by different letters (P < 0.05).

 
CREB Knockdown Reduces Both Basal and GnRH Stimulation of the rFSHβ Gene Promoter
Members of the bZIP family are known to heterodimerize while maintaining transcriptional activity. As the dominant-negative CREB retains its ability to heterodimerize, the observed effects on the GnRH responsiveness of the rFSHβ gene promoter could be mediated either through interference with the activity of CREB and/or with other members of the bZIP family, such as ATF and CRE modulator (CREM) as well as AP1 transcription factors. CREB knockdown studies were performed to further explore the functional significance of CREB on regulation of the rFSHβ promoter. CREB knockdown was achieved using a specific short hairpin RNA (shRNA) construct, which successfully reduced CREB protein levels in two pituitary cell lines (Fig. 11AGo). Cotransfection of the shCREB construct with the –140/+15 rFSHβLuc reporter in LβT2 cells resulted in significantly reduced basal and, in particular, GnRH-stimulated luciferase activity when compared with LβT2 cells transfected with a shRNA construct containing a scrambled control sequence (Fig. 11BGo). These results support an essential role for CREB for maximal GnRH responsiveness of the rFSHβ gene.


Figure 11
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  		Fig. 11. CREB Knockdown Reduces Basal and GnRH-Stimulated Responsiveness of the rFSHβ Gene Promoter

A, Western blot analysis of CREB and actin protein levels in {alpha}T3-1 and GH3 pituitary cell lines transiently transfected with shCREB compared with a shRNA control construct containing a nonspecific scrambled sequence (shScramble). B, LβT2 cells were transiently cotransfected with –140/+15 rFSHβLuc together with a shCREB or shScramble construct. Results from three independent experiments are shown as fold change from basal LβT2 cells tranfected with a shScramble construct. Statistically significant differences are denoted by different letters (P < 0.01).

 
Overexpression of c-Fos, c-Jun, or a Dominant-Negative c-Fos Has No Effect on Transcriptional Activity of the rFSHβ Gene Promoter
Because we detected low levels of AP-1 binding to the cis-regulatory element of rFSHβ, and in light of the previously reported role of AP-1 in the regulation of mFSHβ by GnRH (19), we could not exclude a possible regulatory role of AP-1 in addition to those of CREB and USF in the regulation of rFSHβ transcriptional activity. To assess the possible functional role of AP-1 in rFSHβ gene expression, we expressed c-Jun and c-Fos, or a dominant negative c-Fos construct, A-Fos, with –140/+15 rFSHβLuc, –140/+15 {Delta}CREB{Delta}USF rFSHβLuc, or pXP2 in a transient transfection study. Like A-CREB and A-USF, the basic amino terminus of c-Fos has been replaced by an acidic amphipathic sequence in the dominant-negative mutant, A-Fos. A-Fos binds and sequesters Jun to prevent DNA binding and transactivation (26). No statistically significant change in either basal activity or fold response to GnRH was seen for –140/+15 rFSHβLuc, –140/+15 {Delta}CREB {Delta}USF rFSHβLuc, or pXP2 when either c-Jun and c-Fos or A-Fos were coexpressed (Fig. S1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

GnRH Induces CREB Phosphorylation and Recruits Both pCREB and the Histone Acetyl Transferase CBP to the rFSHβ Promoter
The phosphorylation of CREB regulates its ability to activate gene transcription (36, 37, 38). Therefore, to further characterize the functional significance of CREB in the GnRH stimulation of FSHβ transcription, the effect of GnRH on CREB phosphorylation in LβT2 cells was determined. GnRH specifically induced CREB phosphorylation as early as 5 min after addition of GnRH, a response that was maintained for approximately 2 h, after which pCREB returned to basal levels by 4 h (Fig. 12AGo). There were no apparent changes in total CREB protein or in USF-1/2 protein levels in response to GnRH throughout the time course study, nor in actin, used as a negative control (Fig. 12AGo).


Figure 12
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  		Fig. 12. GnRH Specifically Induces CREB Phosphorylation and Recruits pCREB and CBP to the rFSHβ Promoter

A, Western blot analysis of pCREB, total CREB, USF-1, USF-2, and actin protein levels in LβT2 cells at the indicated times after GnRH stimulation. B, ChIP analysis was performed by specific PCR amplification of rFSHβ promoter in immunoprecipitated DNA from LβT2 cells treated with 100 nM GnRH or vehicle for 1 h and using pCREB and CBP antibodies as indicated, or preimmune IgG as a negative control. Input samples (10-fold diluted) were subjected to PCR as positive controls. End point PCR is shown in the upper panel and quantification of PCR products from quantitative real-time RT-PCR is shown in the graph below. Statistically significant differences between vehicle and GnRH treatments for each respective antibody are denoted by an asterisk (P < 0.05). n.s., Nonsignificant (P > 0.05).

 
CREB phosphorylation enables interaction with the coactivator protein CBP and other histone acetyltransferases, thereby resulting in the transcriptional activation of CREB-responsive genes (39). To determine whether this mechanism is involved in the CREB-dependent activation of rFSHβ transcription by GnRH in vivo, additional ChIP assays were performed in LβT2 cells. Although CREB, USF-1, and USF-2 occupation of the rFSHβ promoter was unchanged after GnRH stimulation (Fig. 7Go), use of a pCREB-specific antibody for chromatin immunoprecipitation followed by PCR amplification of rFSHβ revealed low levels of pCREB occupancy of the rFSHβ promoter in the basal state that increased after 1 h of GnRH stimulation in LβT2 cells, confirmed by quantitative real-time PCR (Fig. 12BGo). Likewise, the use of a CBP antibody for chromatin immunoprecipitation revealed that CBP interaction with the rFSHβ promoter similarly increased after GnRH treatment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The creation of LβT2 cells, a murine gonadotrope-derived cell line, has made the analysis of FSHβ subunit gene regulation possible. This cell line expresses FSHβ and contains necessary transcription factors, receptors, and downstream signaling proteins for basal, activin-, and GnRH-induced expression (10, 40, 41, 42, 43). In this study, we have delineated cis-acting regulatory elements and trans-activating factors necessary for basal and GnRH-induced transcriptional activity of the rat FSHβ gene using the LβT2 cell line.

A previous study by Strahl et al. (16) in 1997 identified two AP-1 sites in the ovine FSHβ (oFSHβ) gene promoter; however, sequence alignment between the ovine and rat FSHβ genes do not show high homology at these sites. Rather, our analysis identified a partial CRE site (–73/–69) and an E-box (–70/–65) in a GnRH-responsive cis-regulatory element within the rat FSHβ promoter, bound by CREB and USF transcription factors, respectively. The identification of an E-box was unexpected, because another overlapping cis-element at –77/–73 has been reported to bind NF-Y in the mFSHβ gene promoter and to be involved in cell-specific FSHβ expression (9). However, based on our EMSA data, the corresponding putative NF-Y binding site in the rFSHβ gene promoter is not occupied by its respective transcription factor, because none of the complexes on probe IV were competed with an NF-Y consensus oligonucleotide or supershifted by an NF-Y-specific antibody, respectively (Fig. 5BGo). The consensus E-box sequence contains the hexanucleotide sequence CANNTG and can be further categorized as Box A (CACCTG or CAGCTG), Box B (CACGTG or CATGTTG), or Box N (CACGCG or CACGAG) depending on the central two nucleotides (33). The Box B E-box in rFSHβ is 86% homologous to CACGTG, whereas both the human and mouse are 71% homologous to CATGTTG. Furthermore, the central nucleotides, and proximal and distal nucleotides surrounding the E-box, can be used to predict which bHLH protein will bind. Group B bHLH proteins include USF, TFE, sterol-regulatory element binding protein, MYC, MAD, HAIRY, G-Box, core-binding factor, and others (44). Both the proximal and distal nucleotides surrounding the rFSHβ E-box suggest the possible binding of Myc, Max, or USF (45). In this study, the addition of c-Myc antibody to either vehicle- or GnRH-treated LβT2 nuclear extracts had no effect. In contrast, antibodies to USF-1 and USF-2 clearly supershifted complexes A and C. USF transcription factors are ubiquitously expressed in mammalian cells and have been shown to form homodimers and heterodimers [USF-1/USF-2 (27, 45)]. Our studies indicate that these USF transcription factors contribute to the formation of complexes A and C by binding to the E box at position –70/–65 of the rFSHβ gene promoter.

The identification of a half-CRE site (–73/–69) in rFSHβ was also unexpected; as mentioned, Strahl et al. (16) previously showed that AP-1 binds to the oFSHβ gene promoter and activates expression in a heterologous cell system, although these AP-1 sites were not necessary for basal and GnRH-induced transcription of the oFSHβ gene in a transgenic mouse model (17) or in LβT2 cells (18). Furthermore, other investigators have described an AP-1 site in the mFSHβ gene promoter necessary for GnRH-induced transcriptional activation (19, 46). However, in this study, we have clearly demonstrated CREB binding to a half-CRE site in the rFSHβ gene promoter, with only minimal AP-1 binding, even after GnRH induction. Collectively, these findings agree that the FSHβ promoter contains an evolutionarily conserved partial CRE/AP1 site that is a major GnRH-responsive element. Furthermore, it appears that this regulatory site is promiscuous in that it has the ability to be bound and regulated by both bZip and AP1 transcription factors. Although the relative importance of these factors may vary in different species, and potentially under different conditions, this observed promiscuity may be important in allowing for transcriptional redundancies in a gene that is essential in maintaining correct reproductive function. Although the human FSHβ promoter contains this partial CRE/AP1 site, studies on this sequence is lacking. Nonetheless, it could be hypothesized that AP1, CREB, and other bZip transcription factors would be essential in mediating the GnRH responsiveness of the human FSHβ gene.

Historically, the CRE site was identified as an 8-bp palindrome, 5'-TGACGTCA-3', but the CRE can also occur as a half-site motif of 5'-CGTCA-3'. Although generally thought to be less active than the full palindrome for CREB binding and cAMP responsiveness (for review, see Ref. 38), a recent in vivo study investigating the ability of CREB to bind to DNA sequences on human chromosome 22 shows that the majority of CREB occupation occurs at half, rather than full, consensus CRE sites (47). The half-site CRE in rFSHβ is 5'-GGTCA-3' and is 100% conserved in human and mouse.

The CREB family of proteins can homodimerize and heterodimerize and belongs to the bZip family along with AP-1. The CREB homodimer is known to bind to the CRE consensus site with high affinity, but ATF2 and ATF3 homodimers (48), C/EBP family members, and AP-1 heterodimers (49) have also been shown to bind to the CRE site with varying affinity. In addition, heterodimerization between bZip family members has also been widely described; it is therefore possible that AP-1 can bind to the half-CRE site in rFSHβ to explain the minor supershift seen with c-Fos antibody. Our complex B appears to be due primarily to the binding of CREB, with possible minor contributions from the binding of AP-1 or a CREB/AP-1 heterodimer. The apparent promiscuity of this region of the FSHβ promoter may explain discrepancies in our EMSA and ChIP data. In EMSA, GnRH-stimulated cell nuclear extracts show increased intensity in complex B compared with controls. However, as measured by ChIP, there is no evident increase in CREB occupation at the FSHβ half-CRE site after GnRH stimulation. The increase in complex B intensity by EMSA may be due to binding of additional bZIP family members.

Because both the E-box and the half-CRE site overlap in the rFSHβ gene, mutagenesis was able to selectively eliminate USF but not CREB binding. Functional studies from transient transfections in LβT2 cells allowed analysis of the contributions of USF and CREB transcription factors in rFSHβ gene expression. USF transcription factors are ubiquitously expressed, yet are involved in expression of several tissue-specific and developmentally regulated genes. Furthermore, USF transcription factors can also function as either activators or repressors depending on the cell and promoter context (50, 27). USF has been shown to bind to {alpha}-GSU at its E-box and has been implicated in mediating cell-specific transcriptional activity (51). In addition, an E-box within the SF-1 promoter is important for basal transcriptional activity of the SF-1 gene (52). SF-1 is essential in pituitary cell development and has a major role in regulating the expression and transactivation of gonadotrope-specific genes, including both LH and FSH β-subunits (28, 53). Consistent with these previous observations, we have seen in this study that overexpression of USF-1 or USF-2 increases activity of the rFSHβ promoter. This underlines the important role USF proteins play in mediating transcription of gonadotrope-specific genes. Surprisingly, overexpression of the dominant-negative A-USF did not cause a statistically significant reduction in basal expression of rFSHβLuc. This observation may be ascribed to the inherently low basal activity of rFSHβ transcription, rendering detection of a further reduction in activity difficult. Likewise, the introduced mutation of the E-box failed to significantly reduce basal FSHβLuc activity.

The ability of CREB and USF to bind to overlapping promoter sequences has been observed previously (54) and indicates that synergy between these transcription factors may not only be limited to the control of FSHβ gene expression but may play a more general role in stabilizing DNA binding proteins to activate transcription. In support of this view, USF and CREB play essential roles in maintaining transcription of the renin gene within the kidney (55) and of brain-derived neutrophic factor within neuronal cells (54). It is possible that CREB and USF synergize to maintain basal FSHβ gene expression, because both trans factors are constitutively bound to the FSHβ promoter as observed by ChIP analysis (Fig. 7Go). Moreover, CREB knockdown causes a reduction in basal FSHβ transcriptional activity (Fig. 11BGo), which could be due to a disruption in CREB and USF synergy. In HeLa cells, USF transcriptional activity absolutely requires a cofactor(s) for USF function (27, 56). Such a cofactor could be a bZip transcription factor such as CREB. In support of this view, a composite element within the {alpha}A-crystalline gene binds both USF and a CREB/CREM family member to activate transcription within the developing lens, whereas in fibroblasts, AP-1, and not CREB, binds to the same composite element to repress gene expression (49). USF transcription factors have been shown to interact physically with TAFII55 and, through this interaction, can recruit the TATA-box binding complex TFIID (57), to form the transcription preinitiation complex at the promoter. USF has also been shown to interact with the histone acetyl transferase CBP (58) that could explain the observation that CBP interacts with the FSHβ promoter at low but detectable levels under basal conditions (Fig. 12BGo) in a mechanism that may maintain constitutive FSHβ transcription.

We have used a potent dominant-negative mutant, A-CREB, which sequesters and prevents activated CREB from binding to CRE sites, to show a significant reduction in GnRH-stimulated transcription of the FSHβ gene. Modest promoter activity remains when the half-CRE site is mutated. This residual activity may be ascribed to an additional CREB binding site located within the proximal FSHβ promoter (data not shown). Because the rFSHβ gene possesses a half-CRE binding site, it has reduced affinity for CREB binding compared with the full palindrome, and it is possible that different homodimers or heterodimers can exist between CREB/ATF/CREM and/or between other bZIP family members in mediating GnRH responsiveness that may equally explain the effect of A-CREB on GnRH stimulation of the rFSHβ gene. To address this issue we conducted CREB knockdown experiments, and we show that reduction in total CREB protein levels reduces maximal GnRH responsiveness of the rFSHβ promoter (Fig. 11Go). This finding supports the functional significance of CREB in mediating GnRH responsiveness of the rFSHβ promoter.

CREB is activated by Ser133 phosphorylation in response to external cellular signals. Although recent studies suggest that CREB binding to target genes in neurons is inducible (59), prevailing views hold that CREB is often bound constitutively to CRE or CRE-like sites in many tissues in the absence of phosphorylation (37, 46, 60), whereas CREB phosphorylation provides the proper electrostatic and structural scaffold for the recruitment of coactivator proteins such as CBP and p300. CBP has intrinsic histone acetyltransferase activity and also serves to recruit additional histone acetyltransferases (e.g. p/CAF, SRC-1, p/CIP) (61). In turn, this induced complex may recruit and stabilize RNA polymerase II at the TATA box (62). We have demonstrated that GnRH stimulates CREB phosphorylation in LβT2 cells (Fig. 12AGo) and increases pCREB occupation of the rFSHβ promoter, resulting in the recruitment of CBP (Fig. 12BGo) to mediate the GnRH transcriptional activation of the rFSHβ gene, as modeled in Fig. 13Go.


Figure 13
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  		Fig. 13. Model Illustrating the Roles of CREB and USF in Basal and GnRH-Stimulated Control of rFSHβ Transcription

Top panel, Under basal conditions, CREB and USF occupy the partial CRE site and E-box of the rFSHβ promoter, respectively, resulting in basal FSHβ gene expression. Bottom panel, GnRH activates CREB by phosphorylation, resulting in the recruitment of the histone acetyltransferase, CBP, to mediate the direct GnRH stimulation of FSHβ transcription, possibly by recruitment of the TATA box-binding proteins (TBP) and other components of the basal transcription machinery.

 
In conclusion, we have identified that the region –72/–65 of the rFSHβ gene is composed of an overlapping half-CRE site and an E-box, and these are bound by CREB and USF transcription factors, respectively, under basal conditions. In addition, we have shown that USF transcription factors are important in mediating basal transcriptional activity of rFSHβ, whereas CREB is necessary for mediating GnRH-induced stimulation of rFSHβ gene transcription by recruiting the histone acetyltransferase CBP. The formation of a multiprotein complex containing USF, CREB, CBP and the transcriptional machinery initiates GnRH stimulation of the FSHβ gene transcription. Future investigations will focus on exploring these in vitro findings in an in vivo context implementing both primary pituitary cells and whole-animal models.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Chemicals used were purchased from either Fisher Scientific (Pittsburgh, PA) or Sigma Chemical Co. (St. Louis, MO). The following antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): ATF 1 (H-60), ATF 2 (N-96), ATF 3 (H-90), CBP (C1, C20), c-Fos (recognizing c-Fos, Fos B, and Fra 1 and 2; K-25), c-Jun (recognizing c-Jun, Jun B, and Jun D; D), c-Myc (C-33), CREB family (25C10G), CREB-1 (sc-240), C/EBP-β (C-19), NF-Y A (H-209), USF-1 (H-86), USF-2 (N-18), and nonspecific IgG. pCREB (05–807) antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY) and β-actin (AC-74) antibody was obtained from Sigma. All oligonucleotides used (see supplemental Table S1) were synthesized by Invitrogen (Carlsbad, CA). The murine gonadotrope-derived LβT2 and {alpha}T3 cell lines were a kind gift from Pamela L. Mellon (University of California, San Diego). GH3 is a rat pituitary cell line generated by Tashjian et al. (63).

Plasmids and shRNA Constructs
The rat FSHβ gene promoter was fused upstream of the luciferase reporter gene in pXP2 (10, 20). Specifically, –2000/+698 rFSHβLuc, –746/+698 rFSHβLuc, –472/+15 rFSHβLuc, –256/+698 rFSHβLuc, –256/+15 rFSHβLuc, –140/+15 rFSHβLuc, –50/+698 rFSHβLuc, and –50/+15 rFSHβLuc were generated as previously described (10, 20, 21). Mutations (2- or 6 bp) were introduced in –140/+15 rFSHβLuc using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s suggested protocol. Oligonucleotides used for mutagenesis and PCR are described in supplemental data (Table S1). Confirmation of mutagenesis was done by bidirectional sequencing using the dideoxynucleotide chain-termination method at the High Throughput Sequencing Service, Brigham and Women’s Hospital (Boston, MA).

For an internal standard, an expression vector encoding β-galactosidase driven by the Simian virus 40 early promoter was used in all transfection studies (SV40βGal; Promega Corp., Madison, WI). The expression vectors for Upstream Stimulatory Factor 1 (USF-1) and Upstream Stimulatory Factor 2 (USF-2) in pcDNA 3.1(–) were a generous gift from Mark Perrella [Brigham and Women’s Hospital (22)] and Michèle Sawadogo [University of Texas, M.D. Anderson Cancer Center, Houston, TX; (23)], respectively. The expression vector for CREB in pCMX was kindly provided by J. Larry Jameson [Northwestern University, Chicago, IL; (24)]. The dominant-negative expression vectors of CREB, Fos, and USF, termed A-CREB, A-Fos, and A-USF, respectively, were generously provided by Charles R. Vinson [National Cancer Institute, NIH, Bethesda, MD (25, 26, 27)]. The cytomegalovirus-driven c-Fos and c-Jun expression plasmids were kind gifts from William L. Miller [(North Carolina State University, Raleigh, NC) (16)].

shCREB and shScramble constructs were purchased from SuperArray Bioscience Corp. (Frederick, MD).

Cell Culture and Transient Transfections
All cell lines were grown and maintained in high glucose DMEM (Hyclone Laboratories, Inc., Logan, UT) supplemented with 10% (vol/vol) fetal bovine serum (Omega, Tarzana, CA), 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate (Invitrogen) in 5% CO2 humidified air at 37 C. For luciferase assays transient transfections were performed as described previously (28). Briefly, LβT2 cells were transfected by electroporation with the indicated rFSHβLuc reporter and SV40βGal, either alone or in combination with an expression vector as indicated. The cells were suspended in 0.4 ml of Dulbecco’s PBS supplemented with 5 mM glucose containing the plasmid DNA to be transfected. The LβT2 cells were exposed to a single electrical pulse of 0.24 V with a total capacitance of 960 µF. After electroporation, the cells were allowed to recover and plated in six-well plates. After 48 h, the cells were treated with vehicle or 100 nM GnRHAg [des-Gly10, [d-Ala6]-LHRH ethylamide, Sigma] for 4 h. Cells were harvested and assayed for both luciferase and β-galactosidase activity. Luciferase activity was normalized to β-galactosidase activity. All transfection experiments were performed in triplicate and repeated a minimum of three times.

{alpha}T3 and GH3 cells were transiently transfected using GenePorter (Genlantis, San Diego, CA) with an shCREB construct or a nonspecific scrambled sequence as a control (shScramble) following the manufacturer’s instructions. After 48 h transfected cells were harvested and total cellular proteins were subjected to Western blot analysis for CREB protein quantification using β-actin as a loading control (further methodological details on Western blot analyses can be found below).

Nuclear Extracts and EMSA
LβT2 cells were grown to approximately 70–80% confluence and treated with vehicle or 100 nM GnRHAg for 1 h. The cells were harvested and nuclear extracts prepared as described previously (29) with the following modifications. The cells were harvested with ice-cold Dulbecco’s PBS containing 0.6 mM EDTA and centrifuged at 2000 x g for 5 min. Cell pellets were suspended in buffer A (10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 250 mM sucrose; 1 mM EDTA; 0.25 mM EGTA; 0.5 mM spermidine; 0.15 mM spermine; 10 mM β-mercaptoethanol; 1 mM phenylmethylsulfonylfluoride; 5 ng/µl leupeptin; and 10 ng/µl each chymostatin, pepstatin, and aprotinin) and pelleted at 2000 x g for 10 min. The pellet was resuspended twice in buffer B [buffer A containing 50 mM sucrose and 0.5% (vol/vol) Triton X-11]. The fraction containing nuclei was pelleted by centrifugation at 2000 x g for 10 min, washed twice with buffer A, and resuspended in buffer C [buffer A with 100 mM NaCl and 10% (vol/vol) glycerol]. This was then gently rocked for 15 min at 4 C and centrifuged at 2000 x g for 10 min. The nuclear fraction was resuspended in buffer D (buffer A supplemented with 400 mM NaCl and 10% (vol/vol) glycerol), gently rocked for 30 min at 4 C, and pelleted at 1000 x g for 20 min. The supernatant containing the soluble nuclear fraction was collected and precipitated by the slow addition of saturated (NH4)2SO4. This was further precipitated by gentle shaking for an additional 30 min at 4 C and centrifuged at 10,000 x g for 20 min. The protein pellet was suspended in dialysis buffer (10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 5 mM MgCl2; 0.1 mM EDTA; 1 mM dithiothreitol; and 20% (vol/vol) glycerol) and dialyzed using a Slide-A-Lyzer dialysis cassette overnight at 4 C. Any precipitate was removed by centrifugation at 12,000 x g for 5 min. Protein yield was assayed as described by Bradford (30) and stored at –80 C.

For EMSA studies, five overlapping oligonucleotides spanning the region of –140/–30 rFSHβ, oligonucleotides containing sequential 2-bp or 6-bp mutations in –80/–51 rFSHβ, and oligonucleotides encoding consensus or mutated AP-1, CREB, USF, MycMax, and NF-Y binding sites were used either as probe or competitor (supplemental Table S1). In all cases, sense and antisense oligonucleotides were annealed in annealing buffer (100 nM NaCl; 10 mM Tris HCl, pH 8.0; 1 mM EDTA, pH 8.0) and purified by polyacrylamide gel electrophoresis. If used as a probe, the oligonucleotide was 5'-end labeled with [{gamma}-32P-ATP] using T4 polynucleotide kinase and purified on a Nick column (Amersham Bioscience). Nuclear extract (5 µg) per sample was used with 200,000 cpm of probe in binding buffer [0.01 µg/µl salmon sperm; 2.15 mM phenylmethylsulfonylfluoride; 5 mM dithiothreitol; 20 mM HEPES, pH 7.9; 60 mM KCl; 5 mM MgCl2; 1 mg/ml BSA; and 5% (vol/vol) glycerol] and incubated for 1 h on ice. In competition experiments, unlabeled oligonucleotides were added 1 h before addition of [{gamma}-32P]-labeled probe. In supershift experiments, antibodies were incubated with nuclear extracts for 1 h before addition of [{gamma}-32P]-labeled probe. The protein-DNA complexes were loaded and electrophoresed on a 5% low-ionic strength nondenaturing polyacrylamide gel in 0.5x Tris-borate EDTA buffer. The gels were dried and exposed to autoradiography.

Western Blot Analyses
LβT2 cells were grown to approximately 70–80% confluence and treated with 100 nM GnRHAg in a time course study using the following time points: 0, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h. Cells were harvested and 10 µg total cellular proteins were subjected to SDS-PAGE separation and transferred onto a PVDF membrane (Millipore Corp., Bedford, MA) for Western blot analyses. The membranes were then blocked with 5% nonfat dry milk in Tris-buffered saline-Tween 20 (TBST) for 1 h at room temperature with shaking. The blots were then incubated with primary antibody, either pCREB (1:10,000), CREB (1:10,000), USF-1 (1:5000), or USF-2 (1:5000), overnight at 4 C with gentle shaking. Blots were subsequently rinsed with TBST and incubated with secondary antibody (for pCREB and CREB: 1:8000 goat antimouse horseradish peroxidase; for USF-1 and USF-2: 1:8000 donkey antirabbit horseradish peroxidase); after rinsing again with TBST, the antibody-antigen complexes were visualized using an enhanced chemiluminescence reagent (PerkinElmer, Wellesley, MA). Blots were stripped (100 mM β-MeOH; 2% sodium dodecyl sulfate; 62.5 mM Tris, pH 6.8) and re-probed with β-actin antibody (1:8000) for normalization purposes.

ChIP Analysis and Quantitative Real-Time PCR
ChIP analysis was performed as previously reported (31). Briefly, LβT2 cells were transfected with –140/+15 rFSHβLuc and incubated for 48 h, with serum starvation for the final 24 h, before treatment with vehicle or 100 nM GnRHAg for 1 h. Cells were subsequently cross-linked with 1% formaldehyde at room temperature for 10 min and terminated with the addition of 125 mM glycine before harvest. Cross-linked DNA was sonicated to fragments ranging from 200–500 bp in length. After sonication, the chromatin solutions were precleared with protein A-agarose (Upstate Biotechnology, Charlottesville, VA) and salmon sperm DNA and subjected to immunoprecipitation (IP) by incubating with CREB, pCREB, USF-1, USF-2, or CBP antibodies, followed by incubation with protein A-agarose-salmon sperm DNA. Preimmune IgG was used as a negative control. After serial washes, precipitated chromatin was eluted, and cross-linking was reversed with 0.3 M NaCl. After protein and RNA removal with proteinase K and ribonuclease (Roche Applied Science, Indianapolis, IN), respectively, chromatin was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative real-time PCR was performed using oligonucleotide primers. The sense primer was directed against the rFSHβ (–117/–93) promoter sequence, and the antisense primer was directed against the pXP2 vector, encompassing the USF- and CREB-binding sites (–72/–65) of the rFSHβ promoter, and cyclophilin primers were used as controls (supplemental Table S1). End-point PCR conditions were performed with a temperature cycle of 30 sec at 94 C, 30 sec at 60 C, and 1 min at 72 C for 30 cycles. Quantitative real-time PCR assays were performed on an ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA) using SYBR green mix (Invitrogen) following the manufacturer’s instructions. Results were analyzed using ABI Prism 7000 SDS software (Applied Biosystems), and data were presented as absolute values as quantified by a standard curve using serial dilutions of –140/+15 rFSHβLuc. PCRs were subsequently electrophoresed on an agarose gel to verify a single band was amplified.

Statistical Analyses
For transfection assays, one-way ANOVA followed by post hoc comparison with Fisher’s protected least significant differences were used to determine statistical difference in pair-wise comparisons. For ChIP real time data, comparisons were made between basal and 1-h GnRH treatment for each respective antibody group using an unpaired t test. The error bars represent SEM. P values < 0.05 were considered statistically significant in all figures.


   FOOTNOTES
 
This research was supported by National Institutes of Health Grants R01 HD33001 and HD19938 (to U.B.K).

Disclosure Summary: The authors have nothing to disclose.

First Published Online June 11, 2008

Abbreviations: AP-1, Activating protein-1; ATF, activating transcription factor; bHLH, basic helix loop helix; bZip, basic leucine zipper; CBP, CREB-binding protein; C/EBP, CCAAT enhancer binding protein; ChIP, chromatin immunoprecipitation; CRE, cAMP response element; CREB, CRE-binding protein; CREM, CRE modulator; {alpha}-GSU, {alpha}-glycoprotein subunit; GnRHAg, GnRH agonist; NF-Y nuclear factor Y; SF-1, steroidogenic factor 1; shCREB, short hairpin CREB; shRNA, short hairpin RNA; SV, simian virus; TBST, Tris-buffered saline-Tween 20; USF, upstream stimulatory factor.

Received for publication October 3, 2007. Accepted for publication May 28, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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