A Novel AP-1 Site Is Critical for Maximal Induction of the Follicle-stimulating Hormone � Gene by Gonadotropin-releasing Hormone*

Received for publication, May 6, 2003, and in revised form, September 29, 2003Published, JBC Papers in Press, October 20, 2003, DOI 10.1074/jbc.M304697200

Djurdjica Coss‡, Suzanne B. R. Jacobs§, Cheryl E. Bender, and Pamela L. Mellon¶

From the Department of Reproductive Medicine, University of California, San Diego, La Jolla, California 92093-0674

Regulation of follicle-stimulating hormone (FSH) syn-thesis is a central point of convergence for signals con-trolling reproduction. The FSH� subunit is primarilyregulated by gonadotropin-releasing hormone (GnRH),gonadal steroids, and activin. Here, we identify ele-ments in the mouse FSH� promoter responsible forGnRH-mediated induction utilizing the L�T2 cell linethat endogenously expresses FSH. The proximal 398 bpof the mouse FSH� promoter is sufficient for response toGnRH. This response localizes primarily to an AP-1 half-site (�72/�69) juxtaposed to a CCAAT box, which bindsnuclear factor-Y. Both elements are required for AP-1binding, creating a novel AP-1 site. Multimers of this siteconfer GnRH induction, and mutation or internal dele-tion of this site reduces GnRH induction by 35%. Thesame reduction was achieved using a dominant negativeFos protein. This is the only functional AP-1 site identi-fied in the proximal 398 bp, since its mutation eliminatesFSH� induction by c-Fos and c-Jun. GnRH regulation ofthe FSH� gene occurs through induction of multiple Fosand Jun isoforms, forming at least four different AP-1molecules, all of which bind to this site. Mitogen-acti-vated protein kinase activity is required for induction ofFSH� and JunB protein. Finally, AP-1 interacts withnuclear factor-Y, which occupies its overlapping site invivo.

Follicle-stimulating hormone (FSH)1 is a key regulator ofreproduction, since it is essential for oogenesis in females andregulates spermatogenesis in males (1). Production of FSH islimited to anterior pituitary gonadotrope cells, which also pro-duce luteinizing hormone (LH). FSH is a heterodimeric glyco-

protein hormone consisting of two subunits: an �-subunit,which is common to LH, thyroid-stimulating hormone, andchorionic gonadotropin (CG), and a unique �-subunit that con-fers specific biological activity (2). Expression of the �-subunitgene is the limiting factor in FSH synthesis, and its transcrip-tion is regulated primarily by gonadotropin-releasing hormone(GnRH), gonadal steroids, and the activin-inhibin-follistatinsystem (3–5).

GnRH is a decapeptide neurohormone, released by a subsetof hypothalamic neurons into the hypophyseal portal system,where it binds its receptor on the pituitary gonadotrope mem-brane. The GnRH receptor belongs to the class of G protein-coupled receptors and, upon ligand binding, activates the pro-tein kinase C and mitogen-activated protein kinase (MAPK)signaling pathways (6). GnRH administration, either to cells inculture or to hypogonadotropic animals, induces transcriptionof the early response genes, c-fos, c-jun, and egr-1 (7–9). Thetranscription factor AP-1, which is composed of Jun/Jun ho-modimers or Jun/Fos heterodimers, has been implicated inGnRH induction of the FSH� gene. Previous studies reportedthat nuclear proteins from GnRH-treated cells bind an AP-1consensus sequence (10), that purified c-Jun protein binds pu-tative AP-1 sites in the ovine FSH� promoter (11), and thatmutation of putative AP-1 sites in this promoter reduces GnRHinduction in heterologous HeLa cells (10). However, in micecarrying a transgene of the ovine FSH� 5�-flanking regionlinked to luciferase in which the same AP-1 sites were mutated,transgene response to GnRH did not differ from the wild-typeovine FSH� promoter (12). Furthermore, one of these AP-1sites is not conserved in the mouse, rat, or human promoters.Therefore, there is a need to examine FSH� regulation of themouse gene, especially in light of the fact that there is a highdegree of conservation between mouse and human FSH� genesand that targeted disruption of the FSH� gene in mice has aphenotype similar to loss-of-function mutations in humans (1).

Until recently, no FSH�-producing cell lines were available.Models of GnRH action using reconstitution of GnRH receptorin non-gonadotrope-derived cell lines may lack signaling mol-ecules or transcription factors necessary for appropriate induc-tion of gonadotrope-specific genes, whereas primary pituitarycell cultures contain only about 5% gonadotropes and are dif-ficult to manipulate in vitro. The gonadotrope-derived L�T2cell line expresses FSH� endogenously (13) and secretes FSHin response to activin (14). These cells also express other mark-ers of pituitary gonadotropes, most notably �-subunit, GnRHreceptor, LH�, and all of the components of the activin systemautocrine loop: activin, follistatin, and activin receptor (13) aswell as inhibin and inhibin receptor (15). Therefore, the L�T2cell line is an excellent model in which to directly study regu-lation of FSH� gene expression.

Indeed, these cells have been used to investigate GnRHsignal transduction (16) and, more recently, the molecular ba-

* This research was supported by NICHD, National Institutes ofHealth (NIH), through cooperative agreement U54 HD12303 as part ofthe Specialized Cooperative Centers Program in Reproduction Research(to P. L. M.). This work was also supported by NIH Grant R37 HD20377(to P. L .M.). The DNA Sequencing Shared Resource, University ofCalifornia San Diego Cancer Center is funded in part by NCI, NIH,Cancer Center Support Grant P30 CA23100. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

‡ Supported by NIH National Research Service Award F32 HD41301and NIH Grant T32 DK07044.

§ Supported in part by NIH Grant T32 DK07451.¶ To whom correspondence and reprint request should be addressed:

Dept. of Reproductive Medicine, University of California, San Diego,2057 Cellular and Molecular Medicine, East, 9500 Gilman Dr., La Jolla,CA 92093-0674. Tel.: 858-534-1312; Fax: 858-534-1438; E-mail:pmellon@ucsd.edu.

1 The abbreviations used are: FSH, follicle-stimulating hormone; LH,luteinizing hormone; GnRH, gonadotropin-releasing hormone; MAPK,mitogen-activated protein kinase; NF-Y, nuclear factor-Y; EMSA, elec-trophoretic mobility shift assay; ChIP, chromatin immunoprecipitation;GST, glutathione S-transferase; MEK, mitogen-activated protein ki-nase/extracellular signal-regulated kinase kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 1, Issue of January 2, pp. 152–162, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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sis for cell-specific expression of FSH�, by comparison with thenon-FSH-producing gonadotrope-derived cell line, �T3–1 (17).In the latter study, we identified specific promoter elementsbinding steroidogenic factor-1, an orphan nuclear receptor thatis specifically expressed in the gonadotrope population andregulates gonadotrope-specific genes within the pituitary. Wealso identified a conserved binding site in the proximal regionof the promoter for nuclear factor-Y (NF-Y), a ubiquitouslyexpressed heterotrimeric transcription factor (18), and showeda role for both NF-Y and steroidogenic factor-1 in gonadotrope-specific expression of the mouse FSH� gene (17).

The goal of the present study is to gain an understanding ofthe molecular mechanisms by which GnRH induces FSH� geneexpression, using the mouse L�T2 gonadotrope cell model. Wedemonstrate that regulation by GnRH is mediated in part byinduction of multiple AP-1 isoforms. These AP-1 isoforms binda novel site that overlaps the element that binds the basaltranscription factor NF-Y. This novel site consists of a half-siteof the AP-1 consensus binding sequence and an adjacentCCAAT box. Furthermore, NF-Y and AP-1 physically interactand, following GnRH stimulation, co-occupy this site in vivo.

EXPERIMENTAL PROCEDURES

Cell Culture and Transient Transfection—L�T2 cells were plated on6-well plates 1 day prior to transfection. Transfection was performed inDulbecco’s modified Eagle’s medium with 10% fetal bovine serum usingFugene 6 reagent (Roche Applied Science) following the manufacturer’sinstructions. Each well was transfected with 1 �g of mFSH�-luc. Plas-mid construction and preparation has been described previously (17).The 398-bp mouse FSH� promoter was PCR-amplified from a genomicclone that was kindly provided by Dr. Malcolm Low and ligated into theSmaI restriction site of the pGL3 luciferase reporter plasmid (Promega,Madison, WI) to generate �398 mFSH�-luc. The �304 and �230 trun-cations were created by digesting the MluI/BglII fragment of �398mFSH�-luc with XmnI and BbvI restriction enzymes, respectively. Thetruncated promoter fragments were then blunt-ended with Klenowfragment (New England Biolabs, Beverly, MA) and ligated into theSmaI restriction site of pGL3. The �129 reporter was created by di-gesting the MluI/BglII fragment of �304 mFSH�-luc with DpnI, blunt-ended with Klenow, and ligated into the SmaI restriction site of pGL3.The �194 plasmid was generated by digesting the MluI/BglII fragmentof �230 mFSH�-luc with the RsaI restriction enzyme and cloning theappropriate promoter fragment into the SmaI and BglII sites of pGL3.The �95 FSH�Luc plasmid was created by PCR-amplifying the pro-moter from �398 mFSH�-luc using a forward primer corresponding tothe sequence of the mouse FSH� promoter from �95 to �77 bp andcontaining a KpnI linker and a reverse primer spanning the HindIIIrestriction site from the pGL3 vector. The PCR product was digestedwith KpnI and BglII restriction enzymes and ligated into the corre-sponding sites in pGL3. The reporter plasmid with the multimerizednovel AP-1 site was created using an oligonucleotide, containing fourcopies of the �78/�67 sequence of the mouse FSH� promoter betweenKpnI and NheI linkers and was ligated into corresponding sites inpGL3, upstream of the �81 bp herpes thymidine kinase promoter,which was ligated between the XhoI and BglII sites. The sequences ofall promoter fragments were confirmed by dideoxynucleotide sequenc-ing performed by the DNA Sequencing Shared Resource, University ofCalifornia San Diego Cancer Center.

An expression plasmid containing �-galactosidase driven by the Her-pesvirus thymidine kinase promoter was co-transfected with mFSH�-luc and used as an internal control. Sixteen h after transfection, thecells were switched to serum-free Dulbecco’s modified Eagle’s mediumsupplemented with 0.1% bovine serum albumin, 5 mg/liter transferrin,and 50 nM sodium selenite. The following day, cells were treated with 10nM GnRH (Sigma) for 6 h, unless otherwise indicated. The cells werethen lysed with 0.1 M potassium phosphate buffer (pH 7.8) with 0.2%Triton X-100. Equal volumes of each lysate were placed in 96-wellplates, and luciferase activity was measured on a luminometer (EG&GBerthold Microplate) by injecting 100 �l of a buffer containing 100 mM

Tris-HCl (pH 7.8), 15 mM MgSO4, 10 mM ATP, and 65 �M luciferin perwell. Galactosidase activity was measured using the Galacto-light as-say (Tropix, Bedford, MA) following the manufacturer’s instructions.All transfection experiments were performed in triplicate and repeatedat least three times. Luciferase values from reporter gene-transfected

cells were consistently at least 100 times higher than values frommock-transfected cells. Results represent the mean � S.E. of all sam-ples analyzed. An asterisk marks a statistically significant differencefrom the control-treated cells, determined by analysis of variance fol-lowed by Tukey-Kramer HSD post hoc multiple range test for individualcomparison with p � 0.05.

Western Blot—Following overnight starvation and GnRH treatment,L�T2 cells were rinsed with phosphate-buffered saline and lysed withlysis buffer (20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 0.5% Nonidet P-40,0.5 mM EDTA, with protease inhibitors: aprotinin, pepstatin, and leu-peptin at 10 �g/ml each and 1 mM phenylmethylsulfonyl fluoride. Pro-tein concentration was determined with Bradford reagent (Bio-Rad),and an equal amount of protein per sample was loaded on SDS-PAGEgel. After proteins had been resolved by electrophoresis and transferred toa polyvinylidene fluoride membrane, they were probed with specific an-tibodies for Fos and Jun proteins (Santa Cruz Biotechnology, Inc., SantaCruz, CA) and NF-YA (Chemicon, Temecula, CA). The bands were de-tected with secondary antibodies linked to horseradish peroxidase andenhanced chemiluminescence reagent (Amersham Biosciences).

Electrophoretic Mobility Shift Assay (EMSA)—After GnRH treat-ment, cells were scraped in hypotonic buffer (20 mM Tris-HCl, pH 7.4,10 mM NaCl, 1 mM MgCl2 with the same protease inhibitors as men-tioned above for the lysis buffer) and allowed to swell on ice. Cells werelysed by passing through a 25-gauge needle, and the nuclei were pel-leted by centrifugation. Nuclear proteins were extracted in hypertonicbuffer (20 mM Hepes, pH 7.8, 420 mM KCl, 1.5 mM MgCl2 with proteaseinhibitors, and 20% glycerol). Two �g of nuclear proteins per samplewas used in the binding reaction (10 mM Hepes, pH 7.8, 50 mM KCl, 5mM MgCl2, 5 mM dithiothreitol, 0.1% bovine serum albumin, 0.1%Nonidet P-40 with 0.5 �g/ml poly(dI-dC) and 2 fmol per reaction ofend-labeled probe). Oligonucleotides were labeled with [�-32P]ATP us-ing T4 kinase. In the competition experiments, competitor oligonucleo-tide was added 10 min prior to the addition of the probe, as were theantibodies in the supershift assays. The Fos, Jun, and NF-Y antibodiesused are the same as in the Western blot, whereas the nonspecific IgGis from Santa Cruz Biotechnology. The reaction was loaded on a 5%acrylamide gel in 0.25� TBE and electrophoresed at 1-V/cm2 constantvoltage. After drying, gels were exposed to autoradiography.

Mutagenesis—Mutagenesis and deletion of the FSH�-luc plasmidwere performed using the QuikChangeTM site-directed mutagenesis kit(Stratagene, La Jolla, CA) according to the manufacturer’s protocol.The oligonucleotide used to mutate the AP-1 half-site (GTCA) was5�-CAGCAGGCTTTATGTTGGTATTGGTCCCGTTAACACCC-3� (topstrand; mutated nucleotides are underlined), and the oligonucleotideused to mutate the NF-Y site (ATTGG) was 5�-CAGCAGGCTTTATGT-TGGTACCGGTCATGTTAACACCC-3� (top strand; again, underlinednucleotides indicate a change from the wild-type sequence). To createan AP-1 consensus, the NF-Y site/AP-1 half-site was mutated to aconsensus AP-1 site using the oligonucleotide 5�-CAGCAGGCTTTAT-GTTGGTATGAGTCATGTTAACACCC-3�. To create a reporter lackingthe AP-1 site, a specific internal deletion was made using the oligonu-cleotide 5�-CAGCAGGCTTTATGTTGGTGTTAACACCCAGTAAATCC-3�. Mutations were confirmed by dideoxyribonucleotide sequencing, asabove.

Chromatin Immunoprecipitation (ChIP) Assay—L�T2 cells werestarved overnight and treated with GnRH for 2 h. Proteins were cross-linked to DNA by the addition of 1% formaldehyde directly to the cellmedium, and, after obtaining the nuclear fraction, chromatin was son-icated to an average length of 1 kb in sonication buffer (50 mM Hepes,pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodiumdeoxycholate, 0.1% SDS). After preclearing with protein A beads, pro-tein-DNA complexes were bound overnight to the same c-Jun, c-Fos,and NF-Y antibodies as were used in the EMSA experiments for super-shift assays and precipitated with protein A beads (Amersham Bio-sciences). After extensive washing (two times each with sonicationbuffer defined above, high salt sonication buffer (500 mM NaCl withother components as defined above), lithium chloride buffer (20 mM

Tris, pH 8, 250 mM LiCl, 1 mM EDTA, 0.1% Nonidet P-40, 0.1% sodiumdeoxycholate), and TE buffer), cross-linking was reversed by the addi-tion of 300 mM NaCl and incubation at 65 °C, and proteins were di-gested by incubation with Proteinase K. DNA was phenol-chloroform-extracted and ethanol-precipitated, and the sequence of interest wasamplified by PCR. Primers used in PCR were 5�-GGTGTGCTGCCAT-ATCAGATTCGG-3� and 5�-GCATCAAGTGCTGCTACTCACCTGTG-3�and spanned the 280-bp sequence in the mouse FSH� gene from �223to �57. The specificity of the product was assessed by the presence of asingle band of the expected size on an ethidium bromide-stained aga-rose gel. For quantification, the PCR product was labeled by including

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[�-32P]dATP in the nucleotide mix used in PCR and run on a 5%acrylamide gel in 0.5� TBE. The gels were dried, subjected to autora-diography, and quantified using a PhosphorImager Optical ScannerStorm 860 (Amersham Biosciences) and the ImageQuant program (Am-ersham Biosciences).

GST Interaction Assay—The glutathione S-transferase (GST)-Jun inthe pGEX vector was kindly provided by Dr. Michael Karin (19),whereas GST-NF-YA and GST-NF-YB were kindly provided by Dr.Sankar Maity (20) and Dr. David Gardner (21), respectively. The c-Junexpression vector was obtained from Dr. Michael Birrer (22), and thec-Fos expression vector was obtained from Dr. Eugene Tulchinsky (23).The NF-Y and green florescent protein expression vectors were pro-vided by Dr. Roberto Mantovani (24) and Dr. Douglass Forbes, respec-tively. 35S-Labeled proteins were produced using the TNT® T7 coupledreticulocyte lysate system (Promega). Bacteria transformed with thepGEX vectors were grown to an OD of 0.6, upon which protein expres-sion was induced by the addition of 0.25 mM isopropyl-�-D-thiogalacto-sidase. Bacterial pellets were sonicated in phosphate-buffered salinewith 5 mM EDTA and 0.1% Triton X-100 and centrifuged, and thesupernatant was bound to glutathione-Sepharose beads (AmershamBiosciences). Beads were washed four times with sonication buffer,followed by equilibration in the binding buffer (below), and split equallybetween different samples and the control. 35S-Labeled proteins wereadded to the beads and bound for 1 h at 4 °C in 20 mM Hepes (pH 7.8),with 50 mM NaCl, 10 mg/ml bovine serum albumin, 0.1% Nonidet P-40,and 5 mM dithiothreitol. After extensive washing, samples were elutedfrom the beads by boiling in Laemmli sample buffer and subjected toSDS-PAGE. Afterward, the gels were dried and autoradiographed.

RESULTS

GnRH Induces FSH� through Proximal Regulatory Sequences—Since GnRH is a major regulator of FSH� synthesis, we sought todetermine the molecular mechanisms by which GnRH inducesFSH� gene expression. A plasmid containing the proximal 398 bpof the mouse FSH� 5� regulatory region linked to a luciferasereporter gene (mFSH�-luc) was transiently transfected into L�T2cells. We chose to study the mouse FSH� regulatory sequencetransfected into this FSH-expressing, murine pituitary gonado-trope cell line to provide a homologous model for analysis of geneexpression. This region of the mouse gene is highly conserved withovine, rat, and human FSH� genes. We have previously shown that398 bp of the 5� regulatory region of the mouse FSH� gene issufficient to provide gonadotrope-specific expression (17). L�T2cells transiently transfected with mFSH�-luc were treated withincreasing concentrations of GnRH to test whether this region ofthe mouse FSH� gene is also sufficient for GnRH responsiveness.Stimulation with GnRH over a range of doses and time periodsrevealed that this short regulatory region contains elements thatallow response to GnRH in a time- and dose-dependent manner.Maximal induction is observed after 6 h of GnRH treatment, andexpression returns to basal level within 24 h of GnRH treatment(Fig. 1A). Increasing doses of GnRH result in increasing activity ofthe mouse FSH� promoter. In the following experiments, cells weretreated for 6 h with 10 nM GnRH, a concentration closer to thephysiological range of GnRH during the estrous cycle.

To identify which promoter elements convey GnRH respon-siveness, we mapped regions of the mouse FSH� gene promoterthat confer GnRH response using truncation deletion analysis.L�T2 cells were transiently transfected with a series of trun-cations of the mouse FSH� gene 5�-flanking region, ranging inlength from 398 to 95 bp upstream of the transcription startsite, and the ability of GnRH to induce transcription was as-sayed (Fig. 1B). Fig. 1B displays the GnRH regulation as -foldinduction over vehicle-treated for each of the truncated pro-moter regions. As we have previously shown, the basal level ofexpression is not significantly changed by truncation throughthe region from �398 to �95 in L�T2 cells (17). However, herewe show that GnRH induction is significantly reduced by se-quential deletion of either of two regions of the mouse FSH�promoter. Significant decreases in GnRH responsiveness werefound when the promoter was truncated from �304 to �230 bp

and in the most proximal region between �95 bp and the startsite of transcription. The reduction due to truncation from�304 to �230 is minor, and an apparent increase in respon-siveness from �127 to �95 is not statistically significant, but itleads to the finding that no statistically significant decrease inresponsiveness exists between truncations �304 and �95. In-deed, a substantial level of induction is retained in the 95-bpmost proximal region, and this is the focus of the followinginvestigation.

An AP-1 Half-site Overlapping the CCAAT Box Is Bound byAP-1 following GnRH Stimulation and Is Essential for Maxi-mal Induction of FSH� by GnRH—Because the proximal 95-bpregion retains 2.6-fold induction by GnRH, whereas the entire398 bp of the regulatory region is induced 3.4-fold, we focusedon this proximal region to determine what transcription factorsconfer GnRH induction. Using the TransFac® data base (25),the proximal region of the promoter was analyzed for putativetranscription factor binding sites. This search revealed a half-

FIG. 1. Localization of the GnRH-responsive region in themouse FSH� promoter. A, the 398-bp mouse FSH� promoter waslinked to a luciferase reporter gene (mFSH��luc) and transientlytransfected into gonadotrope-derived L�T2 cells. The herpes thymidinekinase �-galactosidase reporter was co-transfected as an internal con-trol. After overnight starvation in serum-free Dulbecco’s modified Ea-gle’s medium, the cells were treated with the indicated concentrationsof GnRH for different lengths of time (indicated on the abscissa), afterwhich the luciferase activity in the lysates was measured and normal-ized to �-galactosidase. Results represent the mean � S.E. of at leastthree independent experiments, each performed in triplicate, and arepresented as -fold induction from vehicle control. B, different lengths ofthe mouse regulatory region were transiently transfected into L�T2cells and after overnight starvation, the cells were treated with 10 nM

GnRH for 6 h. The results are represented as -fold induction fromvehicle-treated cells for each truncation. Significantly different induc-tion in the treated cells versus the control cells for each truncation ismarked with an asterisk, whereas a significant drop in induction be-tween the subsequent truncations is marked with a number sign. Re-sults represent the mean � S.E. of four independent experiments, eachperformed in triplicate.

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site for the AP-1 transcription factor adjacent to the CCAATbox, a binding site for the basal transcription factor NF-Y (17).The AP-1 consensus sequence is TGA(C/G)TCA. In the mouseFSH� gene promoter, the first half of the AP-1 inverted repeatis replaced by a CCAAT box (ATTGG on the coding strand), andthe second half of the repeat (GTCA) is juxtaposed to thisCCAAT element. Sequence alignment revealed that the AP-1half-site and the adjacent NF-Y site are conserved in themouse, rat, and human FSH� promoters but absent from theovine and bovine promoters (Fig. 2A).

Next, EMSA was utilized to determine whether NF-Y and/orAP-1 bind to the identified elements and, if so, whether bindingis affected by GnRH treatment. For that purpose, a radiola-beled 35-bp sequence from �99 to �65 bp, which spans thesesites in the mouse gene, was incubated with nuclear extractsfrom L�T2 cells treated with GnRH for different lengths of time(Fig. 2B). Several bands were detected that show altered in-tensity following GnRH treatment. These include complex 1,

which shows increased intensity, and complex 2, which is onlypresent following GnRH treatment. To examine whether thesecomplexes contain AP-1 or NF-Y, we used antibodies that rec-ognize all of the Fos and Jun protein isoforms, respectively, andan antibody directed against the NF-YA subunit, which is themost regulated subunit of the NF-Y heterotrimer (18). The Fosantibody induces a complete supershift of the faster mobilitycomplex 2 and a partial supershift of the slower mobility com-plex 1, reducing the intensity of complex 1 to the untreated (0GnRH) control level, as does the Jun antibody. These resultsindicate that both GnRH-regulated bands contain AP-1 com-plexes, which consist of Fos/Jun heterodimers. The NF-Y anti-body completely supershifts complex 1 in the control extracts,as well as some of complex 1 in GnRH-treated nuclear extracts.We postulated that complex 1 is composed of an AP-1 complexco-migrating with an NF-Y complex and that complex 2 isanother form of AP-1. Attempts to resolve the bands withincomplex 1 were unsuccessful. However, the simultaneous in-

FIG. 2. The �99/�65 region of themouse FSH� gene contains bindingsites for NF-Y and AP-1. A, an NF-Ysite and an adjacent AP-1 half-site wereidentified in the mouse FSH� promoterusing the Transfac® data base. Align-ment of the sequence from �99 to �65 ofthe mouse FSH� gene regulatory regionreveals that the NF-Y site (underline) andthe AP-1 half-site (dashed underline) areconserved in human and rodent speciesbut are absent from the ovine and bovinepromoters. Consensus binding sites arenoted below the alignment. B, EMSAanalysis of nuclear extracts from L�T2cells treated with vehicle (0 h) or 10 nM

GnRH (0.5, 2, or 6 h), using the �99/�65sequence as a probe, are shown. Thelength of GnRH treatment in hours is in-dicated above each lane, and the antibod-ies used in supershift assay are markedabove corresponding lanes. IgG repre-sents a nonspecific antibody used as acontrol. The supershifted bands are indi-cated with ss, whereas 1 and 2 designatecomplexes that change following thetreatment.

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clusion of both NF-Y and AP-1 antibodies supershifts complex1 completely, indicating that this band is composed of twocomplexes, one induced by GnRH (Fos/Jun) and one not af-fected by GnRH (NF-Y). Thus, AP-1 in nuclear extracts fromcells treated for either 2 or 6 h with GnRH, but not fromuntreated cells, bind to this proximal region of the mouse FSH�promoter. NF-Y, on the other hand, binds to its site in bothcontrol and GnRH-treated cells, and the intensity of NF-Ybinding does not change in response to GnRH.

To determine which nucleotides are needed for NF-Y andAP-1 binding, competitions with a 100-fold excess of unlabeledmutant oligonucleotides were utilized (Fig. 3). Two base-pairscanning mutations (mutants A–J) and a one base-pair muta-tion (mutant K), were introduced into the �99/�65 oligonucleo-tide (Fig. 3A). Nuclear extract from vehicle-treated L�T2 cellswas used in EMSA to confirm that NF-Y binds the CCAAT box(Fig. 3B). Oligonucleotides with mutations in the NF-Y element(mutants E, F, G, and to some extent H) cannot bind NF-Y anddo not compete with the labeled wild-type probe. The AP-1consensus sequence (AP-1 lane) cannot compete for NF-Y bind-ing (Fig. 3B). In a similar competition EMSA, using nuclearproteins from cells treated with GnRH for 6 h (Fig. 3C), mu-tants E–I cannot compete successfully for AP-1 binding. Inclu-sion of an NF-Y antibody to induce a supershift of NF-Y allowsvisualization of the AP-1 band, which is normally obscured bytheir co-migration (Fig. 3D). Again, binding of NF-Y, which issupershifted (ssNF-Y), requires bases mutated in E, F, G, andpartially H, whereas binding of AP-1 requires nucleotides mu-

tated in oligonucleotides E–I, which could not compete for AP-1binding. As expected, the AP-1 consensus sequence competesfor AP-1 binding but not for NF-Y binding. These results indi-cate that the AP-1 binding site overlaps the NF-Y binding siteand that both the AP-1 half-site and adjacent CCAAT box arerequired to bind AP-1. Together, these two regulatory elementscreate a novel AP-1 site.

To assess the contribution of these sites to FSH� inductionby GnRH, we introduced selective mutations into mFSH�-luc.We chose mutations that either inhibit both AP-1 and NF-Ybinding in vitro (mutant F in EMSA) or inhibit AP-1 bindingonly (mutant I in EMSA). These mutations reduce GnRH in-duction of luciferase activity by 25 and 35%, respectively, com-pared with the wild-type mFSH�-luc plasmid response (Fig.4A). Approximately the same level of reduction in the responseto GnRH was achieved when putative AP-1 sites were mutatedin the ovine FSH� promoter (10). However, mutation of theNF-Y site/AP-1 half-site to an AP-1 consensus sequence (la-beled AP-1), dramatically increases GnRH induction of lucifer-ase activity to 15-fold, a 6-fold increase over the level of induc-tion of the wild-type sequence containing the novel AP-1 site,indicating that the site in the wild-type promoter is a lowactivity site. Such low activity sites have potentially importantphysiological roles in the regulatory regions of the genes suchas FSH�, which need to be tightly regulated in the course of theestrous cycle, but fluctuate only up to 4-fold in vivo (26).

To address whether the AP-1 element contributes independ-ently to GnRH induction, we introduced a 9-bp deletion, elim-

FIG. 3. Competition EMSA usingoligonucleotides with scanning mu-tations as competitors reveals thebase pairs required for NF-Y andAP-1 binding. A, alignment of wild-typesequence (WT) found in the mouse FSH�promoter �99/�65 and oligonucleotidesused as competitors (labeled A–K) isshown. Scanning mutations were intro-duced into oligonucleotides A–K, andthese changes are underlined. These un-labeled oligonucleotides were used in a100-fold excess in EMSA experiments inB–D, whereas wild-type sequence (WT)was used as a probe. The NF-Y bindingsite is underlined with a solid line in thewild-type sequence, whereas the AP-1half-site is underlined with a dashed line.B, nuclear extracts from control cells weresubjected to EMSA with radiolabeledwild-type probe. Mutated oligonucleotideswere used as competitors in a 100-foldexcess in the corresponding lanes. In thelane labeled AP-1, the AP-1 consensus se-quence was used as a competitor in thesame manner. C, nuclear extracts fromcells treated with GnRH for 6 h were sub-jected to EMSA with the wild-type probe,and the same oligonucleotides with muta-tions as above were used as competitors.D, NF-Y antibody was added to nuclearextracts from the cells treated with 10 nM

GnRH for 6 h, and competition EMSA wasperformed with the wild-type probe andcompetitor oligonucleotides indicatedabove the lanes.

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inating the entire novel AP-1 element (�AP-1) into the �398mFSH�-luc reporter. This deletion reduces responsiveness toGnRH by 25% compared with the wild-type mFSH�-luc re-porter, a similar level of reduction to that obtained with pointmutations in the site (Fig. 4B). Therefore, a reporter plasmidwithout this novel AP-1 site retains a substantial response toGnRH but cannot reach the maximal induction obtained withthe reporter that contains this site. To further characterize thissite and determine whether it is sufficient to confer GnRHresponsiveness to a heterologous promoter, we created a re-porter with four copies of this AP-1 site linked to a minimal�81 bp herpes thymidine kinase promoter driving luciferaseexpression. This reporter was induced 4-fold by GnRH,whereas the control reporter containing only the thymidinekinase promoter was minimally induced 1.4-fold (Fig. 4C). This

result indicates that, in addition to being essential for maximalinduction by GnRH, this site is sufficient for GnRH response.

Multiple AP-1 Isoforms Are Induced by GnRH and Bind tothe Novel AP-1 Site—When we identified AP-1 binding to thisnovel site by EMSA (Fig. 2B), we identified two complexes thatchanged following GnRH treatment. Both were supershiftedwith antibodies against Fos and/or Jun proteins. To determinewhether these complexes differ in their protein components, wefirst tested whether one of these bands is AP-1 protein incomplex with another factor, using in vitro transcribed/trans-lated c-Jun and c-Fos proteins in EMSA. We found that AP-1composed only of c-Jun and c-Fos binds DNA directly at the�99/�65 region as a single band (lane 4), not present in thereticulocyte lysate control (lane 1, Fig. 5A). This c-Jun/c-Foscomplex co-migrates with complex 1 from nuclear extractstreated with GnRH (lane 3), indicating that this upper complex isindeed AP-1 factor alone. Furthermore, this result confirms thatAP-1 can bind directly and does not serve only as a co-factor; nordoes it need to be in complex with NF-Y to bind DNA in vitro.

Since AP-1 does not need another factor to bind to the �99/�65 region, we postulated that the two different complexesobserved following GnRH treatment are composed of differentAP-1 isoforms. To determine which AP-1 isoforms are inducedin L�T2 cells following stimulation with GnRH, Western blotsof whole cell lysates with and without GnRH treatment wereperformed. GnRH selectively induces c-Fos, c-Jun, FosB, andJunB but not JunD in L�T2 cells (Fig. 5B). As expected fromthe EMSA results in which the NF-Y complex intensity did notchange (observable after Fos was supershifted), the amount ofNF-YA in the cells does not change with GnRH treatment.

To examine which of these GnRH-induced AP-1 isoformsbinds to the FSH� promoter, isoform-specific antibodies wereused in EMSA (Fig. 5C). Inclusion of an antibody to c-Fosinduces a supershift in the upper, slower migrating AP-1 com-plex (complex 1), whereas an antibody to FosB supershifts thelower, faster migrating complex (complex 2). Non-isoform-spe-cific antibodies, which recognize all of the Fos or all of the Junisoforms (labeled ns in Fig. 5), supershifted both complexes.Both of the AP-1 bands appear to contain c-Jun and, to a lesserdegree, JunB, since both bands were diminished upon inclusionof antibodies specific for c-Jun and JunB. Thus, the upper band(complex 1) contains c-Fos/Jun heterodimers, whereas thelower AP-1 band (complex 2) contains FosB/Jun heterodimersbinding the AP-1 half-site in the mouse FSH� promoter.

AP-1 Is Necessary and Sufficient for Maximal Induction ofFSH�—To test the role of AP-1 in GnRH induction of themouse FSH� gene, the dominant negative form of c-Fos, namedA-Fos (27), was co-transfected with mFSH�-luc into L�T2 cellstreated with vehicle (control) or GnRH. A-Fos has an acidicextension on the N terminus of the Fos leucine zipper, whichphysically interacts with the Jun basic region, thus preventingthe basic region of the heterodimer from binding DNA (27).Introduction of A-Fos with mFSH�-luc into L�T2 cells has noeffect on the expression of the reporter in untreated controlcells (data not shown). In cells treated with 10 nM GnRH for 6 h,the dominant negative mutant of c-Fos (A-Fos) reduces GnRHinduction of mFSH�-luc by 30% compared with cells trans-fected with the vector control and treated with GnRH (Fig. 6A).The result demonstrates that functional AP-1 protein is neces-sary for maximal induction of FSH� by GnRH. A similar levelof reduction in GnRH responsiveness was observed when theAP-1 site in the promoter was mutated (mutant I, Fig. 4).However, when this mutant I was used in transfection insteadof wild-type mFSH�-luc, A-Fos did not further reduce GnRHresponse (Fig. 6A). These data suggest that the novel AP-1 siteis the only active AP-1 site in the promoter region tested.

FIG. 4. The AP-1 binding site is required for maximal induc-tion with GnRH. A, mutations I and F (see Fig. 3) were introduced intothe mFSH�-luc vector, and transfections were performed using L�T2cells. Cells were treated with 10 nM GnRH for 6 h, after which theluciferase activity was measured and normalized to �-galactosidase.The results are represented as -fold induction from the control cellstransfected with the same reporter vector. The bar labeled AP-1 repre-sents the induction of the reporter with a consensus AP-1 binding siteintroduced into the mFSH�-luc reporter vector in place of the NF-Y/AP-1 site. Significantly different induction in the treated cells versusthe control cells for each reporter is marked with an asterisk, whereasa significant difference in induction of the mutated reporter from in-duction of the wild-type reporter is marked with a number sign. Resultsrepresent the mean � S.E. of four independent experiments, eachperformed in triplicate. B, a 9-bp deletion of the NF-Y/AP-1 site wascreated in mFSH�-luc, and its induction following GnRH treatmentwas compared with the wild-type reporter. Significantly different in-duction in the treated cells versus the control cells for each reporter ismarked with an asterisk, whereas a significant difference in inductionof the mutated reporter from induction of the wild-type reporter ismarked with a number sign. C, a reporter gene with four copies of theNF-Y/AP-1 element upstream of the thymidine kinase promoter wascreated, and its induction was compared with the induction of thecontrol luciferase reporter driven by the thymidine kinase promoter.The activity from GnRH-treated cells was normalized to the activityfrom control cells, and results are represented as -fold induction. Anasterisk marks that the induction of the reporter with multimerizedAP-1 site is significantly different from the induction of the controlplasmid.

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Additionally, c-Fos and c-Jun are sufficient to induce mouseFSH� promoter activity. c-Jun and c-Fos expression vectors,co-transfected with wild-type mFSH�-luc into L�T2 cells, in-duce mFSH�-luc greater than 6-fold over the vector control-transfected cells (Fig. 6B). In contrast, c-Jun and c-Fos expres-sion vectors do not significantly increase luciferase expressionwhen the AP-1 site mutant I is used as a reporter. This againconfirms that the novel AP-1 site we identified is the only sitefor AP-1 binding within the 398-bp regulatory sequence. Fur-thermore, it suggests that the GnRH responsiveness remainingin the �398 FSH� promoter after mutation of the AP-1 site isdue to an activity unrelated to induction of AP-1.

MAPK Is Involved in FSH� Induction by GnRH throughJunB and c-Fos—MAPK is acutely activated following GnRHtreatment of L�T2 cells, and this activation is involved in theinduction of the ovine FSH� promoter (16). To test whetherMAPK plays the same role in the induction of the mouse FSH�promoter by GnRH, we treated L�T2 cells with the MEK in-hibitor, UO126, for 30 min prior to and during GnRH treat-ment. We first established a dose response for MEK inhibitionby UO126 in L�T2 cells, by Western blotting for phospho-MAPK in whole cell lysates following the treatment (data notshown). The minimal concentration of the inhibitor needed tocompletely inhibit phosphorylation of MAPK by GnRH (1 �M)was then used in our experiments. As expected, MAPK signal-

ing plays a role in induction of the mouse FSH� promoter byGnRH, since this induction is reduced by 46% in the presenceof the inhibitor (Fig. 7A).

To test whether levels of AP-1 proteins in L�T2 cells werealtered by inhibition of the MAPK pathway, Western blottingwas performed following GnRH treatment. MEK inhibitionprevents maximal JunB induction by GnRH (Fig. 7B); however,it does not reduce the induction of the c-Jun or FosB isoformsfound to be induced by GnRH in Fig. 5B (data not shown). Inaccordance with previously published results (6), c-Fos levelsare also reduced with MAPK inhibition (date not shown).Therefore, we conclude that the MAPK pathway is involved inGnRH induction of the FSH� gene, in part through the induc-tion of JunB and c-Fos proteins.

AP-1 and NF-Y Interact and Co-occupy the Site in Vivofollowing GnRH Stimulation—Since the AP-1 binding siteoverlaps the NF-Y binding site, as demonstrated in competitionEMSA in Fig. 3, we hypothesized that there are two possibleways that this region can accommodate these transcriptionfactors. One possibility is that AP-1 displaces NF-Y on this sitein vivo following Fos and Jun induction by GnRH. This wastested with a ChIP assay, which allows examination of theproteins binding to this gene region in vivo with and withoutGnRH treatment. After overnight serum starvation, the L�T2cells were treated with GnRH for 3 h. Then, after lysis and

FIG. 5. GnRH induces multiple AP-1 isoforms in L�T2 cells. A, in vitro transcribed and translated c-Fos and c-Jun were used in EMSA tocompare their binding with complexes formed using L�T2 nuclear extract. Lane 1, reticulocyte lysate control; lane 2, control nuclear extracts; lane3, nuclear extracts following 6 h of 10 nM GnRH treatment; lane 4, c-Jun and c-Fos in vitro transcribed and translated in reticulocyte lysate. B,L�T2 cells were treated with vehicle (0 h) or 10 nM GnRH for 1 or 3 h, after which the cells were lysed. Equal amounts of protein from whole celllysates were run on the gel, and after transfer, the membranes were probed with antibodies specific for the indicated proteins. After secondaryantibody, enhanced chemiluminescence was performed, and the blots were exposed to film. C, EMSA using the �99/�65 sequence from the mouseFSH� promoter indicates that at least four different AP-1 isoforms bind the site. The length of GnRH treatment and the isoform specificity of theantibodies used for the supershift assay are indicated above the lanes. Lanes marked ns in the isoform lane included non-isoform-specific antibodiesto Fos and Jun proteins, which therefore interacted with all isoforms of Fos and Jun families, respectively.

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sonication, chromatin was precipitated with antibodies specificto NF-YA, Fos, or Jun. DNA was extracted and subjected toPCR analysis, amplifying the FSH� gene regulatory sequence.The ChIP assay shows that Fos and Jun proteins bind themouse FSH� promoter sequence more intensely followingGnRH treatment, consistent with their induction by GnRH,whereas NF-Y binding to this region does not change (Fig. 8).

This site is the only CAATT box identified in the region ampli-fied with the primers, and, to our knowledge, there are no otherCAATT boxes in the proximity of this site. This assay assessesbinding in a cell population, so if NF-Y were dislodged by AP-1in even a portion of the cells following GnRH treatment, wewould expect the NF-Y antibody-precipitated band to be dimin-ished. Therefore, AP-1 does not appear to displace NF-Y on themouse FSH� promoter.

An alternative mechanism by which these two transcriptionfactors could occupy the same site is if AP-1 and NF-Y physi-cally interact. We tested this hypothesis using GST pull-downassays in which in vitro transcribed and translated c-Fos andc-Jun proteins were tested for their ability to interact withNF-YA-GST fusion protein. In this assay, c-Jun interacts withthe NF-YA subunit (Fig. 9, top panel). 35S-Labeled c-Jun, butnot c-Fos, precipitates with glutathione beads through an in-teraction with GST-NF-YA. Additionally, we performed thereverse experiment in which NF-Y proteins were labeled andsynthesized in vitro and then tested for interaction with c-Jun-GST fusion protein. Labeled NF-YA is retained in the precipi-tate by the interaction with GST-c-Jun (Fig. 9, bottom leftpanel), whereas NF-YB and NF-YC are not (data not shown).No interactions were observed using GST alone; nor did labeledgreen florescent protein, which serves as a control, interactwith any of the used GST fusion proteins (Fig. 9, bottom rightpanel). Thus, NF-Y and AP-1 form heteromeric complexes invitro, through protein-protein interaction between Jun andNF-YA.

FIG. 6. Co-transfections using dominant negative Fos (A-Fos)indicate that AP-1 is necessary for maximal induction either byGnRH (A) or by overexpression of c-Jun and c-Fos (B). A, cellstransfected with wild-type mFSH�-luc or mutation I introduced intomFSH�-luc were treated for 6 h with vehicle (control) or 10 nM GnRH.Cells were co-transfected with an expression vector for dominant neg-ative Fos (A-Fos) or its vector control to assess the necessity for AP-1 inthe inductions by GnRH. Results represent the means of four independ-ent experiments, each performed in triplicate. Results were analyzed byanalysis of variance and Tukey-Kramer post hoc test, and an asteriskindicates a statistically significant difference from the control vehicle-treated cells, whereas a number sign indicates a significant differencefrom GnRH-treated cells co-transfected with wild-type reporter andempty vector control for A-Fos. B, cells transfected with wild-typemFSH�-luc or mutation I introduced into mFSH�-luc were co-trans-fected with c-Jun and c-Fos and with either dominant negative A-Fos orvector control. Twenty-four h after transfection, luciferase activity wasmeasured and normalized to �-galactosidase. None of the vector con-trols for either c-Jun, c-Fos, or A-Fos had any effect on reporter expres-sion (data not shown). Results represent the means of four independentexperiments, each performed in triplicate. An asterisk indicates a sta-tistically significant difference from the control cells, whereas a numbersign indicates a significant difference from cells transfected only withc-Jun and c-Fos.

FIG. 7. Inhibition of the MAPK pathway during GnRH treat-ment prevents maximal induction of FSH� and JunB. A, cellstransfected with wild-type mFSH�-luc were treated for 6 h with vehicle(control) or 10 nM GnRH. Cells were co-treated with the MEK inhibitor,UO126, at 1 �M to assess the necessity of the MAPK pathway ininduction by GnRH. Results represent the mean of five independentexperiments, each performed in triplicate. Results were analyzed byanalysis of variance and Tukey-Kramer post hoc test, and an asteriskindicates a statistically significant difference from the control vehicle-treated cells, whereas a number sign indicates a significant differencefrom GnRH-treated cells without UO126. B, Western blot of JunB inwhole cell lysates of L�T2 cells following 0, 1, or 3 h of GnRH treatmentwith or without the MEK inhibitor, UO126. The experiment was re-peated three times, and a representative gel is shown.

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DISCUSSION

GnRH is a key regulator of FSH� gene expression and there-fore FSH synthesis. Previous studies were limited by a lack ofavailable cell lines that express both FSH subunit genes andrespond to GnRH. The scarce number of gonadotropes in thepituitary precludes performing these studies in primary cells.The genesis of the L�T2 cell line that endogenously expressesFSH�, allows for the dissection of molecular pathways govern-ing its expression (13, 28). In the current study, we have fo-cused on delineating the mechanisms of GnRH induction of themouse FSH� gene.

FSH regulates gonadal development in mammals and isrequired for folliculogenesis (1). Tight regulation of FSH levelsis crucial for the menstrual or estrous cycle. Both FSH proteinin circulation and �-subunit mRNA in the pituitary normallyfluctuate 4-fold during the cycle (26, 29). In mice lackingGnRH, serum FSH levels are 60–90% lower (30). One pulse ofGnRH administered to castrated, testosterone-replaced rats(with low endogenous GnRH) increased FSH� transcription

4-fold (31). This level is comparable with the induction ob-served in our studies using the L�T2 cell line.

We report here that the AP-1 transcription factor, induced byGnRH, can bind a half-site of its consensus sequence, GTCA,when this half-site is juxtaposed to a site involved in basalexpression, in this case a CCAAT box binding NF-Y. Further,we show that this AP-1/NF-Y site is necessary for maximalGnRH induction of the mouse FSH� gene. As shown in Fig. 4,the half-site is, as expected, a low affinity site for AP-1. Spe-cifically, when we mutate the NF-Y site/AP-1 half-site in theFSH� promoter to create a full AP-1 consensus site in thisposition, luciferase expression after GnRH treatment is greaterthan 15-fold higher than the untreated control. This is 6-foldhigher than GnRH induction of the wild-type promoter. Fur-ther, in gel shift assays, an AP-1 consensus competed moreeffectively for AP-1 bands than the nonlabeled wild-type se-quence, and, when used as a probe, the AP-1 consensus re-quires one-tenth of the protein as wild-type probe to observeAP-1 binding (data not shown). Half-sites may have importantphysiological roles despite, or perhaps because of, their lowaffinity. Genes such as FSH� fluctuate only 4-fold, but thisrelatively modest change in expression during the estrous cycleis crucial for normal egg development and selection. A fullconsensus sequence might bring forth an unnecessarily highinduction.

In a recent report describing the regulation of GnRH recep-tor expression by GnRH and activin in L�T2 cells, the promoterelement studied, GTCTAGTCAC, was of special interest (32).The authors conclude that AP-1 binds a novel 6-bp site, AGT-CAC, instead of its usual 7-bp site, whereas activin-regulatedSmad 4 binds a 2-bp site. However, the competition EMSAexperiments shown in that report suggest the alternative ex-planation that Smad 4 binds a Smad half-site GTCT, and AP-1binds its half-site GTCA, which is separated from the Smadhalf-site by only one nucleotide. Thus, in the GnRH receptorgene, in light of our findings, it is possible that AP-1 and Smad4 are stabilized on their respective half-sites by mutual inter-action, and this is the reason both sites are needed for responseto either activin or GnRH. It would be of interest to examinewhether half-sites or low affinity sites are commonly involved

FIG. 8. ChIP reveals that NF-Y binds DNA in the proximalmouse FSH� promoter in both control and cells treated withGnRH for 3 h, whereas AP-1 binds DNA following GnRH treat-ment. A, chromatin was isolated from L�T2 cells treated with vehicle or10 nM GnRH for 3 h and cross-linked with formaldehyde. After sonica-tion, sheared chromatin was precipitated with the antibodies indicatedabove the lanes. The precipitated and purified DNA is then amplified inthe PCR. The antibody specific for NF-Y precipitates the DNA specificfor the sequence in the proximal mouse FSH� promoter, in both controland GnRH-treated cells. Fos and Jun, on the other hand, bind DNA invivo only following the GnRH treatment. In the first two lanes, chro-matin was precipitated with protein A beads only serving as controls. B,chromatin prior to precipitation serves as the control for the amount ofchromatin used for precipitation in the untreated and GnRH-treatedsamples. A serial dilution of the chromatin was performed and thenused in PCR together with precipitated samples. C, four independentexperiments were quantified using a PhosphorImager and then normal-ized to intensity in the control sample precipitated with protein A beadsonly to normalize for any difference in the activity of the [�-32P]dATPused in PCRs. The solid bars represent chromatin immunoprecipitationfrom GnRH-treated cells, whereas open bars represent control samples.

FIG. 9. GST pull-down assays demonstrate that NF-YA caninteract directly with c-Jun. 35S-Labeled proteins, indicated aboveeach panel, were used in the binding assay with GST fusion proteins,labeled above each lane. GST fusion proteins were induced with isopro-pyl-�-D-thiogalactosidase overnight, and the bacterial pellets were son-icated. These proteins were bound to glutathione-Sepharose beads, andin vitro transcribed and translated labeled proteins were added. Afterextensive washing, the precipitates were run on a gel and subjected toautoradiography.

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in the induction of gonadotrope-specific genes that usuallyhave a low level of induction but are very tightly regulatedthroughout the menstrual/estrous cycle.

Another interesting characteristic of the FSH� promoter el-ement is that the AP-1 half-site overlaps an NF-Y site involvedin basal expression. Recently, overlapping NF-Y and YY1 siteshave been identified in the promoter of the Hox4b gene (33);however, this element was in a specialized intronic site able tobind either factor in a mutually exclusive manner. In the FSH�

promoter, AP-1 and NF-Y occupy this element concurrently. InEMSA experiments with control extracts, NF-Y binds this site,and the complex is completely supershifted with antibodies toNF-YA. Competition EMSA in Fig. 3B also shows that aCCAAT box is needed for NF-Y to bind. The ChIP assay indi-cates that NF-Y is present in the complex at the same levelbefore and after GnRH treatment in live cells. AP-1 can alsobind this site in vitro, without other proteins present. In vitrotranscribed and translated AP-1 binds this site, and it co-migrates with the AP-1 complex from GnRH-treated cells.Thus, the FSH� promoter element can be bound by both NF-Yand AP-1.

AP-1 binding to this low affinity site may be stabilized byprotein-protein interactions with the basal transcription factorNF-Y. Our attempts to observe a higher order complex inEMSA were unsuccessful; this is probably due to its expectedsize of 220 kDa, which would be too large to migrate into thegel. Further, the higher order complex would consist of fivedifferent proteins (NF-YA, NF-YB, and NF-YC, which are allnecessary for NF-Y to bind, and Jun and Fos, which form AP-1)and therefore is difficult to reconstitute from recombinant pro-teins. However, we have established that this is a low affinitysite, compared with the AP-1 consensus sequence, and that it isbound by NF-Y prior to and during GnRH treatment, whichleads us to speculate that NF-Y may stabilize AP-1 binding. Atleast three such examples have been reported: SP1, SREBP1,and RF-X. In such cases, NF-Y considerably increases theaffinity of the neighboring factor for DNA, making these com-plexes more stable on the DNA (18). In addition to physicalinteraction with many transcription factors (21, 34, 35), NF-Yinteracts with several components of the basal transcriptionalmachinery (36–38). Therefore, through direct contact with in-duced and/or activated transcription factors and the basal ma-chinery, NF-Y may serve as a transcriptional coordinator orintegrator.

We determined that AP-1 interacts with NF-Y through directprotein-protein contact between c-Jun and NF-YA. This is notsurprising, since there is mounting evidence that NF-YA is theregulatory subunit of the trimeric NF-Y complex (18). c-Junprotein has also been reported to physically interact with othertranscription factors, such as ER� (39), Cbfa1 (40), and Smads(41). How AP-1 is able to circumvent the spatial constraints ofbinding to a half-site directly adjacent to a NF-Y-occupiedCAATT box, without a single nucleotide space, remains to bedetermined. AP-1 has been shown to bind a weak binding sitewhen it cooperatively associates with transcription factors onjuxtaposed sites (42); however, in that case, there were twobase pairs between the binding sites. Fos and Jun heterodimersform a flexible fork, which might permit binding of other tran-scription factors at adjacent sites on the DNA (43). Since themouse FSH� promoter contains an AP-1 half-site, it is possiblethat only one member of the heterodimer, in this case Fos,directly binds the DNA and that Jun binds to NF-Y, as we havedemonstrated in vitro, as well as to Fos, but not directly to theDNA. Alternatively, it is possible that both Fos and Jun pro-teins contact the DNA. We determined that residues TATTG-GTCAT are needed for AP-1 to bind. Conserved residues in the

AP-1 consensus TGAGTCA, are printed in boldface type and/orunderlined for easier observation. The CCAAT element (oppo-site strand: ATTGG) is bound by NF-Y. However, one memberof the AP-1 heterodimer can bind an underlined T, which isconserved in our novel site and the AP-1 consensus, and G (inboldface type), which both Fos and Jun bind according to thecrystal structure (43). The other partner can bind the GTCAhalf of the consensus (in boldface type). Fos and Jun bind theirsite in the major groove, and only four amino acid residuescontact the DNA (43). Vast portions of either protein are foundperpendicular to the DNA. That conformation and the twist ofthe DNA helix may allow enough space for NF-Y to bind. NF-Y,on the other hand, contacts the DNA in the minor groove (44).From our studies, it appears that the NF-Y site has to bepresent for AP-1 to bind, and since NF-Y occupies the site invivo, NF-Y may stabilize low affinity AP-1 DNA interactions.

We used 398 bp of the mouse FSH� regulatory sequence inthese experiments, and this relatively short region has bothhigher expression and greater response to GnRH than themuch longer ovine regulatory sequence used in previous stud-ies (13, 16). Thus, important species-specific differences in FSHregulation exist. The NF-Y/AP-1 site identified comprises se-quences from �76 to �69 in the mouse FSH� promoter and isnot conserved in the ovine or bovine promoters, although it isconserved in the human and rat. Two potential AP-1 sitespreviously identified in the ovine promoter correspond to �69/�63 and �106/�100 sequences in the mouse FSH� promoter(10). When we used oligonucleotides spanning those sites asprobes in EMSA, we did not detect AP-1 binding or any changein binding complexes following GnRH treatment (data notshown). The reports describing those AP-1 sites used purifiedproteins to detect binding to these sites (11) or detected AP-1from extracts of GnRH-treated HeLa cells binding to the AP-1consensus sequence (10). However, this is not surprising, sinceGnRH induces Fos and Jun isoforms. Further, mutations inthese sites do not affect appropriate regulation of FSH bygonadectomy or GnRH antagonist in transgenic animals (12).

Two AP-1 consensus sites exist within the 398 bp of themouse regulatory region, at �10/�4 and �181/�175. As ex-pected, AP-1 from GnRH-treated nuclear extracts can bind theAP-1 consensus sequence. However, based on the result of ourtransfection experiments, we conclude that those sites are notfunctional in the context of the promoter. Namely, when weused a reporter containing a mutation in the AP-1 half-site,mutant I, the induction by GnRH decreased by about the sameamount as when we co-transfected the dominant negative A-Fos in Fig. 6. Notably, dominant negative Fos cannot reducethe induction by either GnRH or overexpression of c-Jun andc-Fos when mutant I is used instead of the wild-type promoter.This finding strongly suggests that this site is required forAP-1 action in this regulatory region. From the truncationanalysis, we determined that there is another region of thepromoter between �230 and �304 responsive to GnRH to alesser degree, since there was a statistically significant drop inthe induction level upon truncation of that region. Further-more, it is possible that another GnRH-responsive site exists inthe region proximal to the start site, since the �95 bp trunca-tion maintains two-thirds of the response, whereas the AP-1site we identified is responsible for one-third of the induction.We are currently investigating the elements in those regionsfor their role in GnRH response.

The GnRH receptor belongs to the class of G protein-coupledreceptors and, upon ligand binding, activates protein kinase Cand downstream MAPK signaling pathways (6). Our resultsshowing the role of MAPK in mouse FSH� induction are inagreement with previously published reports that MAPK is

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involved in ovine FSH� up-regulation and c-Fos induction byGnRH. Our data extend those findings and demonstrate thatMAPK is involved in JunB induction by GnRH but not in c-Junor FosB induction. However, mutation of the AP-1 site or in-troduction of the dominant negative Fos reduces induction ofFSH� by 35 and 30%, respectively, whereas the inhibition ofthe MAPK pathway causes 46% reduction in the FSH� re-sponse to GnRH. Thus, the MAPK pathway probably regulatesother transcription factor(s) in addition to AP-1, either throughactivation by phosphorylation or by induction of their geneexpression.

In this report, we demonstrate that AP-1 binds a novel sitecomposed of an AP-1 half-site and a CCAAT box. This site isinvolved in GnRH induction of the mouse FSH� gene. The AP-1half-site overlaps a site involved in basal expression, which isoccupied by NF-Y in vivo. Direct physical contact between AP-1and NF-Y may stabilize AP-1 on this low affinity site, and NF-Ymay serve as an integrator between hormonal signals and thetranscriptional machinery.

Acknowledgments—We thank Malcolm Low for generously providingthe mouse FSH� genomic clone, David Gardner for the GST-NF-YB plas-mid, Sankar Maity for the GST-NF-YA plasmid, and Roberto Mantovani forthe NF-Y expression vectors. We are grateful to Michael Karin, who pro-vided the GST-Jun vector; to Michael Birrer and Eugene Tulchinsky, fromwhom we obtained the c-Jun and c-Fos expression vectors, respectively; andto Douglass Forbes for green florescent protein expression vector. We alsothank Charles Vinson for the dominant negative A-Fos expression vector.We are appreciative of the time and insight of Mark A. Lawson and thankthe members of the Mellon laboratory for helpful discussions.

REFERENCES

1. Kumar, T. R., Wang, Y., Lu, N., and Matzuk, M. M. (1997) Nat. Genet. 15,201–204

2. Pierce, J. G., and Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465–4953. Kaiser, U. B., Conn, P. M., and Chin, W. W. (1997) Endocr. Rev. 18, 46–704. Vale, W., Rivier, C., and Brown, M. (1977) Annu. Rev. Physiol. 39, 473–5275. Weiss, J., Guendner, M. J., Halvorson, L. M., and Jameson, J. L. (1995)

Endocrinology 136, 1885–18916. Liu, F., Austin, D. A., DiPaolo, D., Mellon, P. L., Olefsky, J. M., and Webster,

N. J. G. (2002) Mol. Endocrinol. 16, 419–4347. Cesnjaj, M., Catt, K. J., and Stojilkovic, S. S. (1994) Endocrinology 135,

692–7018. Padmanabhan, V., Dalkin, A., Yasin, M., Haisenleder, D. J., Marshall, J. C.,

and Landefeld, T. D. (1995) Biol. Reprod. 53, 263–2699. Tremblay, J. J., and Drouin, J. (1999) Mol. Cell. Biol. 19, 2567–2576

10. Strahl, B. D., Huang, H. J., Sebastian, J., Ghosh, B. R., and Miller, W. L. (1998)Endocrinology 139, 4455–4465

11. Strahl, B. D., Huang, H. J., Pedersen, N. R., Wu, J. C., Ghosh, B. R., andMiller, W. L. (1997) Endocrinology 138, 2621–2631

12. Huang, H. J., Sebastian, J., Strahl, B. D., Wu, J. C., and Miller, W. L. (2001)Endocrinology 142, 2260–2266

13. Pernasetti, F., Vasilyev, V. V., Rosenberg, S. B., Bailey, J. S., Huang, H.-J.,Miller, W. L., and Mellon, P. L. (2001) Endocrinology 142, 2284–2295

14. Graham, K. E., Nusser, K. D., and Low, M. J. (1999) J. Endocrinol. 162, R1–R515. Lewis, K. A., Gray, P. C., Blount, A. L., MacConell, L. A., Wiater, E.,

Bilezikjian, L. M., and Vale, W. (2000) Nature 404, 411–41416. Vasilyev, V. V., Pernasetti, F., Rosenberg, S. B., Barsoum, M. J., Austin, D. A.,

Webster, N. J. G., and Mellon, P. L. (2002) Endocrinology 143, 1651–165917. Jacobs, S. B. R., Coss, D., McGillivray, S. M., and Mellon, P. L. (2003) Mol.

Endocrinol. 17, 1470–148318. Mantovani, R. (1999) Gene (Amst.) 239, 15–2719. Xia, Y., Makris, C., Su, B., Li, E., Yang, J., Nemerow, G. R., and Karin, M.

(2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5243–524820. Kim, I. S., Sinha, S., de Crombrugghe, B., and Maity, S. N. (1996) Mol. Cell.

Biol. 16, 4003–401321. Liang, F., Schaufele, F., and Gardner, D. G. (2001) J. Biol. Chem. 276,

1516–152222. Szabo, E., Preis, L. H., and Birrer, M. J. (1994) Cell Growth Differ. 5, 439–44623. Andersen, H., Mahmood, S., Tkach, V., Cohn, M., Kustikova, O., Grigorian, M.,

Berezin, V., Bock, E., Lukanidin, E., and Tulchinsky, E. (2002) Oncogene21, 4843–4848

24. Liberati, C., Ronchi, A., Lievens, P., Ottolenghi, S., and Mantovani, R. (1998)J. Biol. Chem. 273, 16880–16889

25. Wingender, E., Chen, X., Hehl, R., Karas, H., Liebich, I., Matys, V., Meinhardt,T., Pruss, M., Reuter, I., and Schacherer, F. (2000) Nucleic Acids Res. 28,316–319

26. Ortolano, G. A., Haisenleder, D. J., Dalkin, A. C., Iliff-Sizemore, S. A., Land-efeld, T. D., Maurer, R. A., and Marshall, J. C. (1988) Endocrinology 123,2946–2948

27. Olive, M., Krylov, D., Echlin, D. R., Gardner, K., Taparowsky, E., and Vinson,C. (1997) J. Biol. Chem. 272, 18586–18594

28. Alarid, E. T., Windle, J. J., Whyte, D. B., and Mellon, P. L. (1996) Development122, 3319–3329

29. Butcher, R. L., Collins, W. E., and Fugo, N. W. (1974) Endocrinology 94,1704–1708

30. Mason, A. J., Hayflick, J. S., Zoeller, R. T., Young, W. S., Phillips, H. S.,Nikolics, K., and Seeburg, P. H. (1986) Science 234, 1366–1371

31. Dalkin, A. C., Burger, L. L., Aylor, K. W., Haisenleder, D. J., Workman, L. J.,Cho, S., and Marshall, J. C. (2001) Endocrinology 142, 139–146

32. Norwitz, E. R., Xu, S., Xu, J., Spiryda, L. B., Park, J. S., Jeong, K. H., McGee,E. A., and Kaiser, U. B. (2002) J. Biol. Chem. 277, 37469–37478

33. Gilthorpe, J., Vandromme, M., Brend, T., Gutman, A., Summerbell, D., Totty,N., and Rigby, P. W. J. (2002) Development 129, 3887–3899

34. Farsetti, A., Narducci, M., Moretti, F., Nanni, S., Mantovani, R., Sacchi, A.,and Pontecorvi, A. (2001) Endocrinology 142, 3380–3388

35. Ueda, A., Takeshita, F., Yamashiro, S., and Yoshimura, T. (1998) J. Biol.Chem. 273, 19339–19347

36. Bellorini, M., Lee, D. K., Dantonel, J. C., Zemzoumi, K., Roeder, R. G., Tora, L.,and Mantovani, R. (1997) Nucleic Acids Res. 25, 2174–2181

37. Faniello, M. C., Bevilacqua, M. A., Condorelli, G., de Crombrugghe, B., Maity,S. N., Avvedimento, V. E., Cimino, F., and Costanzo, F. (1999) J. Biol.Chem. 274, 7623–7626

38. Currie, R. A. (1998) J. Biol. Chem. 273, 1430–143439. Teyssier, C., Belguise, K., Galtier, F., and Chalbos, D. (2001) J. Biol. Chem.

276, 36361–3636940. D’Alonzo, R. C., Selvamurugan, N., Karsenty, G., and Partridge, N. C. (2002)

J. Biol. Chem. 277, 816–82241. Liberati, N. T., Datto, M. B., Frederick, J. P., Shen, X., Wong, C., Rougier-

Chapman, E. M., and Wang, X. F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96,4844–4849

42. Chen, L., Glover, J. N., Hogan, P. G., Rao, A., and Harrison, S. C. (1998) Nature392, 42–48

43. Glover, J. N., and Harrison, S. C. (1995) Nature 373, 257–26144. Ronchi, A., Bellorini, M., Mongelli, N., and Mantovani, R. (1995) Nucleic Acids

Res. 23, 4565–4572

GnRH Induces FSH� through a Novel AP-1 Site162

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Djurdjica Coss, Suzanne B. R. Jacobs, Cheryl E. Bender and Pamela L. Mellon Gene by Gonadotropin-releasing HormoneβHormone

A Novel AP-1 Site Is Critical for Maximal Induction of the Follicle-stimulating

doi: 10.1074/jbc.M304697200 originally published online October 20, 20032004, 279:152-162.J. Biol. Chem. 

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