Activity of the porcine gonadotropin-releasing hormone receptor gene promoter is partially conferred by a distal gonadotrope specific element (GSE) within an upstream enhancing region, two proximal GSEs and a retinoid X receptor binding site

Sequence for the porcine GnRHR promoter was obtained by inverse PCR [31] utilizing primers specific for 1154 bp of 5′ UTR previously reported by Jiang and colleagues (GenBank Accession No. AF227685) [30]. Briefly, genomic DNA preparations were partially cut by Sau3AI or EcoRI and fragments were self-ligated using a high concentration of T4 DNA ligase (4 U/μl, New England Biolabs, Beverly, MA) and a low concentration of DNA (3 ng/μl). Subsequently, circularized DNA fragments were amplified with the forward primer, 5′-TGG ACT GAC CGT TGA GAC TG-3′ and either the 5′-GTG TAA GTG TTG GAA CCA CAT C-3′ (Sau3AI digested DNA) or 5′-GAG AGC AAT AGC ATT CTC TG-3′ (EcoRI digested DNA) reverse primer to generate the inverse PCR products. The chosen primers were different from traditional PCR in that they were in reverse orientation. Resultant PCR products were subcloned and sequenced at the University of Nebraska-Lincoln (UNL) Genomics Core Research Facility. Sequence data for the 5171 bp of 5′ flanking region for the porcine GnRHR gene has been deposited with GenBank (Accession No. AY166667).

A reporter vector containing 5118 bp of porcine GnRHR promoter (-5118pGL3) was constructed by PCR amplification of the 5′ flanking region for the GnRHR gene from genomic DNA preparations of pigs representing a white crossbred line (Table 1). The PCR product was subsequently subcloned into pBluescript SK- (Stratagene, La Jolla, CA). Promoter inserts were ligated into a reporter vector containing the cDNA encoding luciferase (pGL3, Promega Corp., Madison, WI) at the SacI/EcoRV location of the multiple cloning site. Deletion constructs were made by progressively removing 5′ flanking sequence (approximately 500 bp) via restriction enzyme digests (ApaI, BstXI, NdeI, NsiI, BstAPI, PvuII, SpeI and BlpI) and subsequent intramolecular ligation of the remaining vector backbone. Construction of the deletion constructs in the absence of restriction enzyme digest sites and 100-bp deletion reporter vectors were achieved by amplifying the specified region of the promoter by PCR (Table 1) with a high fidelity Taq DNA polymerase (Bioline, Springfield, NJ). Next, the fragments were subcloned into pBluescript SK- or pCR-Blunt II (Invitrogen, Carlsbad, CA), and then finally into pGL3. Block replacement vectors were constructed via an overlap extension, two-step, site-specific mutagenesis protocol [32] in which altered primers introduced the sequence changes (Table 1). The -5118pGL3 construct served as the template for the two first round PCR reactions where outer forward (OF) or outer reverse (OR) primers were used with the inner mutation containing primer or its complement (Table 1). The single second round PCR used the products from the first reactions as the template and OF and OR as the primer set (Table 1). The μSF1IpGL3, μSF1IIpGL3 and μSF1IIIpGL3 block replacement vectors were constructed by replacing the SF1 binding sites at -179/-171, -315/-310 and -1760/-1753 with NotI, ClaI and NotI sites respectively. The μRXRpGL3, μNF_ΚBpGL3 and μOCT1pGL3 block replacement vectors were constructed by replacing the respective binding sites at -279/-274, -1689/-1684 and -1731/-1707 with PstI, EcoRI and SacI. To verify that the correct mutations had been introduced, vectors were sequenced at the UNL Genomics Core Research Facility before use in the transient transfection experiments. As a transfection efficiency control, luciferase vectors were co-transfected with a plasmid containing the Rous sarcoma virus (RSV) promoter linked to the cDNA-encoding β-galactosidase (RSV-β-gal, Stratagene). A midi plasmid preparation kit (Qiagen, Valencia, CA) was used to isolate transfection quality DNA.

Cultures of αT3-1 (mouse gonadotrope, Dr. Pam Mellon, Salk Institute, La Jolla, CA), CHO (Chinese hamster ovary), Cos-7 (monkey kidney), JAR (human choriocarcinoma), MA10 (mouse Leydig) and PK15 (pig kidney) cells were maintained at 37°C in a humidified 5% CO₂ in air atmosphere. The αT3-1 cells were cultured in high-glucose (4.5 g/L) DMEM (Mediatech, Herndon, VA) supplemented with 5% fetal bovine serum (FBS), 5% horse serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate (Gibco, Grand Island, NY). The CHO, Cos-7 and PK15 cell lines were cultured in high-glucose (4.5 g/L) DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. Finally, JAR and MA10 cells were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640; Mediatech) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate.

Transient transfections were performed using a liposome-mediated protocol (Fugene6; Roche Diagnostics Corp., Indianapolis, IN) following manufacturer’s instructions. The day prior to transfection, cells were plated in 6-well tissue culture plates at the appropriate density (between 2 × 10⁵ and 2 × 10⁶ cells) to result in 40% confluency on the day of transfection. Cells were transfected with a 3:1 Fugene6 to DNA ratio. A total of 1 μg of DNA, 0.9 μg of luciferase test vector and 0.1 μg of RSV-βgal were used per well. Luciferase (Promega Corp.) and β-galactosidase assays (β-gal; Applied Biosystems, Bedford, MA) were performed following manufacturer’s instructions. At 20-24 h post transfection, cells were washed twice with ice-cold PBS and harvested with 200 μl of lysis buffer [100 mM potassium phosphate (pH 7.8), 0.2% Triton X-100 and 1 mM dithiothreitol (DTT)]. Lysates were cleared by centrifugation (14,000 × g) for 2 min at 4°C and 20 μl of lysate was used to perform each of the assays. Luciferase and β-gal values for each sample were determined using a Wallac VICTOR² micro plate reader (PerkinElmer Life Sciences, Boston, MA). To normalize for transfection efficiency, luciferase activity was divided by β-gal values.

Nuclear protein extracts were prepared from αT3-1 cells utilizing the NE-PER® Nuclear and Cytoplasmic Extraction Reagent Kit per manufacturer’s instructions (Pierce Biotechnology, Rockford, IL). Approximately 2.8 × 10⁸ αT3-1 cells were harvested with TNE buffer [10 mM Tris (pH 8), 140 mM NaCl, 1 mM EDTA] and the extracts were treated with protease (catalog no. P8340, Sigma Chemical Co., St. Louis, MO) and phosphatase (catalog no. 524625, Calbiochem, La Jolla, CA) inhibitor cocktails to prevent enzymatic degradation of proteins. The amount of protein present in the extracts was determined using bicinchoninic acid (BCA assay; Pierce Biotechnology). Double-stranded oligonucleotide probes (Table 2) were end-labeled with [γ-³²P]ATP using T4 polynucleotide kinase (MBI Fermentas Inc., Hanover, MD) and purified using sephadex G-25 spin columns (Amersham Biosciences Corp., Piscataway, NJ). Nuclear extracts (5 μg) were incubated with 4 μl of Dignam D buffer (20 mM HEPES, 20% glycerol, 0.1 M potassium chloride, 0.2 mM EDTA, 0.5 mM DTT), 1 mM DTT, 2 μg poly(dI•dC) (Amersham Biosciences Corp.) and, where indicated, rabbit antiserum directed against Ad4BP/SF1 (including the DNA binding domain; Dr. Ken-ichirou Morohashi, Okazaki National Research Institutes, Okazaki, Japan), mouse monoclonal antibodies for RXRα, RXRβ and RXRγ (Dr. Pierre Chambon, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch Cedex, France) or an equal amount of rabbit IgG (catalog no. sc-2027, Santa Cruz Biotechnology, Santa Cruz, CA) at 25°C for 30 min. Following incubation, radiolabeled probe (100,000 cpm) was added and, where indicated, 50-fold molar excess of either homologous, heterologous, consensus or mutant consensus unlabeled competitor. Reactions were incubated for an additional 20 min at 25°C before free probe was separated from bound probe by electrophoresis for 1.5 h at 30 mA in 5% polyacrylamide gels that were pre-run for 1 h at 100 V in 1 X TGE [25 mM Tris (pH 8.3), 190 mM glycine and 1 mM EDTA]. Gels were transferred to blotting paper, dried, and exposed to Biomax MS film (Eastman Kodak Co. Rochester, NY) or RX-B Blue Sensitive autoradiographic film (Marsh BioProducts, Rochester, NY) for 20-24 h at -80°C before being developed.

Initial sequence compilation was performed with the GCG Wisconsin Package (Accelrys Software Inc., San Diego, CA). Additional sequence management and alignment procedures were completed with Vector NTI software (InforMax, Bethesda, MD). Species comparisons were primarily performed utilizing visualization tool for alignment (VISTA) [33] and sequences were aligned with Vector NTI software. Analyses of sequence for transcription factor binding sites were performed with the Patch Public 1.0 program (Biobase, Wolfenbüttel, Germany).

Data were analyzed using the general linear models (GLM) procedure of the Statistical Analysis System software (version 8.2, SAS Institute Inc, Cary, NC). To control for transfection efficiency, the arbitrary light value for each luciferase replicate was divided by the respective β-gal arbitrary light value and this value was then divided by the mean of the empty vector and expressed as fold over pGL3. Transfections were performed in triplicate with three to five replicates containing different plasmid preparations. Individual values (n = 9-15) from all the replicates were used to generate the mean ± SEM. Comparisons between pGL3 and test vectors were analyzed using Dunnett’s t-test. Means for luciferase activity among test vectors were compared using the least significant differences of least squares means.