AN UPSTREAM ENHANCER REGION LOCATED AT -1810/-1666 BP OF 5′ FLANKING SEQUENCE PARTIALLY CONFERS DIFFERENTIAL ACTIVITY OF THE PORCINE GnRH RECEPTOR GENE PROMOTER IN SWINE LINES WITH DIVERGENT OVULATION RATES (2007)

The interaction between GnRH and its receptor is critical to regulation of reproductive function. Additionally, the porcine GnRH receptor (GnRHR) gene is located near a quantitative trait locus for ovulation rate. Thus, the GnRHR gene represents both a physiological and positional candidate for genes influencing ovulation rate. Our laboratory has constructed luciferase reporter vectors containing approximately 5000 bp of the GnRHR gene promoter from 3 lines of swine with divergent ovulation rates; Control, Index and Meishan. Transient transfection of these constructs into gonadotrope-derived alphaT3-l cells revealed significant differences in luciferase activity among promoters from the three pig lines. The Meishan proximal promoter contains two single- bp alterations at -1234 and -844 bp compared to the Control/Index promoters. The alteration at -1234 bp forms a unique binding site for the p65/p52 subunits of NF-kappaB and an Sp1-like protein whereas the alteration at -844 bp binds GATA4. Transient transfection of vectors containing block replacement mutations of these binding sites reduced luciferase activity by 20% compared to the full length Meishan promoter. Additional 5′ deletion studies identified an enhancer region located at -1810/-1666 bp of proximal promoter. Electrophoretic mobility shift assays (EMSA) utilizing radiolabled oligonucleotide probes spanning this region indicated specific binding complexes for oligonucleotides comprising the -1779/-1749, -1748/- 1717, and -1700/-1679 bp regions of the Meishan and Control/Index promoters. Sequence analysis of the region between 1749 and 1779 bp upstream of the translational start site revealed a putative binding site for SF-1. Addition of an antibody directed against SF-1 abrogated binding of the complex. Also, a single bp substitution and deletion, unique to the Meishan promoter, reside in the upstream enhancer region located at - 1748/-1717 bp of 5′ flanking sequence. Sequence analysis indicated two C/EBP and one NF-Y element in the Control/Index promoter. Conversely, the Meishan promoter has two NF-Y and only one C/EBP putative binding site. EMSAs with oligonucleotides spanning the -1748/-1717 bp region from both lines of swine formed specific complexes that were competed with unlabeled consensus oligonucleotides for C/EBP and NF-Y. Within the region between -1700/-1679 bp of 5′ flanking sequence, the Meishan promoter contains a single base pair substitution compared to the Control/Index promoter. Analysis of this region identified potential NF-kappaB and GATA elements in the Meishan and Control/Index promoters. An unlabeled oligonucleotide with consensus GATA binding sites competed the DNA:Protein complex for the Meishan probe whereas the addition of an unlabeled NF-kappaB consensus oligonucleotide abrogated binding of the complex for the Control/Index probe. Next, we added antibodies directed against the p50, p52, and p65 subunits of NF-kappaB, GATA1, GATA2, and GATA4. Antibodies specific for the p52 and p65 subunits of NF-kappaB resulted in a supershift in the Control/Index oligonucleotide whereas the antibody directed against GATA4 resulted in a supershift of the protein complex bound to the Meishan oligonucleotide. Thus, a complex upstream enhancer located at -1810/-1666 bp of proximal promoter, containing both cell- and line-specific elements, contributes to differential activity of the GnRHR gene promoter in swine lines divergent for ovulation rate. (platform)