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Reproductive Biology and Endocrinology

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Open Access 01-12-2015 | Research

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

Authors: Rebecca A Cederberg, Jacqueline E Smith, Emily A McDonald, Chanho Lee, Amy R Perkins, Brett R White

Published in: Reproductive Biology and Endocrinology | Issue 1/2015

Abstract

Background

Regulation of gonadotropin-releasing hormone (GnRH) receptor (GnRHR) numbers on gonadotropes within the anterior pituitary gland represents a critical point for control of reproductive function. Binding of GnRH to its receptor regulates follicle stimulating hormone (FSH) and luteinizing hormone (LH) release and levels of this G-protein coupled receptor on the surface of gonadotropes determines their sensitivity to GnRH pulses. While transcriptional regulation of this gene has been studied in mice, rats, humans and sheep, little is known about its regulation in the pig, an important agricultural species and human research model.

Methods

We isolated 5118 bp of 5′ flanking sequence for the porcine GnRHR gene and generated luciferase reporter vectors. Deletion and mutation constructs were evaluated in gonadotrope-derived alphaT3-1 cells to determine regions important for gene transcription. Additionally, electrophoretic mobility shift assays (EMSAs) were performed to identify transcription factors binding to the GnRHR promoter.

Results

Transient transfections revealed that the GnRHR promoter was functional in alphaT3-1 cells but not in cells of non-gonadotrope origin. Mutation of the highly conserved gonadotrope specific element (GSE) located at -179/-171 of proximal promoter completely ablated luciferase activity, whereas mutation of another GSE at -315/-310 reduced activity by 34%. Consistent with this, EMSAs using alphaT3-1 nuclear extracts and a steroidogenic factor (SF)1 antibody confirmed SF1 binding to both GSEs. EMSAs also demonstrated that a retinoid X receptor (RXR) binding site at -279/-274 binds RXRalpha and RXRbeta and mutation of this site eliminated promoter activity. Transient transfection of alphaT3-1 cells with reporter vectors containing selective removal of 5′ flanking region for the porcine GnRHR gene indicated that the -1915/-1431 segment was important for promoter activity. Definition of this region via transfection assays and EMSAs revealed an upstream enhancing region located at -1779/-1667 that increases porcine GnRHR gene expression in alphaT3-1 cells and includes a SF1 binding site at -1760/-1753.

Conclusions

Porcine GnRHR promoter activity in alphaT3-1 cells is partially conferred by a distal GSE, two proximal GSEs and a RXR binding site. Basal gonadotrope expression of the porcine GnRHR gene uniquely involves three GSEs and RXR is newly identified as a regulator of GnRHR promoter activity.

Clayton RN, Catt KJ. Gonadotropin-releasing hormone receptors: characterization, physiological regulation, and relationship to reproductive function. Endocr Rev. 1981;2:186–209.PubMedCrossRef

Clarke IJ, Cummins JT, de Kretser DM. Pituitary gland function after disconnection from direct hypothalamic influences in the sheep. Neuroendocrinology. 1983;36:376–84.PubMedCrossRef

Hamernik DL, Nett TM. Gonadotropin-releasing hormone increases the amount of messenger ribonucleic acid for gonadotropins in ovariectomized ewes after hypothalmic-pituitary disconnection. Endocrinology. 1988;122:959–66.PubMedCrossRef

Gharib SD, Wierman ME, Shupnik MA, Chin WW. Molecular biology of the pituitary gonadotropins. Endocr Rev. 1990;11:177–90.PubMedCrossRef

Mason AJ, Pitts SL, Nikolic K, Szony E, Wilcox JN, Seeburg PH, et al. The hypogonadal mouse: reproductive functions restored by gene therapy. Science. 1986;234:1372–8.PubMedCrossRef

Wise ME, Nieman D, Stewart J, Nett TM. Effect of number of receptors for gonadotropin-releasing hormone on the release of luteinizing hormone. Biol Reprod. 1984;31:1007–13.PubMedCrossRef

Sealfon SC, Millar RP. The gonadotropin-releasing hormone receptor: structural determinants and regulatory control. Hum Reprod Update. 1995;1:216–30.PubMedCrossRef

Kaiser UB, Jakubowiak A, Steinberger A, Chin WW. Regulation of rat pituitary gonadotropin-releasing hormone receptor mRNA levels in vivo and in vitro. Endocrinology. 1993;133:931–4.PubMed

Turzillo AM, Campion CE, Clay CM, Nett TM. Regulation of gonadotropin-releasing hormone (GnRH) receptor messenger ribonucleic acid and GnRH receptors during the early preovulatory period in the ewe. Endocrinology. 1994;135:1353–8.PubMed

10.

Bauer-Dantoin AC, Weiss J, Jameson JL. Roles of estrogen, progesterone, and gonadotropin-releasing hormone (GnRH) in the control of pituitary GnRH receptor gene expression at the time of the preovulatory gonadotropin surges. Endocrinology. 1995;136:1014–9.PubMed

11.

Clay CM, Nelson SE, DiGregorio GB, Campion CE, Wiedemann AL, Nett TM. Cell-specific expression of the mouse gonadotropin-releasing hormone (GnRH) receptor gene is conferred by elements residing within 500 bp of proximal 5′ flanking region. Endocrine. 1995;3:615–22.PubMedCrossRef

12.

Windle JJ, Weiner RI, Mellon PL. Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. Mol Endocrinol. 1990;4:597–603.PubMedCrossRef

13.

Duval DL, Nelson SE, Clay CM. The tripartite basal enhancer of the gonadotropin-releasing hormone (GnRH) receptor gene promoter regulates cell-specific expression through a novel GnRH receptor activating sequence. Mol Endocrinol. 1997;11:1814–21.PubMedCrossRef

14.

Duval DL, Nelson SE, Clay CM. A binding site for steroidogenic factor-1 is part of a complex enhancer that mediates expression of the murine gonadotropin-releasing hormone receptor gene. Biol Reprod. 1997;56:160–8.PubMedCrossRef

15.

McGillivray SM, Bailey JS, Ramezani R, Kirkwood BJ, Mellon PL. Mouse GnRH receptor gene expression is mediated by the LHX3 homeodomain protein. Endocrinology. 2005;146:2180–5.PubMedCentralPubMedCrossRef

16.

Lents CA, Farmerie TA, Cherrington BD, Clay CM. Multiple core homeodomain binding motifs differentially contribute to transcriptional activity of the murine gonadotropin-releasing hormone receptor gene promoter. Endocrine. 2009;35:356–64.PubMedCrossRef

17.

Jeong KH, Chin WW, Kaiser UB. Essential role of the homeodomain for pituitary homeobox 1 activation of mouse gonadotropin-releasing hormone receptor gene expression through interactions with c-Jun and DNA. Mol Cell Biol. 2004;24:6127–39.PubMedCentralPubMedCrossRef

18.

Kam KY, Jeong KH, Norwitz ER, Jorgensen EM, Kaiser UB. Oct-1 and nuclear factor Y bind to the SURG-1 element to direct basal and gonadotropin-releasing hormone (GnRH)-stimulated mouse GnRH receptor gene transcription. Mol Endocrinol. 2005;19:148–62.PubMedCrossRef

19.

Resuehr D, Wildemann U, Sikes H, Olcese J. E-box regulation of gonadotropin-releasing hormone (GnRH) receptor expression in immortalized gonadotrope cells. Mol Cell Endocrinol. 2007;278:36–43.PubMedCrossRef

20.

Pincas H, Amoyel K, Counis R, Laverriere JN. Proximal cis-acting elements, including steroidogenic factor-1, mediate the efficiency of a distal enhancer in the promoter of the rat gonadotropin-releasing hormone receptor gene. Mol Endocrinol. 2001;15:319–37.PubMedCrossRef

21.

Granger A, Bleux C, Kottler ML, Rhodes SJ, Counis R, Laverriere JN. The LIM-homeodomain proteins ISL-1 and LHX3 act with steroidogenic factor 1 to enhance gonadotrope-specific activity of the gonadotropin-releasing hormone receptor gene promoter. Mol Endocrinol. 2006;20:2093–108.PubMedCrossRef

22.

Schang AL, Granger A, Querat B, Bleux C, Cohen-Tannoudji J, Laverriere JN. GATA2-induced silencing and LIM-homeodomain protein-induced activation are mediated by a bi-functional response element in the rat GnRH receptor gene. Mol Endocrinol. 2013;27:74–91.PubMedCrossRef

23.

Ngan ESW, Cheng KW, Leung PCK, Chow BKC. Steroidogenic factor-1 interacts with a gonadotrope-specific element within the first exon of the human gonadotropin-releasing hormone receptor gene to mediate gonadotrope-specific expression. Endocrinology. 1999;140:2452–62.PubMed

24.

Cheng KW, Ngan ESW, Kang SK, Chow BKC, Leung PCK. Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: role of protein kinase C and activating protein 1. Endocrinology. 2000;141:3611–22.PubMed

25.

Cheng CK, Yeung CM, Hoo RLC, Chow BKC, Leung PCK. Oct-1 is involved in the transcriptional repression of the gonadotropin-releasing hormone receptor gene. Endocrinology. 2002;143:4693–701.PubMedCrossRef

26.

Duval DL, Farris AR, Quirk CC, Nett TM, Hamernik DL, Clay CM. Responsiveness of the ovine gonadotropin-releasing hormone receptor gene to estradiol and gonadotropin-releasing hormone is not detectable in vitro but is revealed in transgenic mice. Endocrinology. 2000;141:1001–10.PubMed

27.

Wilkie PJ, Paszek AA, Beattie CW, Alexander LJ, Wheeler MB, Schook LB. A genomic scan of porcine reproductive traits reveals possible quantitative trait loci (QTLs) for number of corpora lutea. Mamm Genome. 1999;10:573–8.PubMedCrossRef

28.

Braunschweig MH, Paszek AA, Weller JI, Da Y, Hawken RJ, Wheeler MB, et al. Generation and exploration of a dense genetic map in a region of a QTL affecting corpora lutea in a Meishan x Yorkshire cross. Mamm Genome. 2001;12:719–23.PubMedCrossRef

29.

Buske B, Sternstein I, Brockmann G. QTL and candidate genes for fecundity in sows. Anim Reprod Sci. 2006;95:167–83.PubMedCrossRef

30.

Jiang Z, Gibson JP, Archibald AL, Haley CS. The porcine gonadotropin-releasing hormone receptor gene (GnRHR): genomic organization, polymorphisms, and association with the number of corpora lutea. Genome. 2001;44:7–12.PubMedCrossRef

31.

Pang KM, Knecht DA. Partial inverse PCR: a technique for cloning flanking sequences. BioTechniques. 1997;22:1046–8.PubMed

32.

Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989;77:51–9.PubMedCrossRef

33.

Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I. VISTA: computational tools for comparative genomics. Nucleic Acids Res. 2004;32:W273–9.PubMedCentralPubMedCrossRef

34.

Cheng KW, Leung PCK. The expression, regulation, and signal transduction pathways of the mammalian gonadotropin-releasing hormone receptor. Can J Physiol Pharmacol. 2000;78:1029–52.PubMedCrossRef

35.

Barnhart KM, Mellon PL. The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone alpha-subunit gene in pituitary gonadotropes. Mol Endocrinol. 1994;8:878–85.PubMed

36.

Wolfe MW. The equine luteinizing hormone beta-subunit promoter contains two functional steroidogenic factor-1 response elements. Mol Endocrinol. 1999;13:1497–510.PubMed

37.

Keri RA, Nilson JH. A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone ß subunit promoter in gonadotropes of transgenic mice. J Biol Chem. 1996;271:10782–5.PubMedCrossRef

38.

Jacobs SB, Coss D, McGillivray SM, Mellon PL. Nuclear factor Y and steroidogenic factor 1 physically and functionally interact to contribute to cell-specific expression of the mouse follicle-stimulating hormone-beta gene. Mol Endocrinol. 2003;17:1470–83.PubMedCentralPubMedCrossRef

39.

Sugawara T, Kiriakidou M, McAllister JM, Kallen CB, Strauss 3rd JF. Multiple steroidogenic factor 1 binding elements in the human steroidogenic acute regulatory protein gene 5′-flanking region are required for maximal promoter activity and cyclic AMP responsiveness. Biochemistry. 1997;36:7249–55.PubMedCrossRef

40.

Cheng CK, Chow BKC, Leung PCK. Functional mapping of a placenta-specific upstream promoter for human gonadotropin-releasing hormone receptor gene. Endocrinology. 2001;142:1506–16.PubMed

41.

Cheng CK, Yeung CM, Chow BKC, Leung PCK. Characterization of a new upstream GnRH receptor promoter in human ovarian granulosa-luteal cells. Mol Endocrinol. 2002;16:1552–64.PubMedCrossRef

42.

Cho S, Cho H, Geum D, Kim K. Retinoic acid regulates gonadotropin-releasing hormone (GnRH) release and gene expression in the rat hypothalamic fragments and GT1-1 neuronal cells in vitro. Brain Res Mol Brain Res. 1998;54:74–84.PubMedCrossRef

43.

Hoo RL, Chan KY, Leung FK, Lee LT, Leung PC, Chow BK. Involvement of NF-kappaB subunit p65 and retinoic acid receptors, RARalpha and RXRalpha, in transcriptional regulation of the human GnRH II gene. FEBS J. 2007;274:2695–706.PubMedCrossRef

44.

Hong CY, Park KH, Kim J, Im SY, Lee JW, Choi H, et al. Expression of MIS in the testis is downregulated by tumor necrosis factor alpha through the negative regulation of SF-1 transactivation by NF-kB. Mol Cell Biol. 2003;23:6000–12.PubMedCentralPubMedCrossRef

45.

Campion CE, Turzillo AM, Clay CM. The gene encoding the ovine gonadotropin-releasing hormone (GnRH) receptor: cloning and initial characterization. Gene. 1996;170:277–80.PubMedCrossRef

46.

Halvorson LM, Ito M, Jameson JL, Chin WW. Steroidogenic factor-1 and early growth response protein 1 act through two composite DNA binding sites to regulate luteinizing hormone beta-subunit gene expression. J Biol Chem. 1998;273:14712–20.PubMedCrossRef

Title: 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
Authors: Rebecca A Cederberg
Jacqueline E Smith
Emily A McDonald
Chanho Lee
Amy R Perkins
Brett R White
Publication date: 01-12-2015
Publisher: BioMed Central
Published in: Reproductive Biology and Endocrinology / Issue 1/2015
Electronic ISSN: 1477-7827
DOI: https://doi.org/10.1186/s12958-015-0033-0

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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

Abstract

Background

Methods

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

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