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The GnRH promoter: Target of transcription factors, hormones, and signaling pathways
Molecular and Cellular Endocrinology 140 (1998) 151 – 155
The GnRH promoter: Target of transcription factors, hormones, and signaling pathways Shelley B. Nelson, Satish A. Eraly, Pamela L. Mellon * Departments of Reproducti6e Medicine and Neurosciences 0674, The Center for Cellular and Molecular Medicine, Uni6ersity of California, San Diego, 9500 Gilman Dri6e, La Jolla, CA 92093 -0674, USA
Abstract Gonadotropin-releasing hormone (GnRH) is essential for normal reproductive maturation and function. We present a review of the known mechanisms of hypothalamic GnRH transcriptional control through the conserved GnRH promoter. Understanding this promoter region will allow us to comprehend better the complexities of the hypothalamic – pituitary – gonadal axis. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gonadotropin-releasing hormone; POU-homeodomain; Gene expression
1. Introduction Gonadotropin-releasing hormone (GnRH) is expressed in specific hypothalamic neurons and secreted into the hypophyseal portal venous circulation in a pulsatile manner. The secreted hormone travels to the anterior pituitary where it binds to GnRH receptors on the pituitary gonadotrope. Occupation of these receptors activates signal transduction cascades finally causing the release of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH play a role in gonadal maturation, onset of puberty, and ovulation. Changes in GnRH pulse frequency and amplitude regulate gonadotropin release, and GnRH is regulated at the level of transcription, translation, and secretion to produce these physiological effects. Here, we will focus on the mechanism of hypothalamic GnRH regulation at the transcriptional level through the conserved proximal promoter of the gene. GnRH secreting neurons are few in number (B 800 in mice) (Wray et al., 1989) and scattered throughout the hypothalamus. To address the transcriptional con* Corresponding author. Tel.: +1 619 5341312; fax: + 1 619 5341438; e-mail: [email protected]
trol of GnRH, immortalized GnRH-secreting hypothalamic cell lines were created. The GT1-7 cell line was derived in this laboratory by targeting SV40 T antigen to the GnRH-secreting hypothalamic neurons in transgenic mice by placing the 5% flanking region of the rat GnRH (rGnRH) gene upstream of the oncogene SV40 T antigen (Mellon et al., 1990). Cell lines were derived from the resulting hypothalamic tumors that have many of the characteristics of their in vivo counterparts, including neuronal morphology (Liposits et al., 1991), expression of neuronal markers, and secretion of GnRH in a pulsatile manner (Wetsel et al., 1992). Additionally, Radovick and coworkers created the GN10 (NLT) and GN11 cell lines by a similar method (Radovick et al., 1991) using instead the 1100 bp 5% flanking region of the human GnRH (hGnRH) gene. In this instance, tumors were present in the olfactory bulb and these cells proved to be immunopositive for GnRH in culture. Therefore, the GT1-7, GN10 (NLT) and GN11 cells provide model systems for the study of GnRH gene regulation. Within the 5% flanking region of the rGnRH gene, there are two major regions for transcription of the GnRH gene, an enhancer and a promoter. The 300 bp enhancer is located 1.8 kb upstream of the transcriptional start site and confers 50-fold activation of GnRH
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Fig. 1. Footprinted regions of the rGnRH proximal promoter. Regions known to bind particular factors are indicated.
transcription (Whyte et al., 1995) only in GT1-7 cells. It contains binding sites for POU-homeodomain transcription factors and GATA factors (Clark and Mellon, 1995; Lawson et al., 1996). These proteins are important for neuron-specific expression of the GnRH gene. Within the first 173 bases 5% of the transcriptional start site, the rGnRH promoter contains a region that is conserved across species (Eraly and Mellon, 1995). The hGnRH 5% flanking region contains two transcriptional start sites at + 1 and − 579 respectively (Dong et al., 1993). The promoter for the proximal start site (+1) is active in the hypothalamus while the distal promoter is active in other reproductive tissues such as placenta (Dong et al., 1997). Recently, the GnRH gene was isolated from rhesus monkey (Dong et al., 1996). The primate gene is highly homologous to the human gene, conserving the dual promoters. Though little is known about the factors which regulate hGnRH expression, progress has been made in understanding the role of transcription factors binding to and influencing the mouse and rat GnRH promoters.
2. Transcription from the rGnRH promoter The initial analysis of the rGnRH promoter was undertaken to address its function in GT1-7 hypothalamic cells. DNase I footprint analysis of the rGnRH promoter led to the identification of seven footprinted regions (FP1–7) located between −173 and +112 (Fig. 1) (Eraly and Mellon, 1995). Transient transfections in GT1-7 cells revealed that removal of −173 to −126 (FP7 and 6) caused a 2-fold reduction of reporter gene activity. Further deletion of − 126 to − 82 (FP5, 4, and 3) also caused a 2-fold reduction of the reporter gene activity. Additionally, two separate block mutations created within − 110 to − 89 (FP4) show a 60 and 30% reduction in reporter gene activity respectively (Eraly et al., 1998). A block mutation at − 123 to −116 (FP5) showed a 20% reduc-
tion in reporter gene activity while a block mutation at − 85 to − 78 (FP3) showed no significant change. These results are consistent with those presented by Lei et al. showing that a mutation similar to the 3% FP4 mutation resulted in a decrease in reporter gene activity and that a mutation similar to the one within FP3 resulted in an increase in reporter gene activity (Lei and Rao, 1997). In addition, they observed a transcriptional decrease when a region between FP3 and FP4 was mutated. We conclude that the region encompassing FP7–FP3 contains elements necessary for GnRH gene transcription and that the 2-fold decrease in transcription seen with deletion of −126 to − 82 is mostly localized to − 110 to − 89 (FP4). Since the − 70 to − 28 (FP2) deletion caused a 20-fold decrease in activity, we sought to understand its role in gene activation. Because footprint encompasses 51 bases (Fig. 1), seven block mutations were created to dissect its structure. Footprinting of these block mutants led to the conclusion that there are three binding sites within FP2. Transfection of the promoters carrying the block mutations in FP2 into GT1-7 cells revealed that each of the single block mutations resulted in a 40–60% reduction in reporter gene activity. When double mutations were created removing each possible pair of the three potential protein binding sites, an 80% loss in reporter gene activity was seen for each of the three paired combinations. When all three binding sites are present, they have a synergistic activating effect on the reporter gene, compared with individual proteins binding at the doubly mutated FP2.
3. POU-domain transcription factors bind to the rGnRH promoter To identify the proteins binding to the rGnRH promoter, gel mobility shift assays were conducted with oligonucleotide probes comprising the various
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Table 1 Compilation of the transcription factors and hormones/second messengers that regulate the hypothalamic GnRH promoter in GT1-7 cells (or other cell lines where noted) Site Human Factora Oct-1 SCIP/Oct-6/Tst-1 GR hPR Fos/Jun
Transcriptional effect Rat
Mouse
−106/−99, −47/−40 −343/−314, −171/−126, −126/−73
−212/−205 −218/−201, −184/−150
−171/−126, −126/−73, −111/−73 −402/−396
Hormones/Second messengersb Glucocorticoid hCG Estrogen Progesterone TPA IGF-I/TPA −402/−396
−237/−201 −99/−79 −171/−126 −171/−73 −82/−22, −124/−73
+/Rat − −* − + (GN11) − − − − − + (NLT)
a
The site refers to the region known to bind the particular factor in the given species. The site is the region that mediates responsiveness to the hormone/second messenger. * Repression may be mediated through the binding of other factors besides GR.
b
promoter footprinted regions (Eraly et al., 1998). Footprint 5 ( − 128 to − 112) formed one major unidentified complex. Footprint 4 (− 110 to − 89) showed one major low mobility complex which was shown to be Oct-1 by antibody supershift analysis. The use of a full length FP2 ( −82 to −22) gel shift probe results in three specific complexes (Eraly and Mellon, 1995). A smaller oligonucleotide probe encompassing the middle and downstream protein binding sites results in the formation of four specific complexes. Addition of the Oct-1 antibody blocks the formation of the slowest mobility complex, demonstrating that Oct-1 binds to FP2. Mutational analysis indicates that the Oct-1 binding site lies within the downstream region of FP2 (Fig. 1). Thus Oct-1 appears to be involved in GnRH gene regulation by its action on both the promoter and the enhancer (Clark and Mellon, 1995). Wierman et al. illustrated that cotransfection of another member of the POU-domain family of transcription factors, SCIP/Oct-6/Tst-1, can down-regulate rGnRH gene expression (Wierman et al., 1997). Three octamer binding sites are the most relevant to repression, − 343/− 314, − 171/ − 126, and − 126/ − 73 (Fig. 1, Table 1). Though, bacterially expressed SCIP binds these regions in vitro, mutations of the octamer sites do not prevent response to repression by SCIP cotransfection. SCIP may be physiologically relevant to GnRH gene expression since GnRH and SCIP colocalize in the rat hypothalamus. Though SCIP mRNA is present in GT1-7 cells (Clark and Mellon, 1995), the inability of endogenous SCIP protein in GT1-7 nuclear extracts to bind to the octamer sites is a paradoxical observation that remains to be addressed.
4. Hormone and second messenger regulation of GnRH promoter Hormones play a crucial role in GnRH expression and secretion. The proximal promoter contains a majority of the cis-acting elements thus far characterized for hormonal control of GnRH transcription (Table 1). Glucocorticoid repression of GnRH transcription has been localized to the mouse GnRH (mGnRH) promoter by Chandran et al. (1996). Mouse GnRH gene transcription is negatively regulated by dexamethasone in transfections in GT1-7 cells, and glucocorticoid receptor (GR) is present in GT1-7 cells. This effect is conferred by two regions within the mouse promoter termed the proximal and distal negative glucocorticoid responsive elements (nGRE). GR and Oct-1 are present in the complex which binds to the distal nGRE (mouse − 218/− 201) by gel supershift assays. This element corresponds to the element at − 106/− 99 in the rat gene, which also binds Oct-1. In contrast, GR, but not Oct-1, is present on the proximal nGRE (mouse −184/ − 150). This site corresponds to the upstream and middle protein binding sites within the large footprinted region in rat (− 76 to − 26). Oct-1 was not found interacting with this region, in agreement with our localization of Oct-1 binding to the downstream region of the − 76 to −26 footprint. Since GnRH controls the release of LH from the pituitary, it has been hypothesized that LH can also control GnRH production by a short feedback loop. Lei and Rao demonstrated that hCG receptors are found on GT1-7 cells (Lei and Rao, 1994) and that hCG down-regulates rGnRH gene transcription
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through a region (rat − 99 to −79) encompassing FP3 (rat − 85 to − 79) (Lei and Rao, 1997). DNase I footprinting utilizing hCG-treated GT1-7 nuclear extracts shows protection from −99 to − 79 of the rGnRH promoter (Lei and Rao, 1997). Additionally, Lei and Rao identify two specific proteins which bind to the − 126 to − 74 probes by Southwestern screening; the faster migrating protein (95 kDa) increases with hCG treatment. This protein was shown not to be Fos, CREB or Oct-1 by antibody supershift experiments. Steroid hormones control gonadotropin release by a negative feedback mechanism at the level of both the hypothalamus and pituitary. Cotransfection of estrogen receptor and rGnRH promoter with estradiol treatments in GT1-7 cells revealed that the region − 171 to − 126 contains a negative estrogen responsive element (Kepa et al., 1994). Further experiments showed that ER does not bind to this region of the rGnRH promoter. Cotransfections of PR with the rGnRH promoter (Kepa et al., 1996) conferred 50% repression in the presence of progesterone, this effect being mediated through − 171 to −73 of the GnRH promoter. Gel shift assays revealed that purified hPR can bind to three oligonucleotides: − 171 to −126, − 126/ − 73 and − 111/ − 73 (Fig. 1). Since this is a somewhat artificial system, it is not clear how these sites become available for binding by PR when other proteins such as Oct-1 bind to the same regions within the GnRH promoter and PR is not present in GT1-7 cells. PKC down-regulation due to prolonged TPA treatment causes a decrease in rGnRH transcription. This effect was localized to FP2 and FP5, 4, and 3 (Eraly and Mellon, 1995; Bruder et al., 1996). However, the − 124 to −73 fragment in the context of a heterologous promoter showed no repression by TPA or Fos cotransfection, while FP2 does confer repression. Bruder, et al. (1996) have characterized the effect as being Fos dependent but they were unable to detect Fos binding to the GnRH promoter. This suggests that Fos may be regulating the expression of other proteins, such as Oct-1, that bind directly to the promoter. In contrast, under low serum conditions in NLT cells, TPA increases hGnRH reporter gene expression (Zakaria et al., 1996). This effect localizes to a non-conserved AP-1 site at − 402 which is also the site of action of an IGF-I stimulated signal transduction pathway (Zhen et al., 1997). Fos and Jun bind to this site and Fos is up-regulated during IGF-I or TPA stimulation. Thus, activation of the PKC pathway has species specific effects on GnRH gene transcription as suggested by Zakaria et al. (1996).
5. Conclusion The GnRH promoter modulates GnRH gene tran-
scription in response to steroids, hormones and signaling pathways. These agents are likely to act at various stages in development, maturation, and control of reproductive function. Currently, we and other groups have identified proteins which bind to the GnRH promoter and play a role in either basal GnRH expression or in transmission of specific signals. Interactions between the ubiquitous factors, Oct-1, inducible factors, GR, and tissue-specific factors allow GnRH gene expression to be regulated. Further experiments must be undertaken to address how changes in protein binding to the promoter affect GnRH gene regulation and the role the enhancer plays in this process. Acknowledgements We thank Teri Banks, Simon Lee, and Brian Powl for excellent technical assistance, Jeff Ludwig for creating the double block mutation plasmids, Melody Clark and Mark Lawson for scientific discussions and members of the Mellon laboratory for support. This work was supported by the NIH grant DK44838 to P.L.M. S.B.N. was supported by NIH training grants AG00216 and DK07541. References Bruder, J.M., Spaulding, A.J., Wierman, M.E., 1996. Phorbol ester inhibition of rat gonadotropin-releasing hormone promoter activity: role of fos and jun in the repression of transcription. Mol. Endocrinol. 10, 35 – 44. Chandran, U.R., Attardi, B., Friedman, R., Zheng, Z., Roberts, J.L., DeFranco, D.B., 1996. Glucocorticoid repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter elements that are recognized by heteromeric complexes containing glucocorticoid receptor. J. Biol. Chem. 271, 20412 – 20420. Clark, M.E., Mellon, P.L., 1995. The POU homeodomain transcription factor Oct-1 is essential for activity of the gonadotropin-releasing hormone neuron-specific enhancer. Mol. Cell. Biol. 15, 6169 – 6177. Dong, K.-W., Yu, K.-L., Roberts, J.L., 1993. Identification of a major up-stream transcription start site for the human progonadotropin-releasing hormone gene used in reproductive tissues and cell lines. Mol. Endocrinol. 7, 1654 – 1666. Dong, K.W., Duval, P., Zeng, Z., Gordon, K., Williams, R.F., Hodgen, G.D., Jones, G., Kerdelhue, B., Roberts, J.L., 1996. Multiple transcription start sites for the GnRH gene in rhesus and cynomolgus monkeys: a non-human primate model for studying GnRH gene regulation. Mol. Cell. Endocrinol. 117, 121–130. Dong, K.-W., Yu, K.-L., Chen, Z.-G., Chen, Y.-D., Roberts, J.-L., 1997. Characterization of multiple promoters directing tissue-specific expression of the human gonadotropin-releasing hormone gene. Endocrinology 138, 2754 – 2762. Eraly, S.A., Mellon, P.L., 1995. Regulation of GnRH transcription by protein kinase C is mediated by evolutionarily conserved, promoter-proximal elements. Mol. Endocrinol. 9, 848 – 859. Eraly, S.A., Nelson, S.B., Huang, K.M., Mellon, P.L., 1998. Oct-1 binds promoter elements required for transcription of the gonadotrophin-releasing hormone gene. Mol. Endocrinol. 12, 469– 481.
S.B. Nelson et al. / Molecular and Cellular Endocrinology 140 (1998) 151–155 Kepa, J.K., Neeley, C.I., Jacobsen, B.M., Bruder, J.M., McDonnell, D.P., Leslie, K.K., Wierman, M.E., 1994. Estrogen receptor mediated repression of rat gonadotropin releasing hormone (GnRH) promoter activity in hypothalamic cells. Endocrine 2, 947– 956. Kepa, J.K., Jacobsen, B.M., Boen, E.A., Prendergast, P., Edwards, D.P., Takimoto, G., Wierman, M.E., 1996. Direct binding of progesterone receptor to nonconsensus DNA sequences represses rat GnRH. Mol. Cell. Endocrinol. 117, 27–39. Lawson, M.A., Whyte, D.B., Mellon, P.L., 1996. GATA factors are essential for activity of the neuron-specific enhancer of the gonadotropin-releasing hormone gene. Mol. Cell. Biol. 16, 3596 – 3605. Lei, Z.M., Rao, C.V., 1994. Novel presence of luteinizing hormone/ human chorionic gonadotropin (hCG) receptors and the downregulating action of hCG on gonadotropin-releasing hormone gene expression in immortalized hypothalamic GT1-7 neurons. Mol. Endocrinol. 8, 111–1121. Lei, Z., Rao, C.V., 1997. cis-acting elements and trans-acting proteins in the transcriptional inhibition of gonadotropin-releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons. J. Biol. Chem. 272, 14365– 14371. Liposits, Z., Merchenthaler, I., Wetsel, W.C., Reid, J.J., Mellon, P.L., Weiner, R.I., Negro-Vilar, A., 1991. Morphological characterization of immortalized hypothalamic neurons synthesizing luteinizing hormone-releasing hormone. Endocrinology 129, 1575 – 1583. Mellon, P.L., Windle, J.J., Goldsmith, P., Pedula, C., Roberts, J., Weiner, R.I., 1990. Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5, 1–10. Radovick, S., Wray, S., Lee, E., Nicols, D.K., Nakayama, Y., Weintraub, B.D., Westphal, H., Cutler, J., G. B., Wondisford, F.E., 1991. Migratory arrest of gonadotropin-releasing hormone
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neurons in transgenic mice. Proc. Natl. Acad. Sci. USA 88, 3402 – 3406. Wetsel, W.C., Valenc¸a, M.M., Merchenthaler, I., Liposits, Z., Lo´pez, F.J., Weiner, R.I., Mellon, P.L., Negro-Vilar, A., 1992. Intrinsic pulsatile secretory activity of immortalized LHRH secreting neurons. Proc. Natl. Acad. Sci. USA. 89, 4149 – 4153. Whyte, D.B., Lawson, M.A., Belsham, D.D., Eraly, S.A., Bond, C.T., Adelman, J.P., Mellon, P.L., 1995. A neuron-specific enhancer targets expression of the gonadotropin-releasing hormone gene to hypothalamic neurosecretory neurons. Mol. Endocrinol. 9, 467 – 477. Wierman, M.E., Xiong, X., Kepa, J.K., Spaulding, A.J., Jacobsen, B.M., Fang, Z., Nilaver, G., Ojeda, S.R., 1997. Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression? Mol. Cell. Biol. 17, 1652– 1665. Wray, S., Grant, P., Gainer, H., 1989. Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc. Natl. Acad. Sci. USA 86, 8132 – 8136. Zakaria, M., Dunn, I.C., Zhen, S., Su, E., Smith, E., Patriquin, E., Radovick, S., 1996. Phorbol ester regulation of the gonadotropinreleasing hormone (GnRH) gene in GnRH-secreting cell lines: a molecular basis for species differences. Mol. Endocrinol. 10, 1282 – 1291. Zhen, S., Zakaria, M., Wolfe, A., Radovick, S., 1997. Regulation of gonadotropin-releasing hormone (GnRH) gene expression by insulin-like growth factor 1 in a cultured GnRH-expressing neuronal cell line. Mol. Endocrinol. 11, 1145 – 1155.
The GnRH promoter: Target of transcription factors, hormones, and signaling pathways Shelley B. Nelson, Satish A. Eraly, Pamela L. Mellon * Departments of Reproducti6e Medicine and Neurosciences 0674, The Center for Cellular and Molecular Medicine, Uni6ersity of California, San Diego, 9500 Gilman Dri6e, La Jolla, CA 92093 -0674, USA
Abstract Gonadotropin-releasing hormone (GnRH) is essential for normal reproductive maturation and function. We present a review of the known mechanisms of hypothalamic GnRH transcriptional control through the conserved GnRH promoter. Understanding this promoter region will allow us to comprehend better the complexities of the hypothalamic – pituitary – gonadal axis. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gonadotropin-releasing hormone; POU-homeodomain; Gene expression
1. Introduction Gonadotropin-releasing hormone (GnRH) is expressed in specific hypothalamic neurons and secreted into the hypophyseal portal venous circulation in a pulsatile manner. The secreted hormone travels to the anterior pituitary where it binds to GnRH receptors on the pituitary gonadotrope. Occupation of these receptors activates signal transduction cascades finally causing the release of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH play a role in gonadal maturation, onset of puberty, and ovulation. Changes in GnRH pulse frequency and amplitude regulate gonadotropin release, and GnRH is regulated at the level of transcription, translation, and secretion to produce these physiological effects. Here, we will focus on the mechanism of hypothalamic GnRH regulation at the transcriptional level through the conserved proximal promoter of the gene. GnRH secreting neurons are few in number (B 800 in mice) (Wray et al., 1989) and scattered throughout the hypothalamus. To address the transcriptional con* Corresponding author. Tel.: +1 619 5341312; fax: + 1 619 5341438; e-mail: [email protected]
trol of GnRH, immortalized GnRH-secreting hypothalamic cell lines were created. The GT1-7 cell line was derived in this laboratory by targeting SV40 T antigen to the GnRH-secreting hypothalamic neurons in transgenic mice by placing the 5% flanking region of the rat GnRH (rGnRH) gene upstream of the oncogene SV40 T antigen (Mellon et al., 1990). Cell lines were derived from the resulting hypothalamic tumors that have many of the characteristics of their in vivo counterparts, including neuronal morphology (Liposits et al., 1991), expression of neuronal markers, and secretion of GnRH in a pulsatile manner (Wetsel et al., 1992). Additionally, Radovick and coworkers created the GN10 (NLT) and GN11 cell lines by a similar method (Radovick et al., 1991) using instead the 1100 bp 5% flanking region of the human GnRH (hGnRH) gene. In this instance, tumors were present in the olfactory bulb and these cells proved to be immunopositive for GnRH in culture. Therefore, the GT1-7, GN10 (NLT) and GN11 cells provide model systems for the study of GnRH gene regulation. Within the 5% flanking region of the rGnRH gene, there are two major regions for transcription of the GnRH gene, an enhancer and a promoter. The 300 bp enhancer is located 1.8 kb upstream of the transcriptional start site and confers 50-fold activation of GnRH
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Fig. 1. Footprinted regions of the rGnRH proximal promoter. Regions known to bind particular factors are indicated.
transcription (Whyte et al., 1995) only in GT1-7 cells. It contains binding sites for POU-homeodomain transcription factors and GATA factors (Clark and Mellon, 1995; Lawson et al., 1996). These proteins are important for neuron-specific expression of the GnRH gene. Within the first 173 bases 5% of the transcriptional start site, the rGnRH promoter contains a region that is conserved across species (Eraly and Mellon, 1995). The hGnRH 5% flanking region contains two transcriptional start sites at + 1 and − 579 respectively (Dong et al., 1993). The promoter for the proximal start site (+1) is active in the hypothalamus while the distal promoter is active in other reproductive tissues such as placenta (Dong et al., 1997). Recently, the GnRH gene was isolated from rhesus monkey (Dong et al., 1996). The primate gene is highly homologous to the human gene, conserving the dual promoters. Though little is known about the factors which regulate hGnRH expression, progress has been made in understanding the role of transcription factors binding to and influencing the mouse and rat GnRH promoters.
2. Transcription from the rGnRH promoter The initial analysis of the rGnRH promoter was undertaken to address its function in GT1-7 hypothalamic cells. DNase I footprint analysis of the rGnRH promoter led to the identification of seven footprinted regions (FP1–7) located between −173 and +112 (Fig. 1) (Eraly and Mellon, 1995). Transient transfections in GT1-7 cells revealed that removal of −173 to −126 (FP7 and 6) caused a 2-fold reduction of reporter gene activity. Further deletion of − 126 to − 82 (FP5, 4, and 3) also caused a 2-fold reduction of the reporter gene activity. Additionally, two separate block mutations created within − 110 to − 89 (FP4) show a 60 and 30% reduction in reporter gene activity respectively (Eraly et al., 1998). A block mutation at − 123 to −116 (FP5) showed a 20% reduc-
tion in reporter gene activity while a block mutation at − 85 to − 78 (FP3) showed no significant change. These results are consistent with those presented by Lei et al. showing that a mutation similar to the 3% FP4 mutation resulted in a decrease in reporter gene activity and that a mutation similar to the one within FP3 resulted in an increase in reporter gene activity (Lei and Rao, 1997). In addition, they observed a transcriptional decrease when a region between FP3 and FP4 was mutated. We conclude that the region encompassing FP7–FP3 contains elements necessary for GnRH gene transcription and that the 2-fold decrease in transcription seen with deletion of −126 to − 82 is mostly localized to − 110 to − 89 (FP4). Since the − 70 to − 28 (FP2) deletion caused a 20-fold decrease in activity, we sought to understand its role in gene activation. Because footprint encompasses 51 bases (Fig. 1), seven block mutations were created to dissect its structure. Footprinting of these block mutants led to the conclusion that there are three binding sites within FP2. Transfection of the promoters carrying the block mutations in FP2 into GT1-7 cells revealed that each of the single block mutations resulted in a 40–60% reduction in reporter gene activity. When double mutations were created removing each possible pair of the three potential protein binding sites, an 80% loss in reporter gene activity was seen for each of the three paired combinations. When all three binding sites are present, they have a synergistic activating effect on the reporter gene, compared with individual proteins binding at the doubly mutated FP2.
3. POU-domain transcription factors bind to the rGnRH promoter To identify the proteins binding to the rGnRH promoter, gel mobility shift assays were conducted with oligonucleotide probes comprising the various
S.B. Nelson et al. / Molecular and Cellular Endocrinology 140 (1998) 151–155
153
Table 1 Compilation of the transcription factors and hormones/second messengers that regulate the hypothalamic GnRH promoter in GT1-7 cells (or other cell lines where noted) Site Human Factora Oct-1 SCIP/Oct-6/Tst-1 GR hPR Fos/Jun
Transcriptional effect Rat
Mouse
−106/−99, −47/−40 −343/−314, −171/−126, −126/−73
−212/−205 −218/−201, −184/−150
−171/−126, −126/−73, −111/−73 −402/−396
Hormones/Second messengersb Glucocorticoid hCG Estrogen Progesterone TPA IGF-I/TPA −402/−396
−237/−201 −99/−79 −171/−126 −171/−73 −82/−22, −124/−73
+/Rat − −* − + (GN11) − − − − − + (NLT)
a
The site refers to the region known to bind the particular factor in the given species. The site is the region that mediates responsiveness to the hormone/second messenger. * Repression may be mediated through the binding of other factors besides GR.
b
promoter footprinted regions (Eraly et al., 1998). Footprint 5 ( − 128 to − 112) formed one major unidentified complex. Footprint 4 (− 110 to − 89) showed one major low mobility complex which was shown to be Oct-1 by antibody supershift analysis. The use of a full length FP2 ( −82 to −22) gel shift probe results in three specific complexes (Eraly and Mellon, 1995). A smaller oligonucleotide probe encompassing the middle and downstream protein binding sites results in the formation of four specific complexes. Addition of the Oct-1 antibody blocks the formation of the slowest mobility complex, demonstrating that Oct-1 binds to FP2. Mutational analysis indicates that the Oct-1 binding site lies within the downstream region of FP2 (Fig. 1). Thus Oct-1 appears to be involved in GnRH gene regulation by its action on both the promoter and the enhancer (Clark and Mellon, 1995). Wierman et al. illustrated that cotransfection of another member of the POU-domain family of transcription factors, SCIP/Oct-6/Tst-1, can down-regulate rGnRH gene expression (Wierman et al., 1997). Three octamer binding sites are the most relevant to repression, − 343/− 314, − 171/ − 126, and − 126/ − 73 (Fig. 1, Table 1). Though, bacterially expressed SCIP binds these regions in vitro, mutations of the octamer sites do not prevent response to repression by SCIP cotransfection. SCIP may be physiologically relevant to GnRH gene expression since GnRH and SCIP colocalize in the rat hypothalamus. Though SCIP mRNA is present in GT1-7 cells (Clark and Mellon, 1995), the inability of endogenous SCIP protein in GT1-7 nuclear extracts to bind to the octamer sites is a paradoxical observation that remains to be addressed.
4. Hormone and second messenger regulation of GnRH promoter Hormones play a crucial role in GnRH expression and secretion. The proximal promoter contains a majority of the cis-acting elements thus far characterized for hormonal control of GnRH transcription (Table 1). Glucocorticoid repression of GnRH transcription has been localized to the mouse GnRH (mGnRH) promoter by Chandran et al. (1996). Mouse GnRH gene transcription is negatively regulated by dexamethasone in transfections in GT1-7 cells, and glucocorticoid receptor (GR) is present in GT1-7 cells. This effect is conferred by two regions within the mouse promoter termed the proximal and distal negative glucocorticoid responsive elements (nGRE). GR and Oct-1 are present in the complex which binds to the distal nGRE (mouse − 218/− 201) by gel supershift assays. This element corresponds to the element at − 106/− 99 in the rat gene, which also binds Oct-1. In contrast, GR, but not Oct-1, is present on the proximal nGRE (mouse −184/ − 150). This site corresponds to the upstream and middle protein binding sites within the large footprinted region in rat (− 76 to − 26). Oct-1 was not found interacting with this region, in agreement with our localization of Oct-1 binding to the downstream region of the − 76 to −26 footprint. Since GnRH controls the release of LH from the pituitary, it has been hypothesized that LH can also control GnRH production by a short feedback loop. Lei and Rao demonstrated that hCG receptors are found on GT1-7 cells (Lei and Rao, 1994) and that hCG down-regulates rGnRH gene transcription
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through a region (rat − 99 to −79) encompassing FP3 (rat − 85 to − 79) (Lei and Rao, 1997). DNase I footprinting utilizing hCG-treated GT1-7 nuclear extracts shows protection from −99 to − 79 of the rGnRH promoter (Lei and Rao, 1997). Additionally, Lei and Rao identify two specific proteins which bind to the − 126 to − 74 probes by Southwestern screening; the faster migrating protein (95 kDa) increases with hCG treatment. This protein was shown not to be Fos, CREB or Oct-1 by antibody supershift experiments. Steroid hormones control gonadotropin release by a negative feedback mechanism at the level of both the hypothalamus and pituitary. Cotransfection of estrogen receptor and rGnRH promoter with estradiol treatments in GT1-7 cells revealed that the region − 171 to − 126 contains a negative estrogen responsive element (Kepa et al., 1994). Further experiments showed that ER does not bind to this region of the rGnRH promoter. Cotransfections of PR with the rGnRH promoter (Kepa et al., 1996) conferred 50% repression in the presence of progesterone, this effect being mediated through − 171 to −73 of the GnRH promoter. Gel shift assays revealed that purified hPR can bind to three oligonucleotides: − 171 to −126, − 126/ − 73 and − 111/ − 73 (Fig. 1). Since this is a somewhat artificial system, it is not clear how these sites become available for binding by PR when other proteins such as Oct-1 bind to the same regions within the GnRH promoter and PR is not present in GT1-7 cells. PKC down-regulation due to prolonged TPA treatment causes a decrease in rGnRH transcription. This effect was localized to FP2 and FP5, 4, and 3 (Eraly and Mellon, 1995; Bruder et al., 1996). However, the − 124 to −73 fragment in the context of a heterologous promoter showed no repression by TPA or Fos cotransfection, while FP2 does confer repression. Bruder, et al. (1996) have characterized the effect as being Fos dependent but they were unable to detect Fos binding to the GnRH promoter. This suggests that Fos may be regulating the expression of other proteins, such as Oct-1, that bind directly to the promoter. In contrast, under low serum conditions in NLT cells, TPA increases hGnRH reporter gene expression (Zakaria et al., 1996). This effect localizes to a non-conserved AP-1 site at − 402 which is also the site of action of an IGF-I stimulated signal transduction pathway (Zhen et al., 1997). Fos and Jun bind to this site and Fos is up-regulated during IGF-I or TPA stimulation. Thus, activation of the PKC pathway has species specific effects on GnRH gene transcription as suggested by Zakaria et al. (1996).
5. Conclusion The GnRH promoter modulates GnRH gene tran-
scription in response to steroids, hormones and signaling pathways. These agents are likely to act at various stages in development, maturation, and control of reproductive function. Currently, we and other groups have identified proteins which bind to the GnRH promoter and play a role in either basal GnRH expression or in transmission of specific signals. Interactions between the ubiquitous factors, Oct-1, inducible factors, GR, and tissue-specific factors allow GnRH gene expression to be regulated. Further experiments must be undertaken to address how changes in protein binding to the promoter affect GnRH gene regulation and the role the enhancer plays in this process. Acknowledgements We thank Teri Banks, Simon Lee, and Brian Powl for excellent technical assistance, Jeff Ludwig for creating the double block mutation plasmids, Melody Clark and Mark Lawson for scientific discussions and members of the Mellon laboratory for support. This work was supported by the NIH grant DK44838 to P.L.M. S.B.N. was supported by NIH training grants AG00216 and DK07541. References Bruder, J.M., Spaulding, A.J., Wierman, M.E., 1996. Phorbol ester inhibition of rat gonadotropin-releasing hormone promoter activity: role of fos and jun in the repression of transcription. Mol. Endocrinol. 10, 35 – 44. Chandran, U.R., Attardi, B., Friedman, R., Zheng, Z., Roberts, J.L., DeFranco, D.B., 1996. Glucocorticoid repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter elements that are recognized by heteromeric complexes containing glucocorticoid receptor. J. Biol. Chem. 271, 20412 – 20420. Clark, M.E., Mellon, P.L., 1995. The POU homeodomain transcription factor Oct-1 is essential for activity of the gonadotropin-releasing hormone neuron-specific enhancer. Mol. Cell. Biol. 15, 6169 – 6177. Dong, K.-W., Yu, K.-L., Roberts, J.L., 1993. Identification of a major up-stream transcription start site for the human progonadotropin-releasing hormone gene used in reproductive tissues and cell lines. Mol. Endocrinol. 7, 1654 – 1666. Dong, K.W., Duval, P., Zeng, Z., Gordon, K., Williams, R.F., Hodgen, G.D., Jones, G., Kerdelhue, B., Roberts, J.L., 1996. Multiple transcription start sites for the GnRH gene in rhesus and cynomolgus monkeys: a non-human primate model for studying GnRH gene regulation. Mol. Cell. Endocrinol. 117, 121–130. Dong, K.-W., Yu, K.-L., Chen, Z.-G., Chen, Y.-D., Roberts, J.-L., 1997. Characterization of multiple promoters directing tissue-specific expression of the human gonadotropin-releasing hormone gene. Endocrinology 138, 2754 – 2762. Eraly, S.A., Mellon, P.L., 1995. Regulation of GnRH transcription by protein kinase C is mediated by evolutionarily conserved, promoter-proximal elements. Mol. Endocrinol. 9, 848 – 859. Eraly, S.A., Nelson, S.B., Huang, K.M., Mellon, P.L., 1998. Oct-1 binds promoter elements required for transcription of the gonadotrophin-releasing hormone gene. Mol. Endocrinol. 12, 469– 481.
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