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In Vivo Identification of a 107-Base Pair Promoter Element Mediating Neuron-Specific Expression of Mouse Gonadotropin-Releasing Hormone

Section of Reproductive Endocrinology and Infertility (H.H.K., S.C.E., A.L.J., C.N.W.), Department of Obstetrics and Gynecology; and Section of Endocrinology, Department of Pediatrics (H.H.K., A.W., S.R.) and Department of Medicine (R.N.C.), The University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Helen H. Kim, M.D., Section of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, The University of Chicago, 5841 South Maryland Avenue, MC 2050, Chicago, Illinois 60637. E-mail: hkim{at}babies.bsd.uchicago.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
To identify regions of the mouse GnRH (mGnRH) promoter that mediate tissue-specific gene expression, transgenic mice have been generated with fragments of mGnRH promoter fused to the luciferase reporter gene. In this manuscript, we examine transgenic mice, generated with –356/+28 bp and –249/+28 bp of the mGnRH gene. Both fragments of mGnRH promoter target ovarian expression of the luciferase transgene, but neuronal luciferase activity is detected only in the mice bearing the –356-bp fragment, suggesting that the DNA sequences essential for directing neuron-specific expression of the GnRH gene are located between –356 and –249 bp. Two consensus binding sites for Otx2 were identified in this promoter region and were confirmed to be functional. EMSAs demonstrated specific binding of Otx2 to the mGnRH promoter, and overexpression of Otx2 increased transcriptional activity of the mGnRH promoter in transient transfection studies. When both Otx2 binding sites were eliminated, overexpression of Otx2 had no effect. GnRH mRNA expression in immortalized GnRH-secreting cell lines was also found to correlate with Otx2 expression. In addition, transgenic mice, bearing the 356 fragment of the mGnRH gene in which the Otx2 binding sites were eliminated, have significantly lower luciferase activity in the neonatal brain compared with mice generated with intact Otx2 binding sites. Luciferase activity was, however, still present in the ovary. Our findings provide evidence that Otx2 may have a critical role in directing tissue-specific expression of the mGnRH gene to the neuron, but not the ovary.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
APPROPRIATE TISSUE-SPECIFIC expression of GnRH is critical for establishing and maintaining reproductive competence. It has long been recognized that the GnRH decapeptide is released from the hypothalamus and controls gonadal steroidogenesis and ovarian follicular development by regulating the production and release of gonadotropins from the pituitary. The precise coordinated release and expression of GnRH from the hypothalamus is essential for a functioning mammalian reproductive system. More recently, the presence of extrapituitary GnRH has been appreciated. Low levels of GnRH expression have been found in peripheral reproductive tissues, such as placenta (1), breast, ovary, and testes (2).

The extremely low levels of expression in the peripheral tissues, along with the paucity and scattered distribution of GnRH neurons, have limited the in vivo study of GnRH gene regulation. In the adult mouse brain, it has been estimated that GnRH expression is limited to only 800 neurons (3). In the adult, the vast majority of the GnRH neurons are located in the basal hypothalamus and septum, but GnRH neurons have been described along the migratory pathway from the olfactory bulbs, as well as in the cerebral cortex and limbic system (3, 4). A similar anatomic organization is found in all mammals (5).

Several in vitro studies have used mouse-derived immortalized GnRH secreting neuronal cell lines to investigate the molecular mechanisms that regulate expression of the GnRH gene. The GN11 (6) and GT1–7 (7) cell lines were produced by dispersal of tumors that resulted from targeted oncogenesis of the GnRH neuron in transgenic mice using the large T-antigen oncogene. The GN11 cells derived from an olfactory tumor, whereas the GT1–7 cells were derived from a tumor located in the basal forebrain. The GN11 cell line appears to have a phenotype resembling a migrating GnRH neuron with low levels of GnRH secretion, whereas the GT1–7 cells resemble postmigratory neurons, which secrete high levels of GnRH in a pulsatile fashion (8, 9). Transient transfection studies using the rat GnRH (rGnRH) gene promoter identified a 173-bp proximal promoter region (10) and a 300-bp enhancer region located 1.8 kb upstream from the transcription start site that conferred cell-specific expression (11). Both these sites were reported to be important for the correct expression of the rGnRH gene in vitro (12).

In the brain, however, GnRH neurons are dispersed and are influenced by growth factors, steroids, and neurotransmitters secreted by the various adjacent cell types. Because these in vitro studies are unlikely to reflect the elaborate intricacy of in vivo GnRH gene regulation, in vivo models have been developed to study the regulation of the GnRH gene in transgenic mice. Transgenic mice have been generated with gene constructs containing various GnRH promoter deletion constructs fused to reporter genes. By generating transgenic mice with different fragments of the human GnRH (hGnRH) promoter fused to the luciferase (LUC) reporter gene (13), sequences between –1131 and –484 bp of the hGnRH gene (14), were found to mediate neuron-specific expression of hGnRH. Subsequently, the neuron-specific element of the hGnRH gene promoter was further localized between –992 and –795 bp (15).

In our previous study, using different fragments of the mouse GnRH (mGnRH) promoter fused to the luciferase reporter gene (16), we found that the proximal –1005 bp of the mGnRH promoter contains the critical elements for appropriate neuronal expression of mGnRH. Furthermore, data also suggested that an ovarian GnRH repressor element is located in the distal region of the mGnRH promoter between –3446 bp and –2078 bp because deletion of this region unmasks luciferase expression in the ovaries of transgenic mice bearing the mGnRH-LUC transgene. DNA sequences contained within the proximal –1005 bp were also found to be sufficient for directing mGnRH expression to the ovary.

In this manuscript, we have isolated the DNA sequences essential for directing neuron-specific expression of GnRH to a 107-bp region between –356 and –249 bp of the mGnRH gene. We demonstrate that the mGnRH neuron-specific element contains two functional binding sites for Otx2, the vertebrate homolog of orthodenticle (otd), a Drosophila gene that is necessary for normal head development (17). Otx2 protein has been detected in GnRH neurons in both adult (18) and embryonic mice (19). Our studies demonstrate that these putative Otx2 consensus binding sites bind Otx2 protein and transactivate the mGnRH promoter in vitro. Finally, we demonstrate that these Otx2 binding sites are critical for neuronal expression of mGnRH in vivo.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The Proximal –356-bp Region of the mGnRH Gene Promoter Targets Neuronal Luciferase Expression
To define the promoter element necessary to target hypothalamic luciferase expression, a DNA construct containing the region from –356 bp to +28 bp of the mGnRH promoter fused to the luciferase reporter gene was used to generate transgenic mice (–356mGnRH-LUC). Southern blot analysis identified eight separate founder lines that incorporated the luciferase transgene. Because transgenes often integrate into sites that do not permit expression (20), we sought to identify the founder lines that could express the mGnRH-LUC transgene. Luciferase expression was detected in five of eight founder lines.

To identify the founder lines that could express the mGnRH-LUC transgene, whole brain homogenates were obtained from neonatal mice. Luciferase activity was measured as relative light units (RLU) and corrected for background RLU. The corrected RLU reflects the luciferase activity present in a given tissue. A tissue was defined as expressing luciferase if the corrected RLU in the tissue was significantly greater than what was seen in wild-type mice.

Luciferase activity was detected in the brain homogenates obtained from offspring of four of eight founders (nos. 30, 35, 41, and 43). One female, founder no. 36, did not reproduce, but luciferase activity of 54,075 RLU was detected in her hypothalamus. Data from the offspring of the four other transgenic lines, which demonstrated neuronal luciferase activity, are shown in Table 1. The luciferase activity in neonatal mice bearing the luciferase transgene was compared with the levels seen in their wild-type littermates. The luciferase activity ranged from 30,931 ± 11,308 RLU in the highest expressing line (no. 41) to 12,056 ± 4488 RLU in the lowest expressing line (no. 30). As shown in Table 1, the luciferase activity in their wild-type littermates was significantly lower, 40 ± 7 RLU. Offspring from three of eight 8 founders (founder nos. 1–3) did not demonstrate luciferase activity in their brain homogenates despite incorporation of the luciferase transgene, suggesting integration of the transgene into a chromosomal site that prevented expression. In the transgenic offspring from founders 1, 2, and 3, the luciferase activity (9 ± 5 RLU) in their brains did not differ from levels (7 ± 3 RLU) seen in their wild-type littermates (P = 0.79).

The Proximal –356-bp Region of the mGnRH Gene Promoter Targets Both Neuronal and Ovarian Luciferase Expression
The anatomic pattern of luciferase expression is an assay of mGnRH promoter activity, and was examined in the adult offspring from the two founders (nos. 35 and 41) that expressed luciferase at the highest level. As illustrated in Fig. 1A, a similar anatomic pattern of luciferase expression was found in offspring from the founders that expressed the –356mGnRH-LUC transgene. Ten (five female and five male) adult offspring from each of the two founder mice were examined. In the mice bearing the –356mGnRH-LUC transgene, luciferase expression was present only in the hypothalamus and gonads. In the other tissues (cortex, cerebellum, midbrain, heart, lung, kidney, liver spleen), the luciferase activity did not differ from that seen in wild-type mice (P = 0.24).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Luciferase Expression in –356mGnRH-LUC and –249mGnRH-LUC Mice Luciferase was measured from tissue homogenates as RLU. The corrected RLU value was obtained by subtracting the background RLU (seen with lysis buffer alone). A, Anatomic pattern of luciferase expression in tissue homogenates from mice generated with –356/+28 bp of the mGnRH promoter fused to the luciferase reporter gene. Ten reproductive age mice (five male and five female) were examined for each of the two founder lines. In these mice, luciferase expression was detected in the hypothalamus and gonads. Luciferase activity was found at high levels in the ovary and low levels of activity were detected in the testes. These findings suggest that DNA sequences contained in the proximal –356 bp are sufficient to direct both hypothalamic and gonadal mGnRH expression. Luciferase activity in other tissues did not differ from that found in wild-type mice. B, Anatomic pattern of luciferase expression in tissue homogenates from mice generated with –249/+28 bp of the mGnRH promoter fused to the luciferase reporter gene. Data from six male and five female mice from each of the three founder lines are shown. In these mice, luciferase expression was not detected in the hypothalamus, suggesting the sequences contained in the proximal –249-bp fragment of the mGnRH promoter do not contain the sequences necessary to mediate neuronal expression. In contrast, gonadal expression of luciferase is seen, suggesting that the gonadal mGnRH expression is mediated by sequences contained within the proximal –249-bp fragment of the promoter.

Our previous data demonstrated that removal of the distal mGnRH promoter region, between –3446 and –2078 bp, unmasks ovarian luciferase activity in the mGnRH-LUC transgenic mice (16). The region of the mGnRH promoter, between –3446 and –2078 bp, appears to mediate repression of GnRH expression in the ovary. As expected, offspring from the luciferase-expressing founder lines of the –356mGnRH-LUC mice, demonstrated very high levels of luciferase activity in their ovarian homogenates (18,958 ± 3859, and 12,687 ± 3602) as shown in Fig. 1A and Table 2. Compared with wild-type mice (28 ± 9), ovarian luciferase activity was significantly higher in transgenic offspring from both founder no. 35 (P = 0.008) and founder no. 41 (P = 0.025). Our data suggest that the ovary-specific element, as well the neuron-specific element, is located within the proximal –356 bp of the mGnRH promoter.

In the –3446mGnRH-LUC mice, luciferase activity in the testes did not differ from the levels seen in the testes from wild-type males (16 ± 6 RLU, P = 0.085). In the –2078mGnRH-LUC mice, however, testicular luciferase activity was significantly higher than that detected in the wild-type males (207 ± 16 RLU; P < 0.001). These findings demonstrate that the sequences contained in the proximal –356 bp of the mGnRH promoter targets both neuronal and gonadal expression.

The Proximal 249 bp of the mGnRH Gene Promoter Is Not Sufficient to Target Luciferase Expression to the Hypothalamus, but Is Sufficient to Target Gonadal Expression
To define further the promoter element necessary to target hypothalamic luciferase expression, a DNA construct containing the region from –249 to +28 bp of the mGnRH promoter fused to the luciferase reporter gene was used to generate transgenic mice (–249mGnRH-LUC). Southern blot analysis identified seven separate founder mice that incorporated the luciferase transgene. In contrast to our findings in the –356mGnRH-LUC mice, preliminary examination of the neonatal brains did not demonstrate luciferase expression (Table 1).

Because transgenes often integrate into sites that do not permit expression (20), we considered the possibility that lack of neuronal expression might be the result of integration of the 249mGnRH-LUC transgene into a nonexpressing region of the mouse genome. To confirm that lack of neuronal expression reflected an inability of this promoter fragment to target neuronal luciferase, the anatomic pattern of luciferase expression was examined in the founders once they had produced adult offspring. With luciferase expression under the control of –249 bp of the mGnRH promoter, luciferase was not detected in the brain, but gonadal expression of luciferase was detected in four of the seven founders (nos. 15, 17, 97, and 108; data not shown). The ovaries from founder no. 11 could not be assessed because extensive intraabdominal adhesions made her ovaries unidentifiable. The finding of gonadal luciferase expression suggested that in these four founders, the –249mGnRH-LUC transgene had incorporated into a chromosomal site which permitted expression. Because founder no. 108 did not produce enough offspring for analysis, only offspring from founders nos. 15, 17, and 97 were examined in more detail.

As with the –356mGnRH-LUC mice, whole brain homogenates were obtained from neonatal mice. Luciferase activity, measured as corrected RLU, was examined in neonatal mice bearing the –249mGnRH-LUC transgene and compared with that seen in their wild-type littermates. As shown in Table 1, with luciferase expression under the control of the proximal 249 bp of the mGnRH promoter, luciferase activity in neonatal brain homogenates was not significantly different from the luciferase activity (33 ± 10 RLU) seen in their wild-type littermates.

As illustrated in Fig. 1B, a similar anatomic pattern of luciferase expression was found in offspring from all three founders that expressed the –249mGnRH-LUC transgene. Tissues from six male and five female offspring of each founder were examined. Results are shown as corrected RLU. As shown in Table 2, the luciferase activity in the hypothalamus did not differ from that seen in wild-type mice (3 ± 5 RLU). The –249 mGnRH-LUC mice had significantly higher luciferase activity in their testes (301 ± 88 RLU, 517 ± 118 RLU, 204 ± 68 RLU) and ovaries (494 ± 123 RLU, 3492 ± 759 RLU, 1156 ± 360 RLU) compared with wild-type mice. As with the –356 mGnRH-LUC mice, luciferase expression was not found in the other tissues. The luciferase activity in the other tissues did not differ from the levels seen in wild-type mice (P = 0.0.75).

The mGnRH Neuron-Specific Element, between –356 bp and –249 bp of the mGnRH Promoter, Contains Two Consensus Binding Sites for Otx2
Our data demonstrated that mice generated with –356 bp of the mGnRH promoter express neuronal luciferase, whereas mice generated with –249 bp of the mGnRH promoter do not. This would suggest that the sequences necessary to mediate neuronal mGnRH expression are contained between –356 and –249 bp. When this 107-bp region was examined closely (Fig. 2), sequences sharing homology with binding sites for the Otx2 homeodomain were identified. Otx2 has been shown to bind to the TAATCC core element with high affinity but has also been shown to bind with low affinity to TTATC (21). As shown in Fig. 2, the neuron-specific element of the mGnRH gene promoter contains the TTATC sequence at –319 bp and the TAATCC sequence at –257 bp.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Alignment of the mGnRH Neuron-Specific Element with rGnRH and hGnRH Gene Promoter Sequences The mGnRH gene promoter region between –356 to –249 bp, was aligned with sequences from the rGnRH (38 ) and hGnRH (1 ) GnRH gene using the alignment function of the Vector NTI Program (Suite7). The Otx2 binding sites are enclosed in boxes. *, Indicates –173 bp of therGnRH gene (10 ).

Otx2 Consensus Binding Sites within the mGnRH Promoter Bind Selectively to Otx2
The ability of the putative consensus sites within the neuron-specific element to bind Otx2 was examined using EMSAs. ³²P-radiolabeled probes containing the regions of the mGnRH promoter surrounding the putative Oxt2 consensus sites, –268 to –239 and –330 to –301, were generated (Fig. 3A). Additional probes, containing mutations in the Otx2 binding sites were also constructed; the putative low-affinity binding site TTAT (–319 to –316) was mutated to AAGC, and the putative high affinity binding site TAAT (–257 to –254) was mutated to GGCG.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Two Otx2 Binding Sites Are Present in the Proximal mGnRH Gene Promoter A, Probes used for the EMSAs. The mGnRH gene promoter sequences from –330 to –301 and –268 to –239 bp were used as probes. To eliminate binding of Otx2 to the putative binding sites, the core TAAT or TTAT sequences (indicated in boldface) were changed. B, Probes, containing the putative wild-type (WT) and mutated sequences, were incubated with in vitro-translated Otx2. Lanes 1–3 and 7–9 demonstrate increased intensity of binding with the addition of increasing quantities of in vitro-translated Otx2. In lanes 4–6 and 10–12, probes containing mutations in the binding sites were used, and no binding was observed, suggesting that mutations in the sites eliminate this binding. 3C. Unlabeled probes were used to compete for Otx2 binding. In lanes 2 and 8, Otx2 binding is seen with probes containing the wild-type sequence. In lanes 6 and 12, no binding was observed to probes containing mutations in the Otx2 binding site. In lanes 3 and 9, addition of excess cold probe, containing the wild-type sequence eliminated the radiolabeled DNA-protein interaction. In contrast, in lanes 4 and 10, addition of cold probe containing the mutated sequences had minimal effect.

To confirm the specificity of Otx2 binding, we used unlabeled probes to compete for Otx2 binding. As seen in Fig. 3C, Otx2 was again able to bind to both the probes containing the wild-type Otx2 consensus binding sites (lanes 2 and 8), whereas no binding was observed to probes containing mutations in the Otx2 binding site (lanes 6 and 12). Excess unlabeled probe containing the wild-type Otx2 binding sequence eliminated radiolabeled DNA-protein interaction (lanes 3 and 9). In contrast, addition of unlabeled probe containing the mutated Otx2 binding site had minimal effect (lanes 4 and 10).

Otx2 Expression Correlates with GnRH Expression
To determine whether Otx2 expression correlates with GnRH expression, Otx2 and GnRH mRNA expression was assessed in 2 GnRH-secreting cell lines. The GT1–7 cells secrete high levels of GnRH in a pulsatile fashion (22), whereas the GN11 cells secrete low levels of GnRH (23). Reverse transcription (RT) and PCR was performed with RNA from each cell line. A representative experiment, shown in Fig. 4, demonstrates that Otx2 mRNA was detected only in the GT1–7 cells, which also express relatively higher quantities of GnRH mRNA.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Otx2 mRNA Levels Correlate with GnRH mRNA Levels in Immortalized GnRH-Secreting Cells RNA was isolated from GN11 cells and GT1–7 cells. Reverse transcription was performed on 2 µg of total RNA, PCR was performed using 2 µl of the resulting cDNA. The specific primers to exon 1 and exon 3 of the GnRH gene would be expected to amplify a 194-bp fragment. An appropriate size band was detected in RT-PCR product from both the GN11 and GT1–7 cells, but the intensity of the band was lower in the GN11 cells, suggesting that GnRH transcripts are less abundant. The specific primers to exons 2 and 3 of the Otx2 gene would be expected to amplify a 561-bp fragment. An appropriate size band was detected only in RT-PCR product from the GT1–7 cells.

The mGnRH-luciferase reporter constructs were transfected along with expression vectors containing Otx2 or an empty pSG5 expression vector. For each mGnRH-LUC construct, the promoter activity seen with Otx2 overexpression was compared with that seen with empty expression vector, and is reported as fold change in promoter activity. The mGnRH-luciferase reporter constructs were evaluated in multiple, independent experiments. Each independent experiment was performed in triplicate. The mean of five representative experiments are included in this analysis. For each of the experiments, the coefficient of variation (CV) values were 20%. The values represent the mean ± SE of the fold change seen with Otx2 overexpression compared with empty expression vector.

As seen in Fig. 5, overexpression of Otx2 increased GnRH promoter activity in the constructs containing the wild-type Otx2 binding sites. In contrast, Otx2 overexpression did not have any effect on the activity of a minimal GnRH promoter, containing –176 bp. Compared with the GnRH promoter activity seen with the minimal promoter, overexpression of Otx2 resulted in a significant fold induction of GnRH promoter activity in the constructs, containing –1005 bp (P < 0.001) and –356 bp (P = 0.002). Promoter activity was increased 5.79 ± 1.31-fold with –1005 bp of the mGnRH promoter and 5.27 ± 1.48-fold with –356 bp of the promoter. With elimination of a single Otx2 binding site, overexpression of Otx2 still resulted in a significant fold induction of GnRH promoter activity compared with the –176-bp minimal promoter. Promoter activity increased 1.93 ± 0.29-fold with elimination of the Otx2 binding site at –319 to –316 bp (P = 0.011) and 2.32 ± 0.26-fold with elimination of the Otx2 binding site at –257 to –254 bp (P = 0.001). When both Otx2 binding sites are eliminated, however, the addition of Otx2 did not result in a significant increase in promoter activity (P = 0.136) compared with what was seen with the –176-bp minimal promoter.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Overexpression of Otx2 Increases mGnRH Promoter Activity Transient transfection studies were performed in GN11 cells, which had no detectable Otx2 by RT-PCR. The mGnRH-luciferase reporter constructs were transfected along with expression vectors containing Otx2 or an empty PSG5 expression vector. For each mGnRH-LUC construct, the promoter activity seen with Otx2 overexpression was compared with that seen with empty expression vector. Each independent experiment was performed in triplicate. The graph shows the mean ± SE of the fold change from five representative experiments. For each of the experiments, the CV values were 20%. Overexpression of Otx2 did not have any effect on the activity of a minimal mGnRH promoter, containing –176 bp. Compared with the mGnRH promoter activity seen with the minimal promoter, overexpression of Otx2 resulted in a significant fold induction of mGnRH promoter activity in the constructs containing either one or both of the wild-type Otx2 binding sites. No significant increase in promoter activity was seen when both Otx2 binding sites were eliminated. Mutation of either Otx2 binding site (–319 to –316 or –257 to –254) in the context of the –356mGnRH-Luciferase construct reduced the ability of Otx2 to increase GnRH promoter activity, and an even greater effect was seen when both Otx2 binding sites were eliminated. Significant differences from the –176-bp minimal promoter are indicated by the asterisks (*).

These data demonstrate that binding of Otx2 to specific sites in the mGnRH gene promoter is necessary to increase mGnRH expression, and that binding of Otx2 to both sites is necessary for maximal stimulation of the mGnRH promoter.

Elimination of Otx2 Binding Decreases Neuronal GnRH Promoter Activity in Vivo
To determine whether Otx2 binding mediates increases in GnRH promoter activity in vivo, transgenic mice were generated with mGnRH-LUC DNA constructs in which the Otx2 binding sites (–319 to –316 and –257 to –254) were mutated in the context of the –356-bp fragment of the mGnRH gene promoter (Otx2mut-mGnRH-LUC). These mutations were identical with the ones that eliminated Otx2 binding in the EMSAs. Southern blot analysis identified six separate founder mice that incorporated the luciferase transgene.

Examination of the adult offspring suggested that five of six lines were capable of expressing the luciferase transgene. As with the –249mGnRH-LUC mice, luciferase expression was present in the ovary, but not the hypothalamus. Ten female offspring were examined from each founder. Tissue homogenates were examined as for the –249 mGnRH-LUC and –356 mGnRH-LUC mice. As shown in Table 3, the ovarian luciferase activity in the mice bearing the Otx2mut-mGnRH-LUC transgene was significantly greater than that seen in wild-type mice in five of six lines, suggesting that these five lines (nos. 3, 5, 21, 45, and 48) were capable of expressing the luciferase transgene. Furthermore, in each of these lines, the corrected luciferase activity in the hypothalamus did not differ from the levels seen in wild-type mice (Table 3). As expected, in the other tissues, corrected levels of luciferase activity did not exceed that seen in wild-type mice (data not shown).

We also examined whole brain homogenates from neonatal offspring of these five founders, which were able to express the luciferase transgene (Table 4). In offspring from three of five founders (nos. 5, 21, and 45), the luciferase activity in the mice bearing the transgene was not different from the levels seen in their wild-type littermates. In two of five founders (nos. 3 and 48), significantly higher levels of luciferase activity was detected in the mice bearing the Otx2mut-mGnRH-LUC transgene compared with their wild-type siblings. The neuronal luciferase levels in these neonatal mice, however, were dramatically lower than the levels detected in the neonatal mice bearing the –356mGnRH-LUC transgene in which the Ox2 binding sites were intact. Even the Otx2mut-mGnRH-LUC mice from founder no. 48, which expressed neuronal luciferase at the highest level, had significantly lower levels of neuronal luciferase (315 ± 56 RLU) compared with the –356 mGnRH-LUC mice from founder no. 30, which expressed neuronal luciferase at the lowest level (12,056 ± 4489 RLU; P = 0.035). As expected, in neonatal offspring from the nonexpressing founder (no. 2), the luciferase activity in the brain homogenates in the pups incorporating the transgene (2 ± 6 RLU) did not differ from levels seen in their wild-type littermates (P = 0.15).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The Neuron-Specific Element for the in Vivo Expression of mGnRH is between –356 and –249 bp of the mGnRH Gene
In this study, we used various segments of the mGnRH promoter fused to the luciferase reporter gene as a marker for in vivo mGnRH gene expression. The luciferase gene is an extremely sensitive reporter, and because luciferase expression is under the control of the mGnRH promoter, the anatomic pattern and level of mGnRH gene promoter activity can be quantified easily by measuring the luciferase activity in tissue homogenates. Our previous studies (16) have demonstrated that DNA sequences contained within the proximal –1005 bp are sufficient for directing both neuronal and ovarian mGnRH expression.

In these studies, we have isolated further the sequences necessary for directing neuronal expression of mGnRH. We find that the –356/+28-bp 5' fragment of the mGnRH promoter targets neuronal expression of the luciferase transgene, whereas no neuronal luciferase expression is seen in the transgenic mice generated with the –249/+28-bp 5' fragment. Lack of neuronal luciferase expression in the seven separate founders that incorporated the –249mGnRH-LUC transgene would strongly argue that the proximal 249-bp fragment of the mGnRH promoter is not sufficient to target neuronal expression. Although transgenes often integrate at sites that do not permit expression (20), it is extremely unlikely that the lack of neuronal luciferase expression is due to integration into a repressive site. In our previous study, 54.2% (13/24) of the founder lines incorporating the luciferase transgene expressed luciferase (16). In the –356 mGnRH-LUC mice, 62.5% (5/8) of the founder lines, which incorporated the luciferase transgene, expressed luciferase. Given these rates of expression, the likelihood that lack of hypothalamic expression in the –249 mGnRH-LUC mice is due to integration into a repressive site would be much less than 1%. Additionally, the fact that gonadal luciferase expression is found in four of seven transgenic lines bearing –249 bp of the mGnRH promoter suggests that the chromosomal integration site or lower transgene copy number is unlikely to be the cause of the absent hypothalamic expression.

Our study in the mGnRH-LUC transgenic mice demonstrates that sequences contained between –356 and –249 bp of the mGnRH promoter target neuronal expression of mGnRH. Our findings differ from a transgenic mouse study by Pape et al. (24) in which the mGnRH promoter fused to a lacZ reporter localized the critical elements for expression of the mGnRH gene between –2.1 and –1.7 kb of the promoter. In these mice, deletion of mGnRH promoter sequences 5' to 1.7 kb resulted in a complete absence of detectable ß-galactosidase expression within the brain. Subsequently, they reported that the proximal 1.7 kb of GnRH gene was found to target transgene expression to approximately 20% of GnRH neurons when a nuclear localization signal was used (25). Presumably, concentration of ß-galactosidase in the nucleus allowed detection by immunocytochemistry. Compared with methods for detecting ß-galactosidase expression, the measurement of luciferase activity is a more sensitive method of determining and quantifying gene expression. As shown in Table 2, the proximal –356 bp of the mGnRH targeted luciferase to the hypothalamus in the –356mGnRH-LUC mice, but expression levels were lower (2052 ± 583 RLU) compared with the levels seen in the –2078mGnRH-LUC mice (7688 ± 551). The mGnRH promoter region between –2.1 and –1.7 kb, although not critical for targeting hypothalamic expression, may contain sequences essential for enhancing hypothalamic expression of GnRH.

Our previous studies suggested the presence of an enhancer for the in vivo expression of neuronal mGnRH, between –3446 and –2078 bp of the mGnRH gene. As shown in Table 2, the mGnRH promoter fragment containing –2078 bp of 5' sequence targeted luciferase transgene expression to the hypothalamus, but at lower levels than the transgenic mice generated with –3446 bp of mGnRH promoter (49,038 ± 4511 RLU). Previously we examined transgenic mice derived from eight different embryos bearing the –2078mGnRH-LUC transgene, and even in the transgenic line that expressed luciferase at the highest level, luciferase activity in the hypothalamus was lower than was seen in the transgenic mice bearing the –3446mGnRH-LUC transgene (16). In vitro studies, performed in the GT1–7 cells, corroborate our in vivo observations. Deletion analysis of the rGnRH gene promoter in the GT 1–7 cell line identified a neuron-specific enhancer, located between –1863 and –1571 bp (11). This enhancer region of the rat promoter shares 90% homology to the region of the mGnRH promoter located between –2384 and –2081 bp.

An in vivo study of the rGnRH gene generated transgenic mice with the two promoter regions that conferred cell-specific expression in transient transfections studies: the –173-bp proximal promoter along with the 300-bp enhancer region located 1.8 kb upstream from the transcription start site. Using a ß-galactosidase reporter, they demonstrated that these regions of the rGnRH gene are sufficient for appropriate expression of GnRH in the hypothalamus (26). Because the 300-bp upstream region alone did not target GnRH neurons appropriately, this study suggested that the proximal –173-bp regions of the rGnRH promoter contains the important sequences for directing neuron-specific expression of the GnRH gene. This study, however, did not specifically test whether the proximal –173-bp region of the rGnRH promoter was sufficient to direct hypothalamic expression.

The proximal –173-bp region of the rat promoter is highly conserved among the human, rat, and mouse genes, and approximately 80% of the nucleotides are identical (10). The numbering of the promoter, however, is quite different so that –173 bp of the rGnRH promoter is homologous to –278 bp of the mGnRH promoter (Fig. 2). In our studies, we found that the mGnRH promoter region proximal to –249 bp (–143 bp in the rat) was not able to direct neuronal luciferase expression in transgenic mice. It is possible that the 29-bp region between –278 and –249 bp of the mGnRH promoter, containing the proximal Otx2 binding site, is the neuron-specific element, but further studies are needed before this conclusion can be reached. In this paper, we have localized the sequences necessary for neuron-specific expression of the mGnRH gene to a 107-bp region, between –356 and –249 bp.

The mGnRH Neuron-Specific Element, between –356 bp and –249 bp of the mGnRH Promoter, Contains Two Functional Binding Sites for Otx2
Examination of the DNA sequences within the neuron-specific element, between –356 and –249 bp, identified sequences bearing strong homology to the binding site for the Drosophila transcription factor, bicoid (27). The Otx2 homeodomain is very similar to that of bicoid and can bind to the bicoid target sequence and activate gene transcription (28). Otx2 is the vertebrate homolog of otd, a Drosophila gene that is necessary for normal head development (17). Similarly, Otx2 is expressed in the most anterior part of the embryo that ultimately becomes the forebrain and midbrain (29). Knockout of Otx2 in transgenic mice have demonstrated that Otx2 expression is critical for rostral brain development, including the olfactory placode (30, 31, 32). Otx2 protein has previously been detected in GnRH neurons, both in the hypothalamus of adult mice (18) and migrating from the olfactory placode of embryonic mice (19), suggesting Otx2 may have a role in GnRH expression.

Otx2 has been shown to bind to the TAATCC core element with high affinity but has also been shown to bind with low affinity to TTATC (21). Our study has identified a neuron-specific region of the mGnRH gene promoter between –356 and –249 bp. This region of the promoter contains both a low affinity binding site, TTATC (–319 to –315 bp), and a proximal high affinity binding site, TAATCC (–257 to –252 bp). As shown in Fig. 2, the high affinity binding site is conserved in the rat and human promoter and is also contained within the proximal 173 bp of the rGnRH promoter that was found by Lawson et al. (26) to mediate appropriate expression of GnRH in the hypothalamus. The low affinity binding site is located distally and is also found to be conserved between rat and mouse in our alignment. A previous study, using used an older source for the rat gene sequence (33) and a different alignment strategy, did not identify the distal Otx2 binding site in the rat promoter (10).

Our studies suggest that maximal stimulation of mGnRH gene expression requires that both sites are bound by Otx2. The EMSAs demonstrate that Otx2 binds both consensus sites specifically. Functional studies, using the GN11 cells, demonstrated that Otx2 transactivates the mGnRH gene promoter. The introduction of mutations to eliminate either of the Otx2 binding sites reduced the ability of Otx2 to increase mGnRH promoter activity. These data demonstrate that maximal stimulation of mGnRH transcriptional activity by Otx2 requires both Otx2 binding sites to be intact.

Our findings are consistent with previous work performed with the rGnRH gene promoter (18). This group examined the proximal Otx2 binding site in the rGnRH gene promoter and demonstrated that mutation of this site decreased basal transcriptional activity in GT1–7 cells. When Otx2 was overexpressed in GT1–7 cells, a 50% increase in reporter gene activity was seen. This increase was modest compared with the over 5-fold increase seen in our study using GN11 cells, presumably because GT1–7 cells contain endogenous Otx2, whereas GN11 cells do not. Additionally, the reporter construct used by Kelley et al. (18) contained only the proximal Otx2 binding site. Perhaps, a larger increase in transcriptional activity would have been seen with a construct that contained both Otx2 binding sites.

Otx2 Binding to the mGnRH Gene Promoter Increases GnRH Expression and Mediates Neuron-Specific GnRH Expression
We have shown by RT-PCR that Otx2 is present in the GT1–7 cells, whereas it was not detected in the GN11 cells. The GN11 cell line appears to have a phenotype resembling a migrating GnRH neuron with low levels of GnRH secretion, whereas the GT1–7 cells resemble a postmigratory neuron, which secretes high levels of GnRH in a pulsatile fashion (8, 9). On embryonic d 11.5 (E11.5), GnRH mRNA and protein are first detected in neuronal cells in the olfactory pit (3). GnRH neurons then migrate from the olfactory placode to the forebrain, and an adult distribution of GnRH neurons is seen by E16.5 (3). Interestingly, Otx2 protein has been described in the olfactory place of E10 embryos and has also been detected in migrating GnRH neurons (19). Our data, along with evidence from the literature, indicate that the increasing expression of Otx2 may be a critical step in the development of a GnRH neuron.

Previous in vitro findings suggested that Otx2 contributes to the neuron-specific transcription of the GnRH gene in GT1–7 cells (18). Kelley et al. demonstrated that mutation of the proximal Otx2 binding site decreased transcriptional activity in the GT1–7 cells, but not in NIH3T3 cells. Our observations in vivo are consistent with these findings. Mice generated with the –356/+28-bp 5' fragment of the mGnRH promoter targeted neuronal expression of the luciferase transgene, whereas no neuronal luciferase expression was seen in the transgenic mice generated with the –249/+28-bp 5' fragment. The critical region of the mGnRH gene promoter between –356 and –249 bp contains two functional binding sites for Otx2, and elimination of these binding sites dramatically reduced mGnRH promoter activity. These data suggest that Otx2 contributes to neuron-specific expression of mGnRH. Ovarian luciferase expression, however, was detected in mice, bearing the –249 mGnRH-LUC and Otx2mut-mGnRH-LUC transgenes, indicating that Otx2 is not critical for expression of the GnRH gene in the ovary.

Interestingly, in the –356mGnRH-LUC mice, luciferase activity seen in the neonatal (2–8 d old) brain was 10- to 15-fold higher than adult levels. Because we found that the luciferase activity in the whole brain homogenates from the adults did not differ from the activity seen in the hypothalamic homogenates, we are confident that all the luciferase activity in the adult brain is from the hypothalamic region. The finding of higher luciferase activity in the neonatal brain may actually reflect an embryological pattern of GnRH gene expression. Transgenic mice bearing a GnRH-lacZ reporter construct were found also found to express low levels of ß-galactosidase in extrahypothalamic regions of the brain, and further study demonstrated GnRH expression in these nonhypothalamic regions during normal embryological development (34). Others have also postulated that GnRH neurons originate from multiple embryologic sites in the brain (35).

Alternatively, the high neonatal luciferase levels may be reflective of the high GnRH levels seen with neonatal activation of the central reproductive axis. These data suggest that different regions of the promoter may mediate GnRH expression during development. Otx2, and/or other specific proteins present in the neonatal GnRH neuron that interact with sequences contained in the proximal –356 bp of the mGnRH promoter, may be implicated in mediating the neonatal surge of GnRH. In the adult brain, however, appropriate regulation of GnRH may require more distal sequences of the mGnRH promoter. Additional study will be needed to elucidate the mGnRH sequences that are critical for the appropriate expression of GnRH during development.

In conclusion, our study has further defined the promoter sequences necessary for mediating tissue-specific expression of mGnRH in vivo. Previously, we developed a model of tissue-specific expression of neuronal and ovarian GnRH expression (16). Our previous studies suggested the presence of an enhancer for the in vivo expression of neuronal mGnRH, between –3446 and –2078 bp of the mGnRH gene and also identified a region of the mGnRH promoter, between –3446 and –2078 bp, that appear to mediate repression of GnRH expression in the ovary. With our present study, we have updated our model of tissue-specific GnRH expression (Fig. 6). Our results clearly demonstrate that the DNA sequences contained between –356 and –249 bp of the mGnRH promoter direct neuronal mGnRH expression. In contrast, the sequences necessary to direct mGnRH expression to the ovary are contained within the proximal 249 bp of the promoter.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Model of Tissue-Specific Expression of Neuronal and Ovarian GnRH This model represents our understanding of tissue-specific expression of neuronal and ovarian GnRH. Our data demonstrate that the DNA sequences contained between –356 and –249 bp of the mGnRH promoter are sufficient to direct mGnRH gene expression to the neuron. In contrast, the sequences in the proximal –249 bp of the mGnRH promoter were found to be sufficient to direct ovarian, but not neuronal, expression. Our previous data (16 ) also support the presence of an enhancer region for the in vivo expression of neuronal mGnRH in the promoter region between –3446 and –2078 bp. In the ovary, our previous data suggest that an ovarian GnRH repressor element may be located in the distal region of the mGnRH promoter between –3446 and –2078 bp. Thus, we speculate that in the hypothalamus, specific enhancer proteins interact with the enhancer region to increase mGnRH expression and that repressor proteins in the ovary may normally interact with this ovarian GnRH repressor element to repress ovarian GnRH expression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Chemicals and Reagents
Unless otherwise indicated, all chemicals and reagents were obtained from Sigma, Inc. (St. Louis, MO). Restriction enzymes were obtained from New England Biolabs (Beverly, MA) unless otherwise specified.

Construction of the mGnRH Promoter-Luciferase DNA Constructs
A GnRH promoter-luciferase construct containing –3446 bp to +23 bp of the mGnRH promoter fused to pSV0aL5' luciferase was kindly provided by Dr. Donald B. DeFranco (University of Pittsburgh, Pittsburgh, PA). The –1005/+28, –356/+28-, –249/+28-, and –176/+28-bp mGnRH-luciferase DNA constructs were constructed as a HindIII fragment using the PCR and the –3446/+23 mGnRH-LUC construct as template. PCR was performed in a thermocycler (GeneAmp PCR System 9600; PerkinElmer, Foster City, CA), and reaction mixtures contained 5 U Taq polymerase (DisplayTaq; Display Systems Biotech, Vista, CA), and 0.5 mM deoxynucleotide triphosphates. The PCR product was restriction enzyme digested with HindIII and ligated into a HindIII linearized, alkaline phosphatase (Roche Molecular Biochemicals, Indianapolis, IN) dephosphorylated pA3LUC reporter vector (36, 37). Orientation was checked by sequencing constructs using a primer annealing to the 5' end of the luciferase gene.

Construction and Identification of Transgenic Animals
Pvu1 digestion was used to isolate the mGnRH-LUC transgene from the surrounding plasmid. The resulting DNA was electrophoresed, and the linear mGnRH-LUC DNA fragment was excised from the gel and isolated. Transgenic animals were constructed by the Beth Israel Transgenic Facility and The University of Chicago Transgenic Mouse/Embryonic Stem Cell Facility by pronuclear injection. Fertilized mouse oocytes from FVB-N mice were injected with the purified linear mGnRH-LUC DNA fragment. The resulting embryos were transferred into pseudo-pregnant foster mothers. Transgenic animals were identified with Southern blot analysis as described previously (14). Briefly, DNA was isolated from tail snips, restriction enzyme digested with EcoRI, and separated with gel electrophoresis. DNA was then transferred to GeneScreen Plus hybridization transfer membrane (NEN, Boston, MA). A ³²P-labeled 1.2-kb probe for luciferase was used to detect transgenic animals that incorporated the luciferase transgene. For identification of luciferase-expressing transgenic lines, neonatal brains were removed from pups, homogenized, and assayed for the mGnRH-LUC transgene as described below.

Assay of mGnRH-Luciferase Transgene
All procedures were carried out in accordance with the Institutional Animal Care and Use Committee of The University of Chicago.

From adult mice, the olfactory and hypothalamic tissue was dissected in a single fragment consisting of tissue from 1 mm caudal to the mammillary bodies, 1 mm laterally beyond the lateral aspect of the median eminence, and 3 mm dorsally. Small representative sections were taken from the remaining tissues. In the case of the gonads, entire gonads were used due to their small size. Similarly, for examination of neonatal luciferase expression, the entire brain was removed from 2- to 8-d-old pups.

Tissues were placed in 1 ml of lysis buffer (25 mM glycylglycine, 15 mM MgSO₄, 4 mM EGTA, 1% Triton X-100, and 1 mM dithiothreitol), and homogenized with a Polytron tissue homogenizer (Brinkmann Instruments Inc., Westbury, NY). The homogenate was centrifuged at 15,000 x g, and the supernatant was assayed for luciferase activity. Luciferase activity was assayed using a Lumat LB 9507 luminometer (Berthold Systems Inc., Pittsburgh, PA). Samples were injected with 100 µl of 0.75 mM luciferin (Molecular Probes, Eugene, OR), dissolved in lysis buffer; and 100 µl assay buffer (25 mM glycylgycine, 15 mM MgSO₄, 4 mM EGTA, 15 mM KPO₄, 3 mM dithiothreitol, and 3 mM ATP) and luminescence was measured for 20 sec as RLU.

Because the background RLU (the RLU measured with lysis buffer alone) varied from day to day, we corrected for the background in each experiment. On each day, the luciferase activity is measured in several samples of lysis buffer alone and averaged to obtain a background RLU level for the day. This background value is subtracted from the measured tissue RLU to give a corrected RLU. For this reason, it is possible to obtain negative values in some cases. The corrected RLU reflected the luciferase activity of the tissue. A tissue was defined to express luciferase if the luciferase activity detected in the tissue was significantly greater than that found in wild-type mice. Alternatively, luciferase was defined as being nonexpressed if the luciferase activity in the transgenic mice was not significantly different than that found in their wild-type littermates. Two-tailed P values (assuming unequal variance) less than 0.05 were considered statistically significant.

Statistical Methods
Results are expressed as the mean ± SE. Statistical analysis was performed in collaboration with The Biostatistics Laboratory of the Department of Health Studies of The University of Chicago. For analysis of the anatomic data, group means were compared using Student’s t test. Two-tailed P values (assuming unequal variance) less than 0.05 were considered statistically significant. Student’s t test was performed using Microsoft Excel.

For analysis of the pups, the transgenic mice for each founder were compared with their wild-type littermates. For analysis of the adults, the luciferase activity in the organs obtained from transgenic mice was compared with that obtained from wild-type mice. For transient transfection data, the data were analyzed on a log scale using one-way ANOVA, followed by multiple pair-wise comparisons using the Student’s t test.

Alignment of Mouse, Rat, and Human Sequences
The sequences of the GnRH promoter were obtained from GenBank using the following accession numbers: mouse (U29674) (Dong, K. W., Z. W. Zeng, M. Jakubowski, and J. L. Roberts, 1995, Extensive homology between the rat and mouse GnRH promoters; GenBank, direct submission), rat (X62651) (Ref. 38), and human (M34091) (Ref. 1). Using the alignment function of the Vector NTI Program (Suite 7), with the –356/+28-bp fragment of the mGnRH gene promoter as the profile, the proximal fragments of the mGnRH, rGnRH, and hGnRH gene promoters were aligned. The rat sequence, homologous to the mGnRH promoter region between –306 to –281 bp, contained many gaps and the alignment was adjusted to achieve maximal homology.

Isolation and Analysis of RNA
RNA was isolated from both cells types using the TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the protocol provided by the supplier. RT was performed on 2 µg of total RNA per sample using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Two different preparations of RNA were tested from each cell line. PCR was performed five times using 2 µl of the resulting cDNA from three separate RT reactions. For detection of Otx2, primers were designed to anneal to exon 2 (5'-GGCACTGAAAATCAACTTGC-3') and exon 3 (5'-TCCAAGCAATCAGTGGTTGA-3'). These primers have been previously shown to amplify a 561-bp fragment (39). For the amplification of mGnRH, primers were designed to anneal to exon 1 (5'-CCCTTTGACTTTCACATCC-3') and exon 3 (5'-GGTTCTGCCATTTGATCCAC-3') and would be expected to amplify a 194-bp fragment. Thermocycler temperatures settings used were 94 C for 5 min, followed by 35 cycles of 94 C for 30 sec, 58 C for 30 sec and 72 C for 30 sec. A final extension at 72 C for 7 min was then performed. The products were separated by electrophoresis on a 2.5% agarose gel.

Site-Directed Mutagenesis
The QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to introduce mutations into the –356 mGnRH-LUC promoter construct per the manufacturer’s instructions. The sequences of the two Otx2 consensus binding sites were changed to eliminate Otx2 binding to the mGnRH gene promoter. The TTAT sequences, at –319 to –316 bp, were mutated to AAGC, introducing a StuI restriction enzyme site, and generating the –319/316 mutant (MUT). The sequences at –257 to –254, TAAT, were mutated to GGCG, introducing a NarI restriction enzyme site, and generating the 257/254 MUT. The double Otx2 MUT construct contains mutations in both of the Otx2 binding site in the context of the –356mGnRH-LUC reporter construct. The presence of the mutations was confirmed both by restriction enzyme digest and sequencing analysis.

Construction of Otx2 Protein Expression Vectors and in Vitro Translation of Protein
The Otx2 cDNA was obtained from Dr. Siew-Lan Ang (Mount Sinai Hospital, Toronto, Ontario, Canada), and subcloned into the BamHI/EcoRI site of the pSG5 expression vector (Stratagene). Otx2 was in vitro-translated using a coupled transcription/ translation system in reticulocyte lysate (Promega, Madison, WI) with T7 polymerase.

Cell Culture
GN11 cells were maintained at 37 C and 5% CO₂ in DMEM with high glucose, L-glutamine and phenol red (Cellgro, Herndon, VA) and was supplemented with 7% fetal calf serum (BioWhittaker, Inc., Walkersville, MD), 3% newborn calf serum (BioWhittaker) and antibiotic-antimytotic (Invitrogen). GT1–7 cells were maintained similarly to the GN11 cells except that media was supplemented with 10% heat inactivated fetal calf serum.

Transient Transfections and Assay of Luciferase Activity
GN11 cells were grown in 175-cm² Falcon tissue culture flasks (Becton Dickenson, Bedford, MA) until nearly confluent. For transient transfection, cells were passed into 35-mm six-well tissue culture dishes. Lipofectamine (Invitrogen) was used to transfect 0.2 µg of Otx2 or empty pSG5 expression vector along with 0.2 µg of the appropriate mGnRH-luciferase reporter constructs (–1005/+28, –356/+28, –319/316 MUT, –257/254 MUT, double Otx2 MUT, –176/+28). A dual luciferase assay (Dual Luciferase Assay System; Promega), using a thymidine kinase-Renilla luciferase (0.02 µg) reporter plasmid, was performed to normalize for transfection efficiency and cell number.

Luciferase activity was assayed in a luminometer (Lumat LB 9507; Berthold). Tubes containing 100 µl of lysate were injected with 100 µl of LARII reagent. After a 2-sec delay, luminescence was measured for 10 sec as RLU 1. Samples were then injected with 100 µl of Stop & Glo reagent (Promega). After a 2-sec delay, luminescence was measured for 10 sec as RLU 2. Each experiment was performed in triplicate, and the mean relative luminescence (RLU1/RLU2) was calculated. The CV values were always 20% or less. The relative luminescence seen with overexpression of Otx2 was compared with that seen with empty pSG5 vector. A total of five experiments were performed for each mGnRH-luciferase reporter constructs. The data were analyzed on a log scale using one way ANOVA (P < 0.001), followed by pair-wise comparison of groups using the Student’s t test.

EMSA
The ability of the putative consensus sites within the neuron-specific element to bind Otx2 was examined using EMSAs. ³²P-radiolabeled probes containing the regions of the mGnRH promoter surrounding the putative Oxt2 consensus sites, –268 to –239 and –330 to –301 were generated (Fig. 3A). Additional probes containing mutations in the Otx2 binding sites were also constructed; the putative low affinity binding site TTAT (–319 to –316) was mutated to AAGC, and the putative high-affinity binding site TAAT (–257 to –254) was mutated to GGCG. The ³²P-labeled probes were column purified (G50 Sephadex columns; Roche).

For each EMSA, in vitro-translated proteins were mixed with radiolabeled probe for 20 min at room temperature, along with deoxyinosine-deoxycytosine, salmon sperm, and binding buffer [50 mM KCl, 20% glycerol, 20 mM HEPES (pH 7.6–7.8)]. Each sample was then separated by gel electrophoresis on a 5% nondenaturing acrylamide gel, and analyzed by autoradiography. To confirm the specificity of Otx2 binding, excess unlabeled probes were also used to compete for Otx2 binding.

	ACKNOWLEDGMENTS

 
We would like to thank Donald B. DeFranco for providing the mGnRH promoter luciferase vector, Siew-Lan Ang for providing the Otx2 cDNA, and Pamela L. Mellon for the GT1–7 cells. Additionally, we thank Theodore Karrison, Ph.D., for his expertise in statistical methods. Finally, we thank Robyn M. Deneau and Alexis Baria for their excellent technical assistance, and Gail Isenberg and Heidi Vanesky for their assistance preparing this manuscript.

	FOOTNOTES

 
This work was supported by grants from Burroughs Wellcome Fund (to H.K.), National Institutes of Health (NIH) K08 HD43222 (to H.K.), NIH RO1 HD 34551 (to S.R.), U54 HD41859-01 (to S.R.), and Howard Hughes Institute Undergraduate Education Initiative Grant at The University of Chicago (to A.P.). H.H.K. has previously been funded by the Reproductive Scientist Development Program.

Current address for S. R.: Division of Endocrinology, Department of Pediatrics, The Johns Hopkins Medical Institutes, Baltimore, Maryland 21287.

First Published Online November 9, 2006

Abbreviations: CV, Coefficient of variation; E, embryonic day; LUC, luciferase; mGnRH, mouse GnRH; MUT, mutant; hGnRH, human GnRH; rGnRH, rat GnRH; RLU, relative light units; RT, reverse transcription; WT, wild type.

Received for publication May 30, 2005. Accepted for publication November 1, 2006.

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