Identification of a Discrete Promoter Region of the Human GnRH Gene That Is Sufficient for Directing Neuron-Specific Expression: A Role for POU Homeodomain Transcription Factors

Andrew Wolfe, Helen H. Kim, Stuart Tobet, Diane E. J. Stafford and Sally Radovick

Department of Pediatrics (A.W., H.H.K., S.R.), University of Chicago, Chicago, Illinois 60637; The Shriver Center (S.T.), Waltham, Massachusetts 02452; and Department of Medicine (D.E.J.S.), Children’s Hospital, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Dr. Andrew Wolfe, The University of Chicago, Department of Pediatrics, 5839 South Maryland Avenue MC5053, Chicago, Illinois 60637. E-mail: awolfe{at}peds.bsd.uchicago.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human GnRH (hGnRH) gene is expressed, and the GnRH decapeptide produced, primarily in the GnRH neurons of the diencephalon. The molecular elements important for the cell-specific expression and regulation of the hGnRH gene are not well established at this time; therefore, we have used a transgenic mouse model to isolate cis-regulatory elements important for directing gene expression to GnRH neurons in the hypothalamus. Gene constructs consisting of various promoter deletion fragments of the hGnRH gene fused to the luciferase (LUC) reporter gene have been used to create transgenic mouse lines. Cell-specific expression, with the criterion being luciferase expression directed to GnRH neurons of the hypothalamus, was observed when 992 bp, but not 795 bp, of the hGnRH gene promoter were used. Tissue-specific expression was also observed when a deletion construct containing the region from -992 to -763 was fused to a minimal 48-bp promoter fragment fused to LUC. These data indicate that the region between -992 and -795 contains elements both essential and sufficient for targeting gene expression to GnRH neurons. This promoter region was found to contain two DNA-binding sites for the POU class of transcription factors, each of which specifically interacted with the POU homeodomain proteins Brn-2 and Oct-1. Functional studies demonstrated that Brn-2 increased promoter activity of the human and mouse GnRH genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH IS A decapeptide released from the hypothalamus that regulates the synthesis and release of the pituitary gonadotropins. Therefore, the precise coordinated release and expression of GnRH is essential for a functioning mammalian reproductive system. In the mouse, the vast majority of GnRH-containing neurons are located in the basal hypothalamus and septum from the level of the organum vasculosum of the lamina terminalis (OVLT) to the preoptic area (POA), but some GnRH-containing neurons have also been localized in the cerebral cortex, the limbic system, and the olfactory bulbs (1, 2). A similar anatomical organization is seen in all mammals (3). Low levels of GnRH immunoreactivity have also been localized in the placenta, the gonads, and the mammary glands (4, 5). Differential splicing of the human GnRH (hGnRH) gene has been observed in the hypothalamus and in reproductive tissues. The hypothalamic variant has the second exon excised and can therefore not produce GnRH peptide (6). Interestingly, in reproductive tissues such as the mammary gland, and the placenta, the GnRH gene is transcribed from a different promoter initiation site than that used for transcription of the gene in the hypothalamus (7). In addition, the placental variant has the first intron retained, resulting in a large first exon (7, 8). The significance of the extrahypothalamic expression of GnRH, or what factors in the hypothalamus and the placenta direct the differential expression of the splicing of the GnRH gene, is unclear.

To determine the cis-regulatory elements important for directing the hypothalamic cell-specific expression of GnRH, we have created transgenic animals containing gene constructs consisting of various hGnRH promoter deletion constructs fused to the luciferase reporter gene (9). We have previously localized a cell-specific element between -1,131 and -484 bp of the hGnRH gene (9). Here we report a further localization of the promoter region essential for directing cell- specific expression of GnRH to between -992 and -795 of the hGnRH gene. Examination of the DNA sequence within the cell-specific element, between -992 and -795, identified two octamer sites specifically identified as Brn-2 consensus binding sites (10). Brn-2 is a member of the POU homeodomain family of transcription factors, is expressed in the hypothalamus, olfactory tissues, and the septum (11), and has been shown to be important for cell-specific development in another system (12, 13). Oct-1 is another POU homeodomain protein that has been shown to be ubiquitously expressed. It has been reported that Oct-1 may play an important role in the regulation of rat GnRH gene expression (14, 15). Consequently, we provide structural and anatomical evidence that Brn-2 or a Brn-2 related protein may play a role in the regulation of hGnRH gene expression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Delineation of the hGnRH Promoter Elements Essential for Targeting Expression to GnRH-Expressing Neurons
Transgenic animal lines have been created that contain hGnRH promoter fragments fused to the luciferase reporter gene. The promoter fragments were -992 to +5 bp, -795 to +5 bp, or -992 to -763 fused to a minimal 48-bp hGnRH promoter fragment. These animal lines are referred to as -992/+5LUC, -795/+5LUC, and {Delta}-992/+5LUC, respectively. Tissue panels obtained from -992/+5LUC animals (Fig. 1Go, A and B) display relative light units (RLU) as a measure of luciferase levels in various brain and peripheral tissues. Relatively high luciferase levels of 2,200 RLU were observed in the hypothalamus of adult mice from founder 26 relative to all other tissues examined (average of four adult animals for all tissues except hypothalamic values, which are the average of six adult animals; Fig. 1AGo). Higher levels (5,375 RLU) were observed in the hypothalami of adult animals from founder 64 with moderate levels seen in the olfactory bulbs and the cortex (average of three adult animals for all tissues except hypothalamic and olfactory values, which are the average of 16 adult animals; Fig. 1BGo). Very low or background levels of luciferase were observed in all other tissues examined from both of these animal lines. In fact, hypothalamic expression of luciferase was always at least 12 times higher than that observed in all tissues but the olfactory bulbs and the cortex.



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Figure 1. Luciferase Activity Tissue Panels

A, Relative luciferase activity (shown as RLU) of tissues taken from transgenic mice containing the -992/+5LUC transgene. All tissue levels are the mean ± SE of four adult animals from transgenic line 26 except the hypothalamic values, which are the means ± SE of six mice. B, Relative luciferase activity (RLU) of tissues taken from transgenic mice containing the -992/+5LUC transgene. All tissue levels are the mean ± SE of three adult animals from transgenic line 64 except the hypothalamic and olfactory values, which are the means ± SE of 16 mice. C, Averaged data from three tissue panels used in Fig. 1BGo where the values are corrected for protein levels by dividing the RLU by milligrams of protein.

 
To account for differences in tissue amounts, RLU values of some tissue panels were corrected for protein level and, as shown in Fig. 1CGo, did not qualitatively change our results. Therefore, the data are primarily shown as uncorrected RLU because the larger amount of tissue needed to obtain the scattered GnRH neurons in the brain tends to under represent the higher luciferase levels measured in the hypothalamus.

A tissue panel from a representative -795/+5LUC animal, shown in Fig. 2Go, indicates that this promoter deletion fragment does not express luciferase in any tissue examined. Because the site of integration can affect transgene expression, multiple -795/+5LUC founders were examined. More than 80 mice from 8 different founders containing the -795/+5LUC transgene had no detectable transgene expression in any tissue examined (data not shown). A deletion was made between -763 and -48 to ascertain whether the fragment from -992 to -763 was essential for cell-specific expression. Relatively high levels of luciferase activity were observed in the hypothalamus and olfactory bulbs of the {Delta}-992/+5LUC animals when compared with all other tissue examined (eight animals from three separate founders examined) (Fig. 3Go). The hypothalamic luciferase levels in the {Delta}-992/+5LUC animals were lower than the levels seen in the -992/+5LUC animals (a mean of 1,500 RLU for mice from three founder {Delta}-992/+5LUC mice, and a mean of 3,750 RLU for mice from two founder -992/+5LUC mice). This may be due to the deletion of one of the CAAT boxes located in the proximal promoter of the hGnRH gene (8, 16). However, these mice had the same hypothalamic-specific expression pattern seen in the -992/+5LUC mice. A summary of the number of lines and animals, and of the relative luciferase expression in tissues from the three mouse lines, is summarized in Table 1Go.



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Figure 2. Relative Luciferase Activity of Tissues Taken from a Representative Transgenic Mouse Containing the -795/+5LUC Transgene (One of Eight Founders Examined)

 


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Figure 3. Relative Luciferase Activity of Tissues Taken from a Representative Transgenic Mouse Containing the {Delta}-992/+5LUC Transgene (One of Three Positive Founders Examined)

The hypothalamic and olfactory values are the average of animals from all three founders ± SE.

 

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Table 1. Summary of Transgenic Mouse Data

 
To confirm that luciferase expression was confined to GnRH neurons in the -992/+5LUC animals, double fluorescence immunohistochemistry was performed on brain sections of gonadectomized male mice. Figure 4Go shows a neuron exhibiting red fluorescence specific for GnRH peptide (Fig. 4AGo), green fluorescence specific for luciferase peptide (Fig. 4BGo), and an overlay of these two figures with colocalization of GnRH and luciferase appearing yellow (Fig. 4CGo). Fewer than 50% of GnRH neurons contained measurable luciferase peptide. This may be due to the relatively low levels of luciferase in these mice compared with previous lines containing longer promoter constructs and may be indicative of a lack of complete colocalization or a limit of the sensitivity of our luciferase immunohistochemistry. Luciferase protein was not observed in non-GnRH-containing neurons.



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Figure 4. Double Fluorescence Histochemistry of Mouse Brain Sections

Shown is a neuron containing GnRH peptide (shown as red in panel A), luciferase protein (shown as green in panel B). Panel C is an overlay of panels A and B showing colocalization of the two proteins. Cy3 (red) and Alexa Fluoro 488 (green) were visualized using the appropriate filters.

 
Sequence Analysis of Cell-Specific Element Indicates a Cluster of Brn-2 Consensus Binding Sites
An analysis of the DNA sequence of the hGnRH promoter between -1,131 and -700 bp identified two POU protein DNA-binding sites (Fig. 5BGo), one at -925/-916 bp and one at -867/-858 bp indicated as underlined sequence. The identified sites corresponded specifically to a Brn-2 consensus binding site [C/AATnA/TAAA/T where n = 0, 2, or 3 (10)]. Two other Brn-2 consensus binding sites are located proximally to -763, at -148 to -142 and at -111 to -102. There was also an Oct-1 consensus binding site (17) located proximal to -763 at -742 to -735 bp, which is indicated as boldface text on the sequence. A site with homology to the consensus binding site for the STAT (signal transducer and activator of transcription) family of transcription factors (18) was identified between -984 and -976 and is indicated in Fig. 5BGo. No other sites of interest were noted when the sequence was analyzed by the TRANSFAC database (19).



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Figure 5. Protein Interactions with the hGnRH Promoter

A, DNase I footprinting of the hGnRH cell-specific element. A DNA fragment containing -1,131 to -830 was labeled at one end with P32. Radiolabeled probe was incubated with 2 or 8 µg of nuclear extract from NLT cells, Gn11 cells, or 8 µg BSA. Binding reactions were then digested with DNase I (1 ng/tube) for 1 min. DNA was then phenol/chloroform purified, precipitated, and resolved on a 6% acrylamide gel. Regions protected from DNase I digestion are noted at right. Ten-fold excess cold competitor DNA eliminated the footprinted regions (arrow). B, DNA sequence of the hGnRH promoter fragment from -1,131 to -652 upstream of the transcriptional start site. Underlined sequence indicates consensus Brn-2 binding sites [C/AATnA/TAAA/T where n = 0, 2, or 3 (10 )]. An Oct-1 consensus binding site is indicated by the shaded text. A partial consensus binding site for STAT is indicated by the dashed underlined sequence. Arrows delineate the region identified as containing the cis-regulatory elements sufficient and essential for tissue-specific GnRH gene expression.

 
Deoxyribonuclease I (DNase I) Footprint Analysis of the Cell-Specific Element Identified Regions Interacted with Proteins Found in GnRH Neuronal Cell Lines
To identify regions within this promoter segment that interact with GnRH neuronal transcription factors, DNase I footprint analysis was performed (Fig. 5AGo). A single end-labeled probe consisting of the hGnRH promoter fragment from -1,131 to -830 was incubated with nuclear extract obtained from Gn11 or NLT cells. These are two GnRH neuronal cell lines developed in our laboratory. The Gn11 cells have been shown to express low levels of GnRH, whereas the NLT cells have been shown to express relatively high levels of GnRH (6). This probe region was chosen to include the homeodomain consensus binding sites described above and flanking regions. Indicated by the black bars to the right are four regions in which binding interactions were observed when incubated with nuclear extract from NLT cells: from -1,060/-1,040, -975/-930, -922/-907, and -870/-855. These binding interactions were abolished when excess cold competitor was added to the samples. The two most 3'-footprinted regions overlapped the Brn-2 consensus binding sites at -925/-916 and at -867/-858, respectively.

Gel-Mobility Shift Analysis Indicates that Brn-2 Consensus Binding Sites Within the hGnRH Promoter Bind Selectively to in Vitro-Translated Brn-2 and to Binding Elements Present in the Nuclear Extract from GnRH Neuronal Cell Lines
The interaction of the Brn-2 consensus sites within the promoter region of the hGnRH gene with Brn-2 and nuclear extracts from GnRH neuronal cell lines was examined using gel-mobility shift assays. A probe consisting of the region of the hGnRH promoter that contains the Brn-2 consensus sites, -935/-906 (Brn-2 consensus binding site, -925/-916) was used. In Fig. 6Go, a -935/-906 probe was incubated with nuclear extract from our Gn11 and NLT cell lines. Protein elements present in NLT cells strongly interacted with the probe. Protein elements present in Gn11 cells interacted only weakly with this probe. When a -935/-906 probe containing a functional and binding mutation of the Brn-2 consensus (10) was used (indicated by Mut under lane), only a minimal protein DNA interaction was observed with nuclear extract from NLT cells (Fig. 6Go). Interestingly, the relative size of the complex formed by association with NLT nuclear extract was larger than was observed when Brn-2 alone was used (Brn-2 lane vs. NLT lane in Fig. 6Go). Figures 6Go and 7Go show that Brn-2 alone complex formation is interfered with by the addition of a Brn-2 antibody. Neither a Brn-2 antibody nor an Oct-1 antibody interferes with the formation of the complex formed when the probe was incubated with nuclear extract from NLT cells (Fig. 6Go).



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Figure 6. Gel-Mobility Shift Assay of Nuclear Extract from Gn11 and NLT Cells with Oligonucleotides Complimentary to the hGnRH Promoter Region Between -953/-911 bp Containing the Brn-2 Consensus Site at -925/-916

Two shifted complexes are noted (left arrows) when probes are incubated with the NLT nuclear extract, and much weaker interactions are observed when incubated with GN11 nuclear extract. No complex was noted when NLT nuclear extract was incubated with a mutant Brn-2 radiolabeled probe (Mut). A complex formed with in vitro-transcribed and -translated Brn-2 is shown in the second lane (Brn-2). RL, Reticulocyte lysates; Ab, antibody.

 


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Figure 7. Gel-Mobility Shift Assay of in Vitro-Transcribed and -Translated Protein to 32P-Labeled Probe Consisting of the hGnRH Promoter Region Between -953/-911 bp, Which Contains the Brn-2 Consensus Site at -925/-916 (left)

The lanes to the right contain a mutated Brn-2 probe consisting of the same hGnRH region, but with a functional and binding mutation of the Brn-2 consensus site. The arrows indicate complexes formed by the addition of Brn-2 and Oct-1. Addition of reticulocyte lysate and Brn-2 and Oct-1 antibody are indicated at the top. No specific binding interactions were observed when the Brn-2 mutant probe was used. RL, Reticulocyte lysates; Ab, antibody.

 
In vitro-transcribed and -translated Brn-2 and Oct-1 were further used to confirm the identity of the proteins interacting with the Brn-2 consensus sites. Figure 7Go demonstrates that in vitro-transcribed and -translated Brn-2 protein interacts with this probe as indicated by a complex that is not present in the reticulocyte lysate (RL)-only lane (also shown in Fig. 6Go as comparison). This interaction could be interfered with by a Brn-2 antibody, but not by an Oct-1 antibody. Interestingly, this probe also interacted with in vitro-transcribed and -translated Oct-1 protein. This interaction was interfered with by the addition of Oct-1 antibody. When a probe was used that contained a previously reported binding mutation of the Brn-2 consensus site (10), no interactions were observed with either Brn-2 or Oct-1. Similar DNA-protein interactions were observed with a hGnRH fragment probe containing the -867/-858 Brn-2 consensus site (data not shown). This pattern was also observed when a hGnRH fragment probe containing either of the proximal Brn-2 consensus sites was used (-148/-142 and -111/-102; data not shown).

Brn-2 mRNA and Protein Is Present in GnRH-Expressing Cell Lines
To determine whether Brn-2 is found in GnRH neurons, Northern and Western blot analyses were performed on mRNA and nuclear extract obtained from GN11 and NLT cells. As controls for both the Brn-2 expression vector, and the Northern blot procedure, mRNA was also obtained from cells transfected with either the Brn-2pSG5 expression vector or an empty pSG5 expression vector. Figure 8AGo shows a Northern blot of mRNA from these cell types probed with a digoxigenin-labeled Brn-2 riboprobe. A band representing a transcript of 4.5 kb was observed in mRNA obtained from both NLT and GN11 cells transfected with Brn-2pSG5, and in NLT cells transfected with empty pSG5, but no Brn-2 mRNA was observed in Gn11 cells transfected with the empty expression vector. Another group has reported a 4.8-kb Brn-2 mRNA in the hypothalamus (20). Brn-2 protein was observed in NLT cells but not Gn11 cells by Western blot (Fig. 8BGo). A band was observed at about 50 kDa, which is approximately the size of murine Brn-2 as calculated by statistical analysis of protein sequences (21).



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Figure 8. Brn-2 Is in GnRH Neurons

A, Northern blot analysis of mRNA obtained from the GN11 or NLT cell lines transfected with either a Brn-2 expression vector (+) or a control vector (-). A digoxigenin-labeled riboprobe was found to hybridize at a size of 4.5 kb to mRNA of both GN11 and NLT cells transfected with Brn-2pSG5, and to NLT cells transfected with pSG5, but not to GN11 cells transfected with pSG5. B, Western blot of nuclear extract proteins from NLT and GN11 cells. Size markers are indicated on the left. A band is observed in the NLT lane (arrow) that runs at about 54 kDa. No band was observed in the GN11 lane. C, Coronal, whole-mount tissue section of a mouse brain at the level of the OVLT. A black precipitate indicates Brn-2 mRNA, and GnRH peptide is labeled with a Cy-3 phosphoramidite (fluorescing). Brn-2 mRNA is observed in the cell body of the GnRH neuron (arrow). KD, Kilodaltons.

 
Detectable Brn-2 mRNA Is Observed in a Small Number of GnRH Neurons
GnRH immunoreactive neurons were predominately located in rostral regions of the basal hypothalamus, preoptic area, and diagonal band of Broca. However, scattered GnRH neurons were located as far caudally as the rostral portions of the median eminence. Five hundred twenty three brightly fluorescing neurons were counted, with especially dense regions located in the region of the OVLT in the rostral medial POA. The highest levels of Brn-2 mRNA, as indicated by a dark purple precipitate, were observed in the paraventricular nuclei and the supraoptic nuclei. There were also lower levels of Brn-2 mRNA observed in GnRH neuron-containing regions such as the OVLT, the medial POA, and some septal regions. Low levels of Brn-2 mRNA were detected in a small subpopulation (~5%) of GnRH immunoreactive neurons (Fig. 8CGo).

Mouse GnRH mRNA Levels Are Increased in GnRH Neurons Transfected with a Brn-2 Expression Vector
To demonstrate that Brn-2 could increase GnRH mRNA in cultured GnRH neurons, four stable cell lines containing the Brn-2pBKCMV expression vector, and three stable cell lines containing the empty pBKCMV expression vector, were produced by limited dilution cloning of transfected GN11 cells. Mouse GnRH mRNA levels from one of two ribonuclease protection assays (RPAs) performed on the mRNA samples from these cell lines are shown in Fig. 9AGo. Data, averaged from both RPAs, is shown in Fig. 9BGo. Mouse full-length GnRH mRNA levels, when corrected for actin levels, in the Brn-2pBKCMV cell line were between 1.1 and 2.4 (average of 1.52) arbitrary units for the full-length GnRH transcript and were between 1.75 and 2.38 (average of 2) arbitrary units for the truncated splice variant. In the pBKCMV cell lines the levels of mouse GnRH mRNA were between 0.28 and 0.81 (average of 0.49) arbitrary units for the full-length GnRH transcript and between 0.39 and 1.1 (average of 0.73) for the truncated GnRH splice variant. Thus, Brn-2 increased transcription of both the full-length and truncated splice variant GnRH mRNAs an average of nearly 3-fold (ranging from 1.4-fold to 8.6-fold for the full-length species and 1.59-fold to 6.1-fold for the splice variant) when compared with stable lines containing an empty expression vector.



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Figure 9. Brn-2 Stimulated Expression of the Endogenous GnRH Gene

A, RNase protection assay that was performed on mRNA obtained from stable transfection of GN11 cell lines with either the empty pBKCMV expression vector (lanes 5–7) or the Brn-2pBKCMV expression vector (lanes 1–4). GnRH full-length mRNA, the truncated splice variant, and actin mRNA are indicated by arrows to the left. Two separate RPAs were performed on the mRNA, and the values of both the full-length GnRH transcript (FL, solid bars) and the splice variant (SV, shaded bar) averaged and shown in panel B where values are reported as arbitrary units of GnRH corrected for arbitrary units of actin. In panel A the GnRH bands are scanned from a film after exposure for 4 d at -70 C. All quantification of band intensity was performed with the phosphorimager. Background levels for each band were subtracted from band intensity. Images of actin bands are from the phosphorimager to reduce intensity for better visualization and are superimposed on scanned image of film.

 
Brn-2 Stimulates hGnRH Promoter Activity
To confirm that Brn-2 also stimulates hGnRH promoter activity, transient transfections were performed in GN11 cells using either the {Delta}992LUC construct, a m{Delta}992LUC construct in which both Brn2 consensus sites in the GnRH promoter were mutated, or an empty luciferase reporter vector. Cells were cotransfected with various concentrations of Brn-2 expression vector, Oct-1 expression vector, or empty expression vector. Transfection with 0.5 µg of Brn-2pSG5 expression vector stimulated promoter activity of the -992 to -763 bp promoter fragment resulting in expression of luciferase 3.5-fold higher than in cells transfected with the empty pSG5 expression vector (Fig. 10Go). This Brn-2 expression vector did not stimulate the empty pA3LUC reporter construct when compared with empty pSG5 expression vector. Brn-2 (0.5 µg) also stimulated the m{Delta}992LUC reporter, but at only 60% the efficiency that it stimulated the {Delta}992LUC reporter (2.1-fold vs. 3.3-fold, respectively). The stimulation of the mutated promoter fragment by Brn2 may be due to a low level binding at imperfect Brn-2 consensus sites in the tissue-specific element (TSE). An Oct-1 expression vector did not stimulate either the m{Delta}992LUC or the {Delta}992LUC reporters when compared with its effects on the empty pA3LUC reporter (Fig. 10Go).



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Figure 10. Brn-2 Stimulates Promoter Activity of the hGnRH Gene

A, Schematics of constructs used in transient transfection experiments. pA3LUC, {Delta}-992/+5LUC (labeled Delta-992/+5LUC) and Mut Delta-992/+5LUC. M indicates mutated Brn-2 consensus binding sites in the TSE. B, Transient transfections of GN11 cells using Lipofectamine Plus reagent (Life Technologies, Inc.). Cells were transfected with reporter vector [{Delta}-992/+5LUC (labeled Delta-992/+5LUC), Mut Delta-992/+5LUC (the same as {Delta}-992/+5LUC except with mutations of both Brn-2 consensus sites), and the empty pA3LUC] and 0.5 µg of empty pSG5 expression vector, Brn-2pSG5, or Oct-1pSG5. Data are reported as fold stimulation by Brn-2 or Oct-1 as compared with pSG5 ± SE.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It is well established that the appropriate expression and release of GnRH from neurons in the hypothalamus are vital for the elaboration of normal reproductive function in mammals. The murine GnRH gene begins to be expressed on day 11.5 in the olfactory pit of the olfactory placode (1). They migrate during the next few days along the tract of the nervus terminalis through the nasal septum, the rostral forebrain, and into the diencephalon (1, 22). During the migratory process, GnRH neurons appear to be strewn along the migratory pathway; therefore, scattered GnRH neurons are located in anterior regions such as the olfactory bulbs (22). These results were confirmed in our studies in which luciferase expression was observed in the olfactory tissues of the -1,131/+5LUC and -3,828/+5LUC animals (9).

Due to the paucity of GnRH neurons in the brain, and to their scattered distribution, it has been difficult to study the molecular elements involved in regulating hGnRH expression. We have developed an in vivo model with which to study the regulation of the hGnRH gene in transgenic mice (9). These animals contain constructs consisting of various hGnRH gene promoter fragments fused to the luciferase reporter gene. We have previously shown that hGnRH gene promoter segments of 3,828 or 1,131 bp contained elements sufficient to target gene expression to GnRH neurons in the mouse (9). In contrast, the first 484 bp of the promoter did not contain the elements essential for targeting to GnRH neurons (9). These studies also indicated that cis-regulatory elements mediating the gonadal steroid regulation of GnRH gene expression are contained within the first 1,131 bp of the promoter. Therefore, this model is a valuable tool for isolating the cis-regulatory elements important for cell-specific expression and regulation of the hGnRH gene.

In the present study, we have further isolated the promoter elements essential for directing gene expression to GnRH neurons in the hypothalamus. Animals containing a gene with a 992-bp promoter fragment regulating expression of the luciferase gene had relatively high levels of luciferase activity in the hypothalamus (Fig. 1Go) when compared with other brain and peripheral tissues. Luciferase expression was observed in GnRH neurons (Fig. 4Go), indicating that this 992-bp promoter fragment indeed targeted gene expression to GnRH neurons. When 795 bp of the promoter were used, however, no luciferase activity was observed in any of the tissues that were examined (Fig. 2Go). These data indicate that we have isolated a 197-bp region of the hGnRH gene promoter, between -992 and -795 that is essential for the cell-specific expression of the GnRH gene.

To determine whether the cell-specific region that we had isolated was both essential and sufficient, we created the {Delta}-992/+5LUC animal lines. These animals exhibited an anatomical pattern of expression similar to the -992/+5LUC animals (Fig. 3Go). Consequently, we have isolated a tissue-specific element within the hGnRH promoter between -992 and -795 that is both essential and sufficient to target gene expression to GnRH neuron-containing tissues in the hypothalamus and olfactory bulbs. There were clearly lower levels of luciferase expression observed in the brains of the {Delta}-992/+5LUC mice than in the -992/+5LUC mice. This may be due to the deletion of an enhancer element in the proximal promoter. There was also a difference in the relative olfactory/hypothalamic levels between the {Delta}-992/+5LUC mice and the -992/+5LUC mice (50% for the {Delta}-992/+5LUC mice and 10–25% for mice derived from the two founders of -992/+5LUC mice). Perhaps the proximal promoter region contains enhancer elements sensitive to signals in the hypothalamic microenvironment.

Interestingly, the hGnRH tissue-specific region bears little homology to a recently described rat enhancer identified in in vitro studies (14, 15). In fact, the rat enhancer has a 76% homology with a region of the human promoter between -2,884 and -2,586 a region located well outside of the human tissue-specific element [analysis by the Bestfit program (23)]. These differences may be the result of comparing an in vitro model with our in vivo model. However, this may also indicate that there are species differences in the promoter elements essential for the cell-specific expression of the GnRH gene. These differences may reside more within the cis-regulatory elements because we were able to direct expression to mouse GnRH neurons with a human GnRH promoter. Thus, it appears that proteins important for cell-specific expression are shared among species. There have also been efforts by investigators to identify regions of the mouse GnRH promoter that are important for directing expression of the GnRH gene (24, 25, 26). These studies all agree that 3,446 bp of the promoter targets gene expression to GnRH neurons in mice. Pape et al. (26) have further specified that elements located 3' of the gene are required to restrict gene expression to GnRH neurons. Although there is some disagreement over whether this is indeed the case for the mouse promoter, it does not appear as if there is detectable ectopic expression of our reporter under the direction of the hGnRH promoter in any of the lines we have examined (Ref. 9 and present report).

When the DNA sequence of the tissue-specific element was examined, it was noticed that there were two Brn-2 consensus sites (Fig. 5BGo). Other octamer sites were also noted in more proximal regions of the promoter, including an Oct-1 consensus binding site just 3' to the cell-specific element between -742 to -735. Oct-1 has been shown to play a role in regulating the rat GnRH promoter (15) by interactions with the enhancer region reported by Whyte et al. (14). Brn-2 and Oct-1 are members of the POU homeodomain family of transcription factors. The class III POU proteins, of which Brn-2 is a member, have been shown to be important for tissue-specific development in a number of systems (12, 13). In addition, Brn-2 expression is observed in the olfactory bulbs, the septum, and the hypothalamus (Ref. 11 and present study), regions of the brain that contain GnRH neurons (Ref. 22 and present study). Interestingly, it has been reported that Brn-2 is expressed in developing olfactory epithelial cells in the mouse (27), which is the site of the origin of GnRH neurons early in development (1). Northern and Western blot analyses indicated that Brn-2 mRNA and protein were indeed present in GnRH expressing cells (Fig. 8Go, A and B, respectively) and double labeling for Brn-2 mRNA and GnRH protein indicated that a Brn-2 is expressed in a small percentage of GnRH neurons (Fig. 8CGo). Whether this is an indication that only subpopulations of GnRH neurons express Brn-2 or a function of a limit of sensitivity for low levels of Brn-2 mRNA is not clear.

To determine whether transcription factors from GnRH-expressing neurons formed DNA binding complexes within the cell-specific element, DNase I footprint analysis was performed (Fig. 5AGo). These studies demonstrated that there was a binding interaction 5' of the cell-specific element located between -1,060/-1,040 and three interactions within the cell-specific element, at -975/-930, -922/-907, and at -870/-855. The latter two interactions overlapped with the Brn-2 consensus binding sites within the cell-specific element. The other footprinted regions may represent other factors important for expression of GnRH in NLT cells.

To examine further the binding characteristics at the Brn-2 consensus sites, gel mobility shift assays were performed. These data indicated that protein elements present in nuclear extract obtained from NLT cells formed a complex with a Brn-2 consensus site, but not with a mutated Brn-2 consensus site that had previously been shown to be functionally inactive (10). Interestingly, this complex was larger than the complexes formed when the same probe was incubated with in vitro-transcribed and -translated Brn-2 (Figs. 6Go and 7Go). This may indicate that the protein element in GnRH neurons that recognize the octamer element contains Brn-2 and additional cofactors, or is a protein or complex of proteins with Brn-2-like binding characteristics. Additional gel shift assays further demonstrated that Brn-2 formed protein DNA complexes with a probe consisting of hGnRH promoter sequence within the cell-specific element containing either of the Brn-2 consensus sites (Fig. 7Go and data not shown). Brn-2 consensus sites located proximal to the cell-specific element also interact with Brn-2 in gel shift studies (data not shown). It is not clear whether those Brn-2 binding sites external to the tissue-specific element are functionally relevant. These data suggest that Brn-2 or a Brn-2-like protein may regulate hGnRH gene expression by interacting with specific Brn-2 consensus binding sites located within the region of the hGnRH promoter that has been shown to be essential for the tissue-specific expression of the gene. At this time it is unclear whether Brn-2 plays a role in regulating the cell-specific expression of the hGnRH gene. Oct-1 was also found to specifically bind to the Brn-2 consensus site, and not to the mutated Brn-2 consensus site (Fig. 7Go). This is interesting in light of recent reports that Oct-1 may play a role in mediating glucocorticoid repression of the mouse GnRH gene (28, 29). Brn-4 and Pit-1 had no specific interactions with the Brn-2 consensus binding sites in gel shift studies (data not shown).

To assess whether the Brn-2 transcription factor can regulate GnRH gene expression, stable cell lines containing a Brn-2 expression vector were constructed. We examined the expression of both the full-length GnRH mRNA species and the truncated GnRH splice variant (6). Brn-2 stimulated an increase in mouse GnRH mRNA levels of both the full-length transcript and the truncated splice variant nearly 3-fold when compared with cells containing the empty pBKCMV expression vector (Fig. 9Go). Therefore, it appears that the Brn-2 transcription factor regulates endogenous GnRH gene expression in the mouse. It is not clear what role, if any, the seemingly functionally inactive splice variant plays in mouse physiology. The present studies cannot rule out the possibility that Brn-2 may be acting indirectly to stimulate GnRH gene expression, and not via interactions on the GnRH promoter. However, Brn-2 did stimulate the activity of the TSE of the hGnRH promoter in transient transfection studies in a quantitatively similar fashion (Fig. 10Go). Transactivation by Brn-2 was reduced when both of the Brn-2 consensus binding sites in the TSE were mutated. Oct-1 did not stimulate either the wild type or mutated reporter constructs in transient transfections (Fig. 10Go) despite the strong binding interactions observed in gel shifts. In light of the common transcriptional pathways used for directing both mouse GnRH and hGnRH gene expression in vivo, it thus seems likely that Brn-2 might also regulate hGnRH gene expression in vivo.

Recently two groups have produced Brn-2 knockout mice (12, 13). Although not thoroughly examined, Nakai et al. (12) noted that GnRH immunoreactivity was observed in these animals. This, however, does not rule out a possible role for Brn-2 in the regulation of GnRH gene expression. Physiological studies were not performed on these mice due to a lethal phenotype; therefore, Brn-2 may play an important role in the development of a functional GnRH neuron. It is also possible that in the knockout paradigm an alternative molecule can functionally replace the effects of Brn-2. Certainly, POU proteins have shown a certain redundancy in other systems (13, 30), and we have shown that other POU homeodomain proteins can structurally interact with these Brn-2 sites in vitro (Fig. 7Go).

In conclusion, we have isolated a 197-bp region of the hGnRH promoter that is both essential and sufficient to target gene expression to GnRH neurons in the hypothalamus. In addition, evidence has been provided that the POU homeodomain family of transcription factors may play a role in regulating GnRH gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of Transgenic Animals
The procedures used for animal care and study are in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Transgenic animals were constructed by the Beth Israel Transgenic Facility (Boston, MA). Fertilized mouse oocytes from FVB mice were injected with a purified linear DNA fragment containing a GnRH/LUC construct. Construction of GnRH promoter fragment/luciferase constructs is described below. Oocytes were then transferred into pseudopregnant foster mothers. Transgenic animals were identified with Southern blot analysis as described previously (9).

Construction of GnRH Promoter Reporter Constructs
Human GnRH promoter fragments were synthesized using the PCR, and a 3,828-bp hGnRH promoter fragment was used as a template. Promoter fragments were inserted into the pA3LUC reporter vector (31, 32). For the -992/+5LUC and -795/+5LUC constructs, the 5'-primers consisted of a HindIII restriction enzyme site 5' of 20 bp of hGnRH promoter sequence from -992 to -973 or -795 to -776. The 3'-primer for both reactions contained a HindIII site 5' of 26 bp of the promoter sequence from +5 to -20. For the {Delta}-992/+5LUC construct, the same 3'-primer was used as above, and the 5'-primer consisted of a KpnI site 5' of sequence from -48 to -29. A -992/-763 fragment with KpnI ends was constructed in the same manner using a 5'-primer with a KpnI restriction site 5' of 20 bp of hGnRH promoter sequence from -992 to -973 and a 3' primer containing a KpnI site 5' of 20 bp of complementary hGnRH promoter sequence from -763 to -744. This fragment was then cloned into the -48/+5LUC construct. Orientation was confirmed by sequencing constructs using a primer annealing to the 5'-end of the luciferase gene.

Construction of POU Homeodomain Protein Expression Vectors
A murine Brn-2 cDNA was isolated from PCR of the mass excision product of a neonatal whole brain library (Stratagene, La Jolla, CA) using as primers the published sequence of Brn-2 (20). The PCR product was ligated into the pGEM T easy vector (Promega Corp., Madison, WI; Brn-2pGEM T), excised with EcoRI, and ligated into the pSG5 expression vector (CLONTECH Laboratories, Inc., Palo Alto, CA). A Brn-4 cDNA was obtained from Dr. Franz Cremer (Nijmegen, The Netherlands) and excised with EcoRI and cloned into pSG5. The pCGOct-1 vector was obtained from Dr. Winship Herr (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), and was excised with XbaI and BamHI and inserted into pBS SK+. The Oct-1 cDNA was then excised with SacI and BamHI and inserted into a modified pSG5 vector that contains a SacI site in the multiple cloning site. A Pit-1pSG5 expression vector was obtained from Dr. Laurie Cohen (33). All plasmids were sequenced and orientation was confirmed.

Assay of Luciferase Activity In Tissues
Tissues were collected and prepared for assay as previously described (9). Luciferase assays were done using a Lumat LB9501 luminometer (Berthold Systems Inc., Pittsburgh, PA). Samples were injected with 100 µl of luciferin (0.75 mM in lysis buffer: obtained from Molecular Probes, Inc., Eugene, OR) and 100 µl of assay buffer (25 mM glycylcycline, 15 mM MgSO4, 4 mM EGTA, 15 mM KPO4, 3 mM dithiothreitol, and 3 mM ATP), and luminescence was measured for 20 sec in relative light units.

Cell Culture
Cells were maintained at 37 C and 5% CO2 in DMEM (Life Technologies, Inc., Gaithersburg, MD), with high glucose, and phenol red supplemented with 7% FCS (Life Technologies, Inc.), 3% newborn calf serum (BioWhittaker, Inc., Walkersville, MD), and antibiotic/antimytotic (Life Technologies, Inc.) (referred to as DMEM+++). Cells were grown in 750-mm Falcon tissue culture flasks (Becton Dickinson and Co., Bedford, MA) until nearly confluent. Cells were plated in 150-mm tissue culture dishes (for data shown in Fig. 8AGo). Cells were plated in six-well tissue culture dishes for transient transfections and were transfected as previously described (34).

DNase I Footprinting
Single end-labeled DNA probe was constructed in the following manner. A -1,131 to -810 hGnRH promoter fragment was created using the PCR as described above. The primer set was constructed with an EcoRI restriction enzyme site at the 5'-end and a HindIII restriction enzyme site on the 3'-end. The promoter fragment was then subcloned into PGEM4Z (Promega Corp.), and the resultant plasmid is referred to as 1131/830PGEM4Z. The 1131/830PGEM4Z plasmid was then digested with HindIII, dephosphorylated with alkaline phosphatase (Roche Molecular Biochemicals, Indianapolis, IN), and electrophoresed on a 1.4% agarose gel, and the 300-bp promoter fragment was excised and gel purified. The promoter fragment was then 5'-end labeled with {gamma}32P, gel purified, digested with XbaI, and then again electrophoresed, excised, gel purified, and used at a specific activity of 20,000 cpm/lane. Probe was incubated with nuclear extract for 20 min in a binding buffer consisting of 0.5 mM dithiothreitol, 25 mM Tris HCl (pH 8), 50 mM KCl, 6 mM MgCl2, and 4% glycerol. To binding solution, 50 µl of a 10 mM MgCl2, 5 mM CaCl2 solution was added, followed by addition of 0.1 U of DNase I for 80 sec. The digestion was terminated by the addition of 100 µl of a solution consisting of 12.5 mM EDTA, 0.1% SDS, 125 µg/ml tRNA, 12.5 µg/ml proteinase K, and 0.3 M sodium acetate. DNA was then phenol/chloroform extracted, precipitated, and electrophoresed on a 6% denaturing polyacrylamide gel, run in 1x TBE buffer (0.9 mM Tris-boric acid, 10 mM EDTA), and then dried and exposed to film overnight at -70 C.

Gel-Mobility Shift Assay
32P-labeled probe consisting of 20-bp hGnRH promoter fragments was created using PCR and antisense primers homologous to the template.

Probes were column purified (G50 Sephadex columns, Roche Molecular Biochemicals, Indianapolis, IN) and used at an activity of 1,000,000 cpm/14 lanes. Ten microliters of a probe mix consisting of 0.28 U dIdC/14 lanes and 126 µl probe + binding buffer (binding buffer; 50 mM KCl, 20% glycerol, 20 mM HEPES, pH 7.6–7.8) was added to each tube and incubated at 4 C with binding proteins for 20–30 min. One microliter of Brn-2 or Oct-1 antibody (Santa Cruz Biochemicals, Inc., Santa Cruz, CA) was added to some tubes before incubation. Each sample was then separated by gel electrophoresis on a 5% nondenaturing acrylamide gel containing 5% glycerol.

Nuclear Extract Preparation
Cells were plated on 150-mm diameter plates, and cellular nuclear extracts were obtained by the method of Schreiber et al. (35). Nuclear extract was centrifuged at 14,000 rpm for 5 min and stored at -70 C. Protein assays were done using a Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay and BSA standards.

Northern Blot Analysis
mRNA was isolated from Gn11 and NLT cells using the Invitrogen Fast Track kit. mRNA (1–2 µg) was separated with gel electrophoresis on a 1.4-% agarose gel containing 2% formaldehyde and 0.05% ethidium bromide. Gel was washed for 40 min in a 0.05 M NaOH, 0.15 M NaCl solution and then 40 min in a 0.1 M Tris HCl (pH 7.5), 0.15 M NaCl solution. RNA was then transferred to GeneScreen Plus hybridization transfer membrane (NEN Life Science Products, Boston, MA) by Northern blot using 20x SSC as the transfer solution. The membrane was washed and the RNA was UV cross-linked to the membrane followed by prehybridization [50% deionized formamide, 800 mM NaCl, 1 mM EDTA, 50 mM sodium phosphate buffer (pH 7.4), 2% SDS, and 2.5x Denhardt’s solution] overnight at 42 C. For analysis of Brn-2, mRNA digoxigenin-labeled riboprobes were produced with the Roche Molecular Biochemicals DIG RNA labeling kit using the recommended protocol. For the Brn-2 riboprobe, SP6 RNA polymerase was incubated with a SmaI linearized Brn-2pGEM T vector at 37 C for 2 h. Samples were then treated with DNAse I for 15 min and precipitated with LiCl and 100% ethanol. Probe was used at a concentration of 0.5 µg/ml and was added to blots with 100 µg/ml salmon sperm DNA. After hybridization overnight at 42 C, the blot was washed at 65 C with a 2x SSC/1% SDS solution. The blot was washed with Tris-buffered saline (TBS), pH 7.4, and incubation for 1 h at room temperature with antidigoxigenin FAB fragment conjugated to alkaline phosphatase (Roche Molecular Biochemicals) diluted 2,000:1 with TBST (TBS, pH 7.4, and 1% Tween 20) and 1% sheep serum. The blot was washed and then developed with TBS, pH 9.5, 0.05 M MgCl2, and a 1:50 dilution of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate stock solution (Roche Molecular Biochemicals). The reaction was stopped by washing with TBS, pH 7.4.

Western Blot Assay
Proteins in nuclear extract preparations were separated by SDS-PAGE and transferred to nitrocellulose membrane by semidry transfer. Blots were blocked in 1x Towbin with 10% dried milk for 1 h and then incubated with 1:1,000 Brn-2 antibody (Geneka Biotechnology, Inc., Montréal, Québec, Canada) in 1x Towbin and 5% dried milk. Blots were washed 4 x 15 min in 1x Towbin and then incubated with horseradish peroxidase-conjugated mouse antirabbit antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in 1x Towbin and 2% dried milk. Blots were then washed three times for 10 min in 1x Towbin. Antibody binding was visualized using the ECL Plus reagent (Amersham Pharmacia Biotech, Piscataway, NJ).

RPA
GN11 cells were transfected with either the Brn-2pBKCMV expression vector or the empty pBKCMV expression vector using the Lipofectamine Plus reagent (Life Technologies, Inc.). Stable transformants were obtained by treating cells with 400 µg/ml genaticin. Individual cell lines were then obtained by performing limited dilution cloning into six-well plates. Cell lines were expanded and mRNA was obtained from GN11 cells as described above. Ribonuclease protection assay procedures were performed as previously reported (36). Briefly, 32P-labeled antisense mouse GnRH and mouse actin riboprobes were synthesized in vitro using 32P-UTP and T7 polymerase (Maxiscript T7 kit, Ambion, Inc., Austin, TX) and a mouse GnRH cDNA template obtained from our Gn11 cell line, a GnRH-expressing neuronal cell line (6). Riboprobe was gel purified, and 1,000,000 cpm of the GnRH probe and 20,000 cpm of the actin probe were hybridized to 1 µg of mRNA in 10 µl of hybridization solution at 45 C overnight (RPA III kit, Ambion, Inc.). After RNAse A and T1 digestion, the labeled products were resolved on an 6% denaturing polyacrylamide gel. Gels were analyzed using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Arbitrary units of GnRH darkness were then divided by arbitrary units of actin darkness to correct for total mRNA loaded on the gel. This value was then multiplied by 1,000 to give integer values.

Histology
Double labeling of brain sections for Brn2 and GnRH was performed using in situ hybridization for Brn-2 mRNA, followed by immunocytochemistry for GnRH peptide. Fluorescent histochemistry double labeling was used to colocalize GnRH and luciferase.

Fixation
All mice were fixed in situ. The brains were stored in fixative solution until use. For Brn2/GnRH colocalization, adult FVB mice were used. Brains were then embedded in 5% agarose, blocked, and 75-µm coronal brain sections were cut using a Vibratome and were collected in PBS for in situ hybridization. For double fluorescence histology, 30-µm frozen sections were obtained using a sliding microtome.

In Situ Hybridization
Sections were treated with a 6% H2O2/PBST solution for 1 h, and then with 10 µg/ml proteinase K in PBST for 15 min, washed with 2 mg/ml ultrapure glycine in PBST, and then postfixed with 4% paraformaldehyde and 0.2% glutaraldehyde. Sections were washed and transferred to prehybridization solution (50% formamide, 5x SSC, 50 µg/ml yeast tRNA, 50 µg/ml heparin, and 0.3% SDS) at 60 C and incubated for 1 h before the addition of riboprobe. Riboprobe, produced as described above for Northern blot analysis, was allowed to anneal overnight. Sections were then washed with 50% formamide, 5x SSC, pH 4.5, and 1% SDS, followed with 50% formamide and 2x SSC, pH 4.5. Sections were washed and blocked with 10% sheep serum in TBST at room temperature. Sections were then incubated overnight at 4 C with antidigoxigenin FAB fragment conjugated to alkaline phosphatase (Roche Molecular Biochemicals) diluted 1/2,000 with TBST and 1% sheep serum. Sections were then washed for 36 h. Sections were washed with NTMT (100 mM NaCl, 100 mM Tris-HCL, pH 9.5, 50 mM MgCl2, and 1% Tween) and then incubated with reaction mixture [10% polyvinyl alcohol, 100 mM Tris-HCL, pH 9.5, 100 mM NaCl, 5 mM MgCl2, and 1:50 dilution of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate stock solution (Roche Molecular Biochemicals)] overnight at 37 C in a dark humid chamber. Reaction was stopped by washing with NTMT, then with PBST, pH 5.5, and finally with PBST, pH 7.4.

Immunocytochemistry After in Situ Hybridization
Tissue sections were blocked for 1 h at room temperature in PBS with 0.3% Triton X-100 (Tx) and 1% BSA. Sections then incubated overnight at 4 C with a 1:10,000 dilution of GnRH antiserum (LR1 rabbit anti-GnRH antibody kindly provided by Dr. Robert Benoit) in blocking solution. Sections were washed and then incubated at room temperature for 2 h with biotinylated goat antirabbit IgG (6.75 µg/ml, Vector Laboratories, Inc., Burlingame, CA). Sections were then washed overnight. Sections were incubated with 2 µl/ml Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.) for 90 min at room temperature, and then washed and mounted.

Double Fluorescence Histology
Sections were incubated overnight in 2 µg/ml goat antiluciferase antibody (Cortex Biochemical, San Leandro, CA). Sections were washed and then incubated with 6.75 µg/ml biotinylated antigoat IgG (Vector Laboratories, Inc.) for 2 h. Sections were washed and then incubated with 5 µg/ml streptavidin-conjugated Alexa Fluor 488 (Molecular Probes, Inc.) for 2 h. Sections were then incubated overnight at 4 C with a 1:5,000 dilution of GnRH antiserum (LR5 rabbit anti-GnRH antibody kindly provided by Dr. Robert Benoit) in blocking solution. Sections were washed and incubated with Cy3-conjugated donkey antirabbit IgG for 2 h. Sections were washed and mounted.

Microscopy
Tissue sections were mounted and coverslipped with Vectashield mounting media (Vector Laboratories, Inc.). In situ sections were analyzed on an Eclipse E800 microscope (Nikon, Melville, NY), and photomicrographs were taken using a U111 camera (Nikon). Double fluorescence histology sections were analyzed on an Axiovert SS100TV inverted fluorescent microscope (Carl Zeiss, Thornwood, NY), and images were captured with a Carl Zeiss video camera with a charge-coupled device chip.


    ACKNOWLEDGMENTS
 
The authors would like to thank Elizabeth Ross, Geary Smith, and Robyn Deneau for their excellent technical assistance, Marjorie Zakaria for support with the RPAs, and Fredric Wondisford for his general recommendations on troubleshooting the DNase I footprinting assays and the gel-mobility shift assays.


    FOOTNOTES
 
This work was supported by NIH Grants R01HD-34551 and KO1DK-02730.

Abbreviations: DNase, Deoxyribonuclease; hGnRH, human GnRH; OVLT, organum vasculosum of the lamina terminalis; PBST, phosphate-buffered saline with 1% Tween; POA, preoptic area; RLU, relative light units; RPA, ribonuclease protection assay; TBS, Tris-buffered saline; TBST, TBS, pH 7.4, and 1% Tween 20; TSE, tissue-specific element.

Received for publication April 6, 2000. Accepted for publication November 14, 2001.


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