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.), Childrens 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.
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ABSTRACT |
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INTRODUCTION |
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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.
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RESULTS |
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A tissue panel from a representative -795/+5LUC animal, shown in Fig. 2, 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
-992/+5LUC animals when compared with all other tissue examined (eight animals from three separate founders examined) (Fig. 3
). The hypothalamic luciferase levels in the
-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
-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 1
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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. 6, 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. 6
). 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. 6
). Figures 6
and 7
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. 6
).
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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 8A 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. 8B
). 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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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. 9A. Data, averaged from both RPAs, is shown in Fig. 9B
. 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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DISCUSSION |
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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. 1) when compared with other brain and peripheral tissues. Luciferase expression was observed in GnRH neurons (Fig. 4
), 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. 2
). 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 -992/+5LUC animal lines. These animals exhibited an anatomical pattern of expression similar to the -992/+5LUC animals (Fig. 3
). 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
-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
-992/+5LUC mice and the -992/+5LUC mice (50% for the
-992/+5LUC mice and 1025% 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. 5B). 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. 8
, 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. 8C
). 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. 5A). 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. 6 and 7
). 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. 7
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. 7
). 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. 9). 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. 10
). 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. 10
) 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. 7).
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.
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MATERIALS AND METHODS |
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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 -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. 8A). 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 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 7.67.8) was added to each tube and incubated at 4 C with binding proteins for 2030 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 (12 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 Denhardts 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 ,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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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