Homeobox Protein Gsh-1-Dependent Regulation of the Rat GHRH Gene Promoter

Noriko Mutsuga, Yasumasa Iwasaki, Minako Morishita, Atsushi Nomura, Etsuko Yamamori, Masanori Yoshida, Masato Asai, Nobuaki Ozaki, Fukushi Kambe, Hisao Seo, Yutaka Oiso and Hidehiko Saito

First Department of Internal Medicine (N.M., M.M., A.N., E.Y., M.Y., M.A., N.O., Y.O., H.S.), Department of Clinical Laboratory Medicine (Y.I.), Nagoya University School of Medicine and Hospital, Nagoya, Japan 466-8560; and Research Institute of Environmental Medicine (F.K., H.S.), Nagoya University, Nagoya, Japan 464-8601

Address all correspondence and requests for reprints to: Yasumasa Iwasaki, M.D., Ph.D., Department of Clinical Laboratory Medicine, Nagoya University School of Medicine and Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan, E-mail: iwasakiy{at}med.nagoya-u.ac.jp


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although GHRH is known to play a pivotal role in the regulation of the GHRH-GH-IGF-I axis, the molecular mechanism of GHRH gene expression has not yet been examined. Here we studied the transcriptional regulation of the GHRH gene 5'promoter using an in vitro experimental model system. We especially focused on the role of homeobox transcriptional factor Gsh-1, because a dwarf phenotype and abolished GHRH expression was observed in Gsh-1 knockout mice. First, we cloned human Gsh-1, which showed 87.3% homology with mouse Gsh-1 at the nucleotide level. When the 5'-promoter region of the rat GHRH gene was introduced into the human placental cell line JEG-3, in which we found the endogenous expression of Gsh-1 as well as GHRH mRNA, substantial transcriptional activity of the promoter was recognized. Promoter activity was further enhanced by overexpression of Gsh-1 protein, whereas it was substantially reduced by elimination of Gsh-1 binding sites. EMSA confirmed the actual binding of Gsh-1 on the multiple binding sites of GHRH gene promoter. Finally, coexpression of CREB-binding protein significantly enhanced the Gsh-1-induced GHRH gene expression, suggesting the cooperative role of the coactivator protein. Because Gsh-1 is found to be expressed in the hypothalamus of the adult rat, our data provide evidence that the Gsh-1 homeobox protein plays a key role in the expression of the GHRH gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SOMATIC GROWTH IN the mammalian organism is controlled by a complex regulatory system that originates in the hypothalamus, from which two peptides important for the regulation of pituitary somatotroph cells are released: GHRH and somatostatin (1). Of these, GHRH facilitates GH secretion from the pituitary gland, which in turn enhances the production of IGF-I in the liver or other tissues, mediating a variety of somatic and/or metabolic effects of GH (2). Thus, GHRH is a key regulatory factor in the positive control of the function of GH-IGF-I axis. GHRH also plays an important role in the proper proliferation and differentiation of the somatotroph through the development of the pituitary gland (3).

GHRH was first isolated from human pancreatic tumors (4, 5), and since then intensive physiological studies regarding the hormone have been carried out. Its cDNA and gene in various species have been cloned as well (6, 7, 8). Nevertheless, there has been virtually no study attempting to elucidate the transcriptional regulation of GHRH gene expression. This may be due, in part, to the lack of availability of appropriate host cells that would allow the expression of the GHRH gene promoter. Recently, however, it has been reported that genetic ablation of a homeobox gene Gsh-1 showed a dwarf phenotype in the mouse, in which no expression of the GHRH gene in the arcuate nucleus of the hypothalamus was reported (9, 10). This raises the possibility that Gsh-1 is an indispensable transcriptional factor for the tissue-specific expression of the GHRH gene, and those cells expressing Gsh-1 may be adequate for studying the GRH gene expression.

In this study, we first investigated the transcriptional regulation of the GHRH gene, using the JEG-3 human placental cell line. We found that endogenous Gsh-1 as well as GHRH mRNA was expressed in the cells, and that a substantial level of GHRH gene promoter activity was observed when the GHRH 5'-promoter-luciferase fusion gene was introduced. Furthermore, we obtained evidence showing that Gsh-1 is indeed a key transcription factor for maintaining the expression of the GHRH gene.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of the Human Gsh-1 cDNA
We first cloned the human homolog of Gsh-1 cDNA. By screening the human fetal brain cDNA library with mouse Gsh-1 gene as a probe, we isolated the cDNA clone of human Gsh-1. When the obtained sequences were compared with the mouse cDNA, 87.3% and 96.6% homology were observed at the nucleotide and protein levels, respectively (Fig. 1Go, A and B; GenBank accession nos. AB044157 and AB044158 for human Gsh-1 cDNA and gene). The gene structures (two exons and one intron) were also similar between the mouse and human (data not shown), indicating that the homeobox protein Gsh-1 is a highly conserved transcriptional factor among the species.



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Figure 1. Comparison of the Sequences of Mouse and Human Gsh-1 cDNA and Proteins

A, Alignment of the human and mouse Gsh-1 cDNA. Numbers in the right column correspond to nucleotide residues. Asterisks indicate residues identical to human Gsh-1. B, Alignment of the predicted protein sequences of human and mouse Gsh-1. Numbers in the right column correspond to amino acid residues. Asterisks indicate residues identical to human Gsh-1.

 
Analyses of GHRH and Gsh-1 mRNA Expression
We then screened the expression of Gsh-1 and GHRH mRNA by RT-PCR in various endocrine cell lines and adult rat tissues. We found that both mRNAs were coexpressed in the human placental cell line JEG-3 (Fig. 2AGo) and in the hypothalamus (Fig. 2BGo) and the testis (not shown in the figure). Although Gsh-1 expression was detected in some other cell lines and tissues without GHRH expression, we could not see any GHRH mRNA without Gsh-1 expression (Table 1Go). These results suggest that the homeobox gene Gsh-1 is expressed in adult as well as fetal tissues, and that the presence of Gsh-1 is necessary, although not sufficient, for GHRH expression. In addition, the intrinsic expression of both Gsh-1 and GHRH in JEG-3 cells suggests that the cell line is suitable for studying the transcriptional regulation of the GHRH gene.



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Figure 2. Expression of Gsh-1 and GHRH mRNA in Human Placental Cell Line JEG-3 and the Rat Hypothalamus Analyzed by RT-PCR

A, The amplified DNA fragments with the predicted lengths (381 bp and 331 bp for the human Gsh-1 and GHRH mRNA, respectively) derived from JEG-3 cells using specific primer sets for the human Gsh-1 and GHRH. B, The amplified DNA fragments with the predicted lengths (381 bp and 214 bp for the rat Gsh-1 and GHRH mRNA, respectively) derived from the adult rat hypothalamic tissues using specific primer sets for the rat Gsh-1 and GHRH. M refers to DNA markers. Control reactions without RT showed no amplifications (data not shown).

 

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Table 1. RT-PCR Analyses of the Expression of GRH and Gsh-1 mRNA

 
Transcriptional Activity of GHRH Gene 5'-Promoter in JEG-3 or Neuronal Cell Lines Expressing Gsh-1
When the rat GHRH gene 5'-promoter (-472 to +71; +1 designates the hypothalamic transcription start site)-luciferase fusion gene was introduced into the JEG-3 cells, substantial promoter activity was observed compared with that of the promoterless plasmid (Fig. 3AGo). To confirm whether the promoter activity may be attributable to the presence of Gsh-1, we examined the expression of the promoter in mouse hypothalamic progenitor cell lines with or without Gsh-1 (Gsh+/+ and Gsh-/-; kindly provided by Dr. Potter) (14). The Gsh+/+ cell line, which intrinsically expresses Gsh-1, was derived from the hypothalamus of Gsh-1-SV40 T-antigen transgenic mice, and the Gsh-/- cell line, which lacks Gsh-1, was derived from the hypothalamus of Gsh-1 knockout mice (14). Again, the GHRH gene promoter activity was more than 4-fold higher in Gsh+/+ cells than in Gsh-/- cells (Fig. 3BGo). Furthermore, when Gsh-1 protein was coexpressed with GHRH in JEG-3 cells, the promoter activity was further enhanced in a dose-dependent manner (Fig. 4Go). Altogether, our data strongly suggest that the homeobox protein Gsh-1 plays a key role in the expression of the GHRH gene.



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Figure 3. Gsh-1-Dependent Expression of the GHRH Gene 5'-Promoter

A, JEG-3 cells were transfected with GRH472Luc or a promoterless plasmid (pA3Luc), and the promoter activities were determined by luciferase assay. *, P < 0.01 vs. the promoterless plasmid. B, Gsh-1+/+ and Gsh-1-/- cells were transfected with GRH472Luc, and the promoter activities were determined by luciferase assay. *, P < 0.001 vs. Gsh-1-/- cells. In both experiments, ß-galactosidase expression vector was used as an internal control.

 


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Figure 4. Dose-Dependent Effect of Gsh-1 Overexpression on the GHRH Gene 5'-Promoter in JEG-3 Cells

JEG-3 cells were transfected with GRH472Luc and various amounts of the Gsh-1 expression vector, and the GHRH 5'-promoter activities were determined by luciferase assay. *, P < 0.05 vs. the control group (C) without Gsh-1 coexpression. In both experiments ß-galactosidase expression vector was used as an internal control.

 
Binding of Gsh-1 Protein to the 5'-Promoter Region of GHRH
An analysis of the nucleotide sequences of the GHRH gene 5'-promoter region revealed five putative binding sites of Gsh-1 [consensus sequence: GCT/CA/C ATTA G/A] (9) (see legend of Fig. 5Go). To see whether the Gsh-1 protein binds to some or all of the sequences, we carried out an EMSA using glutathione-S-transferase (GST)-Gsh-1 fusion protein. As shown in Fig. 5Go, a DNA-protein complex was observed when Gsh-1 protein was treated with the DNA fragments containing four (G2–G5) of the five sequences analyzed (lanes 3, 6, 9, and 14). No binding was obtained with GST protein without Gsh-1 (lanes 2, 5, 8, and 13). All of the gel shift entities formed by GST-Gsh-1 fusion protein were antagonized when incubated with 100 molar excess of each unlabeled DNA fragment, but not with a mutated sequence (M). Furthermore, 32P-labeled probe M failed to bind to GST-Gsh-1 (lane 12). Addition of antibody to Myc-tag caused a supershift and/or displacement, confirming the specificity. Altogether, these data suggest that Gsh-1 indeed binds to at least four sites in the 5'-promoter region of the GHRH gene.



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Figure 5. Binding Analysis of Gsh-1 Protein to the 5'-Promoter Region of the Rat GHRH Gene

A, Localization of the Gsh-1 binding sites in the rat GHRH 5'-promoter. The boxed sequences indicate the putative binding elements based on the Gsh-1 consensus sequence (GCT/CA/C ATTA G/A) (9 ). Sequences below each box are the mutated one used in EMSA or in promoter analysis (Fig. 6Go). B, EMSA of Gsh-1 binding sites. In the left panel, the 32P-labeled fragments spanning approximately 20 bp of each Gsh-1 binding site were incubated with GST (lanes 2, 5, 8, and 13) or GST-Gsh-1 fusion protein (lanes 1, 3, 4, 6, 7, 9–12, and 14–23). The mutated probe of G4 (M) was used in lanes 11 and 12. Nonlabeled fragments were used as a competitor (lanes 4, 7, 10, and 15). In the right panel, each reaction mixture was incubated for another 60 min with anti-Myc tag antibody (lanes 17, 19, 21, and 23).

 
Deletion/Mutation Analyses of the Gsh-1 Binding Sites of the GHRH Gene Promoter
To confirm that some or all of the Gsh-1 binding sites are functional, transcriptional activity of deleted or mutated promoter was analyzed in JEG-3 cells. As shown in Fig. 6AGo, the GHRH promoter activity significantly decreased when all the Gsh-1 binding sites were eliminated (GRH47Luc), whereas deletion of G1 to G4 had no effect. Furthermore, introduction of mutation in an individual binding site (mG1–mG5) caused no effect (Fig. 6BGo), whereas the promoter activity dramatically decreased when all binding sites were mutated (mG1–5) (Fig. 6CGo). These results suggest that alternative binding of one or more Gsh-1 proteins contribute to maintain the transcriptional activity of the GHRH gene.



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Figure 6. Deletion/Mutation Analyses of the GHRH Gene Promoter

A, JEG-3 cells were transfected with full-length (GRH472Luc) or deleted promoter constructs (GRH304Luc, GRH247Luc, GRH187Luc, GRH133Luc, GRH47Luc) with a constant amount of Gsh-1 expression vector (RSV-Gsh-1). G1–G5 represents putative Gsh-1 binding sites. *, P < 0.05 vs. GRH472Luc. B, JEG-3 cells were transfected with a full-length construct (GRH472Luc) or constructs mutated in each of the Gsh-1 binding sites (mG1, mG2, mG3, mG4, and mG5) with a constant amount of RSV-Gsh-1. X represents the mutated sites. C, JEG-3 cells were transfected with wild-type (GRH472Luc) or the mutant construct in which all of the putative Gsh-1 binding sites were mutated (mG1–5). *, P < 0.001 vs. GRH472Luc. In all experiments, ß-galactosidase expression vector was used as an internal control.

 
Functional Cooperation of Gsh-1 with CREB-Binding Protein in the Regulation of the GHRH Gene
Finally, we examined whether Gsh-1 functions by interacting with some other transcription factors or cofactor(s). Specifically, we focused on the coactivator protein CBP, because some of the transcription factors determining tissue-specific expression (such as Pit-1) have been shown to interact with CBP (15). We found that again, expression of Gsh-1 markedly enhanced the promoter activity of GHRH. Furthermore, coexpression of CBP significantly enhanced the effect of Gsh-1, although expression of CBP alone had no effect (Fig. 7AGo). In the mG1–5 construct in which all of the Gsh-1 binding sites are mutated, CBP had no enhancing effect either, suggesting that the binding of Gsh-1 is necessary for Gsh-1/CBP synergism (Fig. 7BGo). In addition, when each individual Gsh-1 binding site (G2–G5) was located in a heterologous promoter (thymidine kinase minimal promoter), no synergistic effect of Gsh-1 and CBP was observed (Fig. 8Go).



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Figure 7. Synergistic Activation of Gsh-1 and CBP on the GHRH Gene Promoter

A, JEG-3 cells were cotransfected with GRH472Luc and the Gsh-1 and/or CBP expression vectors, and the GHRH 5'-promoter activities were determined by luciferase assay. B, JEG-3 cells were cotransfected with the mG1–5 construct and the Gsh-1 and/or CBP expression vectors, and the GHRH 5'-promoter activities were determined by luciferase assay. ß-Galactosidase expression vector was used as an internal control. *, P < 0.01 vs. GRH472Luc alone. **, P < 0.05 vs. GRH472Luc+Gsh-1.

 


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Figure 8. Effects of Gsh-1/CBP Coexpression in Gsh-TK-Luc Constructs

A, Structure of the Gsh-TK-Luc construct. Each Gsh-1 binding site (G2–G5) in the GHRH 5'-promoter was isolated and located in front of the TK minimal promoter-luciferase gene. B, JEG-3 cells were cotransfected with each Gsh-TK-Luc construct and the Gsh-1 and/or CBP expression vectors, and the promoter activities were determined by luciferase assay. ß-Galactosidase expression vector was used as an internal control. *, P < 0.05 vs. each Gsh-TK-Luc activity without CBP/Gsh-1.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although more than 10 yr have passed since the GHRH gene with its promoter region was cloned (6, 7), virtually no report has emerged dealing with its transcriptional regulation of GHRH. In fact, previous efforts have been hampered mainly by the lack of a homologous cell line that would be appropriate for studying the tissue-specific regulation of the GHRH gene. Recent findings of the Gsh-1 transcription factor of the homeobox type (9, 10), however, prompted us to search for appropriate host cells expressing the homeobox gene, and we finally found the GHRH gene promoter to be well expressed in some placental and neuronal cell lines in which Gsh-1 is endogenously expressed.

Tissue specificity of the expression of the hypothalamic or pituitary hormone genes is, in many cases, determined by the existence of specific transcription factors. For example, the expression of GH, PRL, and TSH genes are largely dependent on the POU domain transcription factor Pit-1 (16, 17, 18), and CRH and vasopressin by homeobox protein Brain-2 (19, 20). Regarding GHRH, the work by Potter and associates (9, 10) using homologous recombination techniques strongly suggests that Gsh-1 is the key determinant of GHRH gene expression. Our data obtained in this study support the notion, showing that Gsh-1 is a prerequisite for maintaining the transcriptional activity of the GHRH gene. Indeed, the promoter activity was well maintained in the JEG-3 and Gsh+/+ hypothalamic cell lines, both of which express Gsh-1 (14), whereas much less activity was observed in the Gsh-/- cell line. Overexpression of Gsh-1 in JEG-3 further enhanced the promoter activity, indicating the dependency of GHRH gene expression on the homeobox protein. Moreover, RT-PCR analysis showed Gsh-1 mRNA expression to be present in any cell line or tissue that expressed GHRH mRNA. On the other hand, expression of Gsh-1 is not always accompanied by that of GHRH, and, in fact, short-term expression of Gsh-1 alone did not rescue the promoter activity of GHRH gene in Gsh-/- or other cell lines (data not shown). We assume that some additional Gsh-1-dependent transcriptional factor(s) is also needed to maintain the expression of the GHRH gene.

In the 5'-promoter region of the GHRH gene we examined, five putative Gsh-1 binding sites were recognized through the homology search technique. The results from the EMSA method showed that Gsh-1 actually binds to most of the consensus sequences in the promoter. The binding characteristics of each site may be somewhat different, because a clear supershift was observed in G2 and G3 whereas the binding was disrupted in G4 and G5. In any event, the functional significance of the binding was supported by the deletion analysis, which showed a marked decrease in the basal transcriptional activity when all the binding sites were eliminated. Induction of mutation into each binding site, however, did not affect the promoter activity, whereas induction into all sites markedly decreased the promoter activity of the GHRH gene, suggesting that alternative Gsh-1 binding is necessary and sufficient to maintain the promoter activity. A similar regulatory pattern is reported in the rat PRL gene promoter, in which four Pit-1 binding sites exist and function to maintain the transcriptional activity (21, 22).

In this study, we also showed the positive role of CBP, a coactivator protein that is ubiquitously expressed in the nucleus of most cells. Specifically, the promoter activity of the GHRH gene was found to be maximal when CBP and Gsh-1 were coexpressed, suggesting the positive cooperation between the two factors. Cohen et al. (15) reported the direct interaction between Pit-1 and CBP in the regulation of the GH gene promoter, and that a similar mode of regulation may be occurring in the promoter of GHRH. Our efforts, however, have so far failed to demonstrate the protein-protein interaction between the two factors using GST pull-down assay or EMSA (using full-length, in vitro translated CBP and GST-Gsh-1 fusion protein), or the yeast two-hybrid system (using fragments of CBP and Gsh-1) (data not shown). Thus, some additional unknown factor(s) may be necessary to reconstitute the CBP interaction to Gsh-1 in vitro. This hypothesis is supported by our data showing that Gsh-1/CBP synergy was not observed when each Gsh-1 binding site alone was located in the heterologous promoter.

Homeobox proteins usually play an important role in development and organogenesis during the embryonic stage. In fact, Gsh-1-deficient mice show agenesis of GHRH neurons in the arcuate nucleus with the dwarf phenotype (10). However, our results showing the expression of Gsh-1 mRNA in the hypothalamus of the 7-month-old rat strongly suggest that Gsh-1 protein exists and plays an important role in the brain of mature animals.

We carried out most of the experiments in this study using the JEG-3 placental cell line. Although the cells have endogenous GHRH mRNA and peptide, expression of GHRH is believed to be regulated mainly by placenta-specific alternative promoter (23). However, the construct we used in our experiments includes only the hypothalamic promoter region and does not contain the placenta-specific region, indicating that the regulation observed does not reflect the placenta-specific promoter. Instead, the results are likely to show the hypothalamic mode of regulation because of the Gsh-1-dependent regulation occurring in the hypothalamic promoter region.

Finally, the dwarf phenotype of Gsh-1 knockout mice suggests that some human familial dwarfism may be caused by mutation of the Gsh-1 gene. Since we cloned the human homolog of the Gsh-1 gene, the nucleotide sequence of which is now available, it is an additional possible candidate for disease-causing genes in dwarf patients in which no mutation is found in GHRH, GHRH receptor, or GH genes. Further research may add a novel type of genetic dwarfism in the future.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RT-PCR
Expression of Gsh-1 and GHRH in various cell lines and adult rat tissues (see Table 1Go) was examined by the RT-PCR method. Total RNA was isolated from the monolayer culture of the cells or tissues of the adult Sprague Dawley (Chubu Science Materials, Nagoya, Japan) rat using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD), and 1 µg each was applied for each reaction with specific primer sets: sense, 5'-GACAAGAAGGCTCCGGAGGG-3', and antisense, 5'-CAGCTGGTTCGAGCTGCTGT-3' for the human/rat Gsh-1 mRNA; sense, 5'-GGGTGTTCTTCTTTGTGATCC-3', and antisense, 5'-TCACAGGAGGAATCTTCATC-3' for the human GHRH mRNA; sense, 5'-TATGCAGATGCCATCTTCACC-3', and antisense, 5'-TCAAGCCTCCGCTGAAAGCT-3' for the rat and mouse GHRH mRNA. The primer sets for GHRH span exons 3–5 and thus amplify generic GHRH transcripts. An RNA sample without reverse transcriptase was used as a negative control.

Plasmid Constructions
The 5'-flanking region of the rat GHRH gene (-472 to +71; +1 designates the transcription start site), kindly provided by Dr. Kelly E. Mayo (Northwestern University, Evanston, IL) (7), was fused with the luciferase reporter gene in the pA3Luc plasmid (GRH472Luc). Deletion mutants of various lengths of the promoter were constructed as well (GRH304Luc, GRH247Luc, GRH187Luc, GRH133Luc, GRH47Luc). The mutated GRH472Luc constructs (mG1, mG2, mG3, mG4, mG5, and mG1–5) (see Fig. 5Go for details) were constructed by the PCR-based site directed mutagenesis technique (24). The Gsh-TK-Luc constructs (G2-TK-, G3-TK-, G4-TK-, and G5-TK-Luc) were constructed by insertion of annealed oligonucleotides in the pFlashII plasmid (SynapSys Co., Burlington, MA) containing thymidine kinase (TK) minimal promoter: sense, 5'-GGCCGAAATATAATTACTAGG-3', and antisense, 5'-GATCCCTAGTAATTATATTTC-3' for G2; sense, 5'-GGCCGATTCAACATTATATTG-3', and antisense, 5'-GATCCAATATAATGTTGAATC-3' for G3; sense, 5'-GGCCGGGTCTAAATTAGTGGG-3', and antisense, 5'-GATCCCCACTAATTTAGACCC-3' for G4; sense, 5'-GGCCGTTTCCCCATTACTTTG-3', and antisense, 5'-GATCCAAACTAATGGGGAAAC-3' for G5. Mouse Gsh-1 cDNA was isolated from mouse genomic DNA and subcloned into pRC/RSV (Rous sarcoma virus) mammalian expression vector (Invitrogen, Groningen, The Netherlands). Sequence analyses were performed for all constructs made by PCR. Dr. Toshihiro Nakashima (University of Tsukuba, Tsukuba, Ibaragi, Japan) kindly provided the CBP expression vector (RSV-CBP).

Cell Culture and Transfection
The human placental cell line JEG-3 and the mouse hypothalamic neuronal cell lines (Gsh+/+, Gsh-/-) were grown in DMEM supplemented with 10% FBS. Cultures were maintained at 37 C in a humid atmosphere of 5% CO2. In each experiment, cells were plated in 3.5-cm dishes with 70% confluency (~2 x 106 cells per dish), and the plasmids used (3 µg of test plasmid(s) and 0.3 µg of ß-galactosidase expression vector as an internal control per dish) were transfected with TransIT LT-100 (Mirus, Madison, WI). In Gsh-1 or CBP coexpression studies, 0.3–0.6 µg of Gsh-1/CBP expression plasmids or empty vector (promoter alone) was used so that the total amount of expression vector was equal.

Forty-eight hours after transfection, cells were harvested with lysis buffer containing 1% Triton X-100 and centrifuged, and the supernates were used for luciferase and ß-galactosidase assays. Luciferase assay was performed as previously described (25). Briefly, 100 µl of each supernatant were added to 400 µl of reaction buffer, and the reactions were started by the injection of 200 µl of luciferin solution containing 0.2 mM D-luciferin (Sigma, St. Louis, MO). Light output was measured for 20 sec at room temperature using a luminometer (Lumat LB 9501, EG&G Berthold, Oak Ridge, TN). ß-Galactosidase assay was determined using commercially available assay kits (Galacto-Light Plus Kit, Tropix, Inc., Bedford, MA) based on the manufacturers’ instructions. Luciferase activities were divided by the ß-galactosidase activities to correct for the transfection efficiency.

Preparation of Fusion Protein
Mouse Gsh-1 cDNA was incorporated into pcDNA 3.1/myc-His (+) A plasmid (Invitrogen). After the myc-tagged cDNA was subcloned into pGEX 4T-2 expression vector (Amersham Pharmacia Biotech, Piscataway, NJ) to produce GST- and myc-tagged fusion protein, the protein product was subsequently purified using glutathione-sepharose 4B beads (Amersham Pharmacia Biotech). Western blot of the tagged protein using anti-Myc antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) showed a single band in the appropriate size. The purified fusion protein was used in EMSA as shown below.

EMSA
To assay protein binding, synthetic double-stranded oligonucleotides encompassing the Gsh-1 binding sequences of the rat GHRH 5'-promoter region (G1: sense, 5'-TGAACAAAACAGAATTATAGGG-3', antisense, 5'-AGTTCCCTATAATTCTGTTTTG-3'; G2: sense, 5'-TGGTAAATATAATTACTAGA-3', antisense, 5'-AGAGTCTAGTAATTATATTT-3'; G3: sense, 5'-TAGTCATTCAACATTATATTC-3', antisense, 5'-TGAACAGAATATAATGTTGAATG-3'; G4: sense, 5'-GTATGGGTCTAAATTAGTGGG-3', antisense, 5'-TGCCCCCACTAATTTAGACCC-3'; G5: sense, 5'-CGACTTTTCCCCATTAGTTTG-3', antisense, 5'-CACGCAAACTAATGGGGAAAA-3') were labeled with [{alpha}32P] deoxy-CTP (Amersham Pharmacia Biotech) and DNA polymerase (Klenow enzyme, New England Biolabs, Inc., Beverly, MA). The labeled fragment (2–5 fmol, 40,000 cpm) was then incubated with 1–2 µg of GST gsh-1-Myc fusion proteins in the EMSA binding buffer (40 mM HEPES, pH 7.9, 75 mM KCl, 0.2 mM EDTA, 10% glycerol, 1 mM dithiothreitol) and 2 µg poly dI-dC (Amersham Pharmacia Biotech) for 15 min on ice. Pure GST protein was used as a control. For competition assays, a 100-fold molar excess of nonlabeled probe was added to the reaction solution. For supershift analysis, anti-Myc antibody (Santa Cruz Biotechnology, Inc.) was added to the reaction and incubated for additional 60 min on ice. The mixture was then subjected to 4% nondenaturing polyacrylamide gel, and the electrophoresis was run at 160 V with cooling for 4 h. The gels were dried and subjected to autoradiography by exposing the gel to Kodak X-AR film (Eastman Kodak Co., Rochester, NY).

Data Analysis
Samples in each group of the experiments were in triplicate. All experiments were repeated three times at independent times, and the data are presented as mean ± SE of the averaged data from the three experiments. When statistical analyses were performed, data were compared by one-way ANOVA with Fisher’s protected least significant difference test, and P values of <0.05 were considered significant.


    ACKNOWLEDGMENTS
 

We are grateful to Drs. Kelly E. Mayo (Northwestern University, Evanston, IL) and Steven S. Potter (University of Cincinnati, Cincinnati, OH) for providing the rat GHRH gene and Gsh+/+, -/- cells, respectively. We also thank Dr. Toshihiro Nakashima (Tsukuba University, Tsukuba, Japan) for providing the CBP expression vector.


    FOOTNOTES
 
This work was supported in part by the Novo Nordisk Growth Award (to Y.I.), Japan.

Abbreviations: CBP, CREB-binding protein; GST, glutathione-S-transferase; RSV, Rous sarcoma virus; TK, thymidine kinase.

Received for publication December 29, 2000. Accepted for publication August 29, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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