Regulation of the Rat Thyrotropin Receptor Gene by the Methylation-Sensitive Transcription Factor GA-Binding Protein

Norihiko Yokomori, Masato Tawata, Tukasa Saito, Hiroki Shimura and Toshimasa Onaya

Third Department of Internal Medicine Yamanashi Medical University Tamaho, Yamanashi 409–38, Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GA-binding protein (GABP), a transcription factor with a widespread tissue distribution, consists of two subunits, {alpha} and ß1, and acts as a potent positive regulator of various genes. The effect of GABP on transcription of the TSH receptor (TSHR) gene in rat FRTL-5 thyroid cells has now been investigated. Both deoxyribonuclease I footprint analysis and gel mobility-shift assays indicated that bacterially expressed glutathione S-transferase fusion proteins of GABP subunits bind to a region spanning nucleotides (nt) -116 to -80 of the TSHR gene. In gel mobility-shift assays, nuclear extracts of FRTL-5 cells and FRT cells yielded several specific bands with a probe comprising nt -116 to -80. Supershift assays with antibodies to GABP{alpha} and to GABPß1 showed that GABP was a component of the probe complexes formed by the nuclear extracts. Immunoblot analysis confirmed the presence of both GABP subunits in the nuclear extracts. A reporter gene construct containing the TSHR gene promoter was activated, in a dose-dependent manner, in FRTL-5 cells by cotransfection with constructs encoding both GABP{alpha} and GABPß1. Both GABP binding to and activation of the TSHR gene promoter were prevented by methylation of CpG sites at nt -93 and -85.

These CpG sites were highly methylated (>82%) in FRT cells and completely demethylated in FRTL-5 cells, consistent with expression of the TSHR gene in the latter, but not the former. These results suggest that GABP regulates transcription of the TSHR gene in a methylation-dependent manner and that methylation of specific CpG sites and the methylation sensitivity of GABP contribute to the failure of FRT cells to express the endogenous TSHR gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The transcription factor GA-binding protein (GABP) is composed of two subunits, {alpha} and ß1. GABP{alpha} is a low-affinity DNA-binding protein with an Ets domain, whereas GABPß1 contains a Notch-related structural motif (1, 2). Both subunits show a widespread tissue distribution (2). GABP is thought to act as a transcription factor for various genes, including the aldose reductase gene, the adenovirus E4 gene, the ß2-integrin gene, the folate-binding protein gene, and the cytochrome c oxidase subunit IV gene (3, 4, 5, 6, 7). We also recently showed that GABP acts as a methylation-sensitive transcriptional activator of the male-specific cytochrome P-450 gene Cyp 2d-9 (8).

Many genes are regulated by specific combinations of widely expressed factors and tissue-specific factors. The TSH receptor (TSHR) gene is regulated by thyroid transcription factor-1 (TTF-1), a tissue-specific factor that binds to the region spanning nucleotides (nt) -189 to -175 of the 5'-flanking region of the TSHR gene and activates transcription (9, 10). The TSHR gene promoter also contains an octameric cAMP response element (CRE)-like sequence between nt -139 and -132. A 10-nt tandem repeat sequence between nt -162 and -141, immediately 5' to the CRE, acts as a repressive element with regard to constitutive CRE enhancer activity. This decanucleotide tandem repeat sequence, which interacts with single-stranded DNA-binding proteins, modulates the interaction of the CRE with CRE-binding proteins (11). The sequence GGAA, which is the core binding site for several members of the Ets family of transcription factors, is present within the TSHR gene minimum promoter (nt -220 to -1) (9, 10, 11). Ikuyama (10) previously showed that CpG sites at nt -93 and -85 are methylated in nonfunctioning FRT and proposed the importance of the methylation to TSHR gene expression by comparing methylation at these nucleotides in FRT and FRTL-5 thyroid cells. FRT cells are nonfunctioning thyroid cells that do not express the TSHR, while FRTL-5 cells are functioning and do express the TSHR. We have now therefore examined the effect of GABP and CpG site methylation on expression of the TSHR gene in FRTL-5 rat thyroid cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Direct Binding of Bacterially Expressed GABP to the 5'-Flanking Region of the TSHR Gene
Deoxyribonuclease I (DNase I) footprint analysis revealed that bacterially expressed GST-GABP{alpha} and GABPß1 bound to nt -116 to -80 of the TSHR gene sense strand and nt -116 to -86 of the TSHR gene antisense strand, respectively, but only in the presence of both proteins (Fig. 1AGo). Subsequent gel mobility-shift assays showed that GST-GABP{alpha} bound to the sequence nt -116 to -80 only in the presence of GST-GABPß1 (Fig. 1BGo).



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Figure 1. Direct binding of Bacterially Expressed GABP to the TSHR Gene Promoter as Revealed by DNase I Footprint Analysis and Gel Mobility-Shift Assays

A, Footprints were obtained by DNase I digestion of sense and antisense strand templates (nt -199 to -36) in the absence (control), or presence of bacterially expressed GST-GABP fusion proteins (1 µg each), as indicated. The protected regions are indicated by solid bars showing the 5'- and 3'-positions. B, Gel mobility-shift assays were performed with probes encompassing nt -116 to -80 and 100 ng of GST-GABP fusion proteins ({alpha}, ß1, or {alpha} and ß1) as described in Materials and Methods. Arrows indicate probe-protein complexes.

 
Binding of GABP Present in FRT and FRTL-5 Cell Nuclear Extracts to the 5'-Flanking Region of the TSHR Gene
Gel mobility-shift assays were also performed with crude nuclear extracts prepared from FRT and FRTL-5 cells. Several specific bands were observed from FRT cell nuclear extracts with the probe comprising nt -116 to -80; supershift assays showed that the specific complex indicated by the arrow contained GABP{alpha} and GABPß1 (Fig. 2AGo). The same complex of GABP was also observed with crude nuclear extracts prepared from FRTL-5 cells (Fig. 2BGo). To confirm the presence of GABP{alpha} and GABPß1 in FRT and FRTL-5 cells, we subjected crude nuclear extracts from these cells to immunoblot analysis. Antibodies to GABP{alpha} and to GABPß1 detected 60- and 52-kDa proteins, respectively (data not shown), sizes consistent with those of the corresponding antigens.



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Figure 2. Supershift Analysis with Antibodies to GABP of TSHR Gene Promoter Complexes Formed with FRT and FRTL-5 Cell Nuclear Proteins

Crude nuclear extracts (5 µg) from FRT (A) and FRTL-5 cell (B) were incubated, where indicated, with antibodies to either GABP{alpha} or GABPß1 (1 µl) before the addition of radioactive probes comprising nt -116 to -80. Preimmune serum (1 µl) was added to the reaction mixture for control. A 100-fold molar excess of the corresponding unlabeled probe (wild type) or of an Oct1 consensus sequence was used to assess binding specificity. Arrows indicate specific complexes that are supershifted by antibodies.

 
Differential Methylation of the TSHR Gene in FRT and FRTL-5 Cells
Genomic DNAs were prepared from FRT and FRTL-5 cells and treated with sodium bisulfite. The TSHR gene promoter was amplified and sequenced to determine the methylation status of the CpG/-190, CpG/-154, CpG/-142, CpG/-93, CpG/-85, CpG/-72, CpG/-38, CpG/-36, and CpG/-31 sites by the method of Frommer et al. (12). Some examples of sequences are depicted in Fig. 3Go, and Fig. 4Go summarizes the methylation levels at each site in FRT and FRTL-5 cells. The TSHR gene is expressed in FRTL-5 cells but not in FRT cells (10), and, consistent with this cell-specific expression, all of these CpG sites of the TSHR gene were completely demethylated in FRTL-5 cells. In contrast, the CpG/-154, CpG/-142, CpG/-93, and CpG/-85 sites were highly methylated (>71%), and the CpG/-190, CpG/-72, CpG/-38, CpG/-36, and CpG/-31 sites were methylated to a lesser extent (<32%) in FRT cells.



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Figure 3. DNA Sequences of Bisulfite-Treated Promoters

The promoter sequences with different methylation patterns from FRT cells and FRTL-5 cells are shown. Three examples of sequences from FRT cells (clone 1–3) and one from FRTL-5 cells are shown. The arrows indicate CpG/-31, CpG/-38, CpG/-72, CpG/-85, CpG/-93, CpG/-142, and CpG/-154 in TSHR gene. The sequencings were carried out using another 25 clones in FRT cells and 27 clones in FRTL-5 cells, and the rate of methylation at each site was calculated as shown in Fig. 4Go.

 


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Figure 4. Level of CpG Methylation

The levels of methylation are shown as percentages of the total number of sequences (indicated by N). The values were generated from the two independent bisulfite treatments and the two separate amplifications from each bisulfite-treated DNA sample. SEs were obtained from four independent experiments.

 
Effect of DNA Methylation on GABP Binding
We recently showed that GABP is a methylation-sensitive transcription factor, binding to the Cyp 2d-9 and the Slp genes only when the CpG site within the consensus binding site is not methylated (8, 13). The region of the TSHR gene encompassing nt -116 to -80 contains two CpG sites, at nt -93 and -85, that are highly methylated (>82%) in FRT cells and completely demethylated in FRTL-5 cells. Although, these CpG sites are not within the consensus binding elements of GABP, they are within the footprinted binding site, nt -116 to -80, containing two GABP-binding elements at nt -110 to -105 and -101 to -96; we, therefore, examined the effect of CpG/-93 and CpG/-85 methylation on GABP binding to the TSHR gene by gel mobility-shift assay. For these experiments, we used three probes: a wild-type probe (nt -116 to -80) and probes in which both CpG sites had been methylated with the use of either HpaII methylase or 5-methyl deoxycytidine CED phosphoamidite. As shown in Fig. 5AGo, GABP did not bind to either methylated probe. Furthermore, whereas unlabeled wild-type probe competed with the labeled wild-type probe for the binding of GABP, unlabeled methylated probe [prepared with 5-methyl deoxycytidine CED (ß-cyanoethyl-N,N-diisopropylamino) phosphoramidite] had no effect on GABP binding to the labeled wild-type probe (Fig. 5BGo). We further examined the effect of methylation on GABP binding to the TSHR gene promoter by DNase I footprint analysis (Fig. 5CGo). Nucleotides -116 to -86 of the TSHR gene were protected by GST-GABP{alpha} and GST-GABPß1 with a probe subjected to mock methylation but not with a probe that had been methylated by HpaII methylase.



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Figure 5. Effect of Methylation on GABP Binding to the TSHR Gene Promoters

A, Gel mobility-shift assay of the reaction mixture containing GABP{alpha} and GABPß1 fusion proteins together with a wild-type probe (nt -116 to -80) or a probe in which both CpG sites had been methylated with the use of either HpaII methylase or 5-methyl deoxycytidine CED phosphoamidite (5-methyl-C type). B, Gel mobility-shift assay of a reaction mixture containing both GST-GABP fusion proteins, 32P-labeled wild-type probe (nt -116 to -80), and the indicated molar excesses of unlabeled, double-stranded wild-type, methylated (5-methyl-C type), or Oct1 probes as competitors. Arrows in panels A and B indicate specific complexes. C, Footprint analysis of an HpaII-methylated or unmethylated (mock methylated) DNA template (nt -199 to -36) in the absence (control) or presence of GST-GABP{alpha} and GST-GABPß1 (1 µg each). The protected region is indicated by the solid bar showing the 5'- and 3'-positions.

 
Effect of Methylation on TSHR Gene Transcription
Finally, we investigated the effect of methylation on TSHR gene transcription by chloramphenicol acetyltransferase (CAT) assay (Fig. 6Go). FRTL-5 cells were transfected with mock-methylated or HpaII-methylated plasmids in the absence or presence of GABP{alpha} and GABPß1 expression vectors. The basal activity of the HpaII-methylated pTRCAT5'-146 plasmid was reduced by 60% compared with that of the mock-methylated plasmid. Furthermore, whereas GABP increased transcription of the mock-methylated pTRCAT5'-146 plasmid, it had no effect on the activity of the HpaII-methylated plasmid. This activation of the mock-methylated plasmid by GABP was dose dependent (data not shown). GABP had no effect on the activity of either mock-methylated or HpaII-methylated plasmid pTRCAT5'-90, which does not contain the binding site for GABP. The basal activity of pTRCAT5'-90 or of the promoterless plasmid p8CAT was not affected by methylation.



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Figure 6. Effect of Methylation on TSHR Gene Transcription

FRTL-5 cells were transfected with 20 µg of the pTRCAT5'-146 plasmid or pTRCAT5'-90 plasmid after it had been subjected to methylation with HpaII or to a mock-methylation reaction. Where indicated, cells were also transfected with 10 µg each of expression vectors encoding GABP{alpha} and GABPß1. CAT activity of cell lysates was measured, normalized to the amount of ß-galactosidase activity (from a cotransfected plasmid) in each extract, and expressed relative to the normalized CAT activity of cells transfected with mock-methylated p8CAT. SEs were obtained from four independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The heteromeric transcription factor GABP has been shown to activate several genes (3, 4, 5, 6, 7, 8). In addition to its role as an activator, however, GABP can act as a repressor of gene expression by interacting with other transcription factors. Rosmarin et al. (14) showed that GABP and PU.1 compete for binding to the promoter of the ß2-integrin gene, yet cooperate to increase gene transcription. GABP also acts as a repressor of mouse ribosomal protein gene transcription (15), apparently by interfering with the formation of the transcriptional initiation complex. The hexanucleotide 5'-CGGAA(A or G)-3' was identified as the GABP-binding site in the herpes simplex virus (HSV) promoter (1, 2). This sequence is repeated in the HSV promoter, and such repetition appears to be essential for GABP binding. The affinity of GABP for DNA containing duplicated binding sites is 10–20 times higher than for DNA with a single binding site (16). The corresponding binding sequence, however, is not repeated in the Cyp 2d-9 promoter or in the adenovirus E4 promoter or E1A core enhancer (8, 17, 18, 19). We have now shown that GABP binds to nt -116 to -80 of the TSHR gene by both DNase I footprint analysis and gel mobility-shift assays. This region contains two copies of the reverse complement of the GABP-binding site (5'-CTTCCT and 5'-TTTCCT, nt -110 to -105 and -101 to -96, respectively), with the exception that the 3'-nucleotide G is substituted with a T, and was shown to mediate activation of the TSHR gene by GABP. The potential GABP-binding sites in this region overlap the transcription start sites and are near the CRE (nt -139 to -132). A Y-box protein binds to nt -162 to -151, immediately 5' to the CRE, of the TSHR gene and acts as a repressor by decreasing the constitutive CRE enhancer activity (20). This Y-box protein also binds to nt -120 to -113 of the TSHR gene, immediately 3' to the CRE, in a region that overlaps the GABP-binding region (nt -116 to -80). Thus, protein-protein interactions among GABP, the Y-box protein, the CRE-binding protein, and the transcriptional initiation complex may contribute to regulation of the TSHR gene.

DNA methylation is an important mechanism by which gene expression is regulated during growth and development (21, 22, 23, 24). In general, DNA methylation is associated with inhibition of gene expression. A high degree of DNA methylation can result in cell transformation (25), whereas demethylation of MyoD or another regulatory gene results in the conversion of fibroblasts to myoblasts (26). The reduced thyroglobulin gene expression in Ras-transformed FRTL-5 thyroid cells is also associated with methylation of the gene promoter (27); treatment of the transformed cells with the DNA demethylating agent 5-azacytidine reactivates the thyroglobulin gene promoter (28). Various methylation-sensitive transcription factors, including activator protein-2, CRE-binding protein/activating transcription factor, and nuclear factor-{kappa}B, have been described (23). Thus, methylation of the CRE of the human proenkephalin gene prevents activator protein-2 binding and stimulation of transcription (29, 30). Myeloid-specific transcription of the mouse M lysozyme gene is also regulated by a single CpG methylation site within the enhancer (31).

We recently added GABP to the list of factors sensitive to methyl-CpG (8). The CpG sites in the promoters of the sex-specific P-450 genes exhibit sex-specific patterns of methylation related to expression of the genes in the liver of mice. The CpG site at nt -97 in the promoter of the male-specific Cyp 2d-9 gene is preferentially demethylated in male mice. GABP transactivates the male-specific Cyp 2d-9 promoter through direct binding to the regulatory element 5'-TTC-97CGGGC; GABP does not bind to the promoter when the CpG/-97 site is methylated. Thus, we proposed that DNA demethylation and the methylation-sensitive transcription factor GABP underlie the sex-specific transcription of the Cyp 2d-9 gene. We have now shown that the methylation of CpG/-93 and CpG/-85 abolishes the binding of GABP to the TSHR gene promoter and reduces basal TSHR gene transcription. Thus, CpG sites located outside of the consensus-binding site of GABP affect the binding of GABP to the promoter of the TSHR gene.

The TSHR gene is expressed in FRTL5 cells but not in FRT cells, which are derived from rat thyroid (32) and have the characteristics of epithelial cells. FRT cells do not secrete thyroglobulin and do not express thyroperoxidase. Although they express Pax-8, they do not express TTF-1 (33, 34), which may largely explain the failure of FRT cells to express the endogenous TSHR gene. We have now also shown that CpG/-93 and CpG/-85 in the TSHR gene are highly methylated (>82%) in FRT cells, whereas these sites are completely demethylated in FRTL-5 cells consistent with the pattern of TSHR gene expression. It appears, therefore, that GABP does not transactivate the TSHR gene promoter when these CpG positions are methylated in FRT cells, and that GABP stimulates transcription of the TSHR gene when these CpG positions are demethylated in FRTL-5 cells. Thus, methylation of the promoter and the methylation-sensitive transcription factor GABP may also contribute to the failure of FRT cells to express the endogenous TSHR gene. All of the the CpG islands in the TSHR gene between -190 and -31 were completely demethylated in FRTL-5 cells, although only two of them appear relevant for GABP. Therefore, it remains possible that the other CpG sites, especially CpG/-154 and CpG/-142, which were also highly methylated in FRT cells in the TSHR gene promoter, and other methylation-sensitive transcription factors may also contribute to the regulation of this gene. In conclusion, the heteromeric transcription factor GABP can bind to, and thereby transactivate, the TSHR gene promoter. Moreover, the binding of GABP is sensitive to methylation of CpG sites at nt -93 and -85, and transcription of the TSHR gene is decreased by methylation of these CpG sites. Therefore, GABP may regulate the transcription of the TSHR gene in a manner dependent on the methylation status of the CpG sites at nt -93 and -85.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
FRTL-5 cells were kindly provided by Dr. L. D. Kohn (NIH, Bethesda, MD) and maintained in Coon’s modified Ham’s F-12 medium supplemented with 5% calf serum and a six-hormone mixture (6H) containing insulin (10 µg/ml), somatostatin (10 ng/ml), hydrocortisone (10 nM), transferrin (5 µg/ml), glycyl-L-histidyl-lysine acetate (10 ng/ml), and bovine TSH (10 mU/ml); all 6H components were obtained from Sigma (St. Louis, MO). FRT cells were also provided by Dr. L. D. Kohn and maintained in Coon’s modified Ham’s F-12 medium supplemented with 5% calf serum.

Plasmids
The plasmids pTRCAT5'-146, and pTRCAT5'-90, containing 146 and 90 bp, respectively, of the 5'-flanking region of the rat TSHR gene, upstream of a CAT reporter gene, were kindly provided by Dr. L. D. Kohn (10, 11). The promoterless CAT plasmid, p8CAT, was also provided by Dr. L. D. Kohn. The in vitro methylation of plasmid DNA was performed using HpaII methylase according to the instructions of the supplier (New England Biolabs, Beverly, MA). Unmethylated control plasmids (mock-methylated plasmids) were prepared identically without addition of methylase. Before transfection, the methylated and mock-methylated plasmids were phenol-extracted and ethanol-precipitated. Complete methylation was verified by digesting the DNA with an excess of HpaII restriction enzyme.

Sequencing of the Sodium Bisulfite-Treated Promoter
Genomic DNAs were prepared from FRT and FRTL-5 cells using the SDS/proteinase K method, digested with PstI, and then subjected to a sequential reaction to determine CpG methylation pattern according to Frommer et al. (12). The oligonucleotide primers were synthesized based on the reported sequences of the TSHR genes (10). The top strand of promoter sequence (-220/-1) of the TSHR gene was amplified using 10 µl of the bisulfite-reacted DNA as a template, and the oligonucleotides 5'-GGGGAAGCTTTTTGTTTGGATGGAGAGTTG and 5'-GGGGTCTAGATTTCCAAAAAACCTCCAATA as the 5'- and 3'-primers, respectively. The underlined regions indicate that a HindIII site and a XbaI site were added at each end of the amplified DNAs. Amplified DNAs were digested with HindIII and XbaI and then cloned into M13 mp19 vectors for DNA sequencing.

Nuclear Extracts
Nuclear extracts were prepared from FRT and FRTL-5 cells. Cells were harvested, washed with Dulbecco’s modified PBS without Mg2+ and Ca2+ (pH 7.4), and, after centrifugation at 500 x g, suspended in five pellet volumes of buffer A [10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, leupeptin (2 µg/ml), and pepstatin A (2 µg/ml) ] containing 0.3 M sucrose and 2% (vol/vol) Tween-40. The cells were then frozen, thawed, and gently homogenized, and nuclei were isolated by centrifugation of the homogenate at 25,000 x g through a 1.5 M sucrose cushion prepared in the same buffer. Nuclei were lysed in buffer B [10 mM HEPES-KOH (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 10% (vol/vol) glycerol, 0.5 mM phenylmethylsulfonyl fluoride, leupeptin (2 µg/ml), and pepstatin A (2 µg/ml)], and the lysate was centrifuged at 100,000 x g for 1 h. The resulting supernatant was dialyzed for use in DNase I footprint analysis or gel mobility-shift assays.

Bacterial Expression of GABP
The pCRII vector containing the mouse GABP{alpha} or GABPß1 coding sequence was digested with EcoRI and ligated with EcoRI-digested pGEX-2T (Pharmacia Biotech, Piscataway, NJ). Ten milliliters of an overnight culture of transformed bacteria were inoculated in 1 liter of LB medium supplemented with ampicillin (100 µg/ml) and glucose (0.2%). After incubation for 3 h at 37 C, isopropyl-ß-D-thiogalactopyranoside was added to a final concentration of 0.1 mM, and the cells were incubated for an additional 2 h. The bacterial cells were then harvested and the glutathione S-transferase (GST) fusion protein was purified on a glutathione-Sepharose 4B column (Pharmacia Biotech).

DNase I Footprint Analysis
We performed DNase I footprint analysis with a Sure Track footprinting kit (Pharmacia Biotech). The AvrII-BssHII (nt -199 to -36) fragments of the rat TSHR gene were end-labeled with [{gamma}-32P]ATP (>5000 Ci/mmol; Amersham, Arlington Heights, IL) with T4 polynucleotide kinase, and then purified by agarose gel electrophoresis. Methylated DNA was prepared using HpaII methylase according to the supplier’s protocol and then end-labeled. Labeled DNA fragments (30,000 cpm) were incubated with recombinant mouse GABP{alpha} and/or GABPß1 in 50 µl of 10 mM Tris-HCl buffer (pH 7.5) containing 2.5 µg of poly(deoxyinosinic-deoxycytidylic)acid, 50 mM NaCl, 2.5 mM MgCl2, 1 mM DTT, 0.5 mM EDTA, and 5% glycerol for 30 min at room temperature. Then the DNAs were digested by 1 U of DNase I for 30 sec, extracted with phenol-chloroform, and precipitated with ethanol. As the sequence markers, the corresponding DNA fragment was chemically cleaved at nucleotides G and A by the method of Maxam and Gilbert (35). Finally, the digested DNA samples were electrophoresed on an 8% polyacrylamide-7 M urea gel, and the gel was then dried, exposed to an imaging plate, and analyzed with a Bas 2000 image analyzer (Fuji, Tokyo, Japan).

Gel Mobility-Shift Assay
Each oligonucleotide was annealed to its complement and labeled by using [{alpha}-32P]dATP (>6000 Ci/mmol; Amersham) and DNA polymerase Klenow fragment. Methylated oligonucleotides were prepared by including 5-methyl deoxycytidine CED phosphoramidite (Pharmacia Biotech) during the appropriate cycle of synthesis or with the use of HpaII methylase. Each radioactive probe was incubated with 5 µg of nuclear proteins or 0.1 µg of GST fusion proteins of GABP{alpha} and GABPß1 in 10 µl of 20 mM Tris-HCl (pH 7.5) containing 1 µg of poly(deoxyinosinic-deoxycytidylic)acid, 50 mM NaCl, 0.1 mM DTT, and 10% glycerol at room temperature. In experiments using antiserum to GABP, nuclear extracts were incubated with the antiserum (1 µl) in the same buffer for 30 min at room temperature before adding the labeled probes and processing above. The following oligonucleotides were used in the studies as the probes:-116CTCCTCCTTCCTCCCTTTCCCTCCGGCACCCCGGTCT-80, and -116CTCCTCCTTC-CTCCCTTTCCCTCm5CGGCACCCm5CGGTCT-80. An Oct1 consensus oligonucleotide, 5'-AATTGCATGCCTGCAGGTGGACTCTAGAGGATCCATGCAAATGGATCCCCGGGTACCC-AGCTC, was also used as a nonspecific competitor.

Transient Expression Analysis
FRTL-5 cells were grown to 80% confluency in 6H medium, shifted to 5H medium for 1 day, and then returned to 6H medium for 1 day before transfection by electroporation (300 V; capacitance, 960 µfarad) (Gene Pulser; Bio-Rad, Richmond, CA). Cells were harvested, washed, and suspended at 1.5 x 107 cells/ml in 0.8 ml PBS and cotransfected with 20 µg of the pTRCAT plasmids or p8CAT plasmids, 10 µg of GABP{alpha} (pCR3-{alpha}), and GABPß1 (pCR3-ß1) expression plasmids, and 5 µg of the ß-galactosidase expression plasmid pCH110. The total amount of transfected DNA was adjusted to 45 µg by adding carrier DNA. The cells were pulsed, then plated, and cultured for 72 h. To measure CAT activity, the cells were lysed by freezing and thawing and the lysate (30 µg of protein) was incubated with [14C]chloramphenicol according to the method of Gorman et al. (36).


    ACKNOWLEDGMENTS
 
We thank Dr. L. D. Kohn (National Institutes of Health, Bethesda, MD) for the gift of the plasmids pTRCAT and p8CAT. We also thank Dr. M. Negishi (National Institutes of Environmental Health Sciences, Durham, NC) for the gift of GABP{alpha} and GABPß1 cDNAs and anti-GABP antiserum.


    FOOTNOTES
 
Address requests for reprints to: Toshimasa Onaya, M.D., Ph.D., Professor and Chairman, Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Yamanashi 409–38, Japan. E-mail: onayat{at}res.yamanashi-med.ac.jp

Received for publication December 31, 1997. Revision received March 30, 1998. Accepted for publication April 10, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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