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 40938, Japan
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ABSTRACT
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The GA-binding protein (GABP), a transcription
factor with a widespread tissue distribution, consists of two subunits,
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
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
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.
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INTRODUCTION
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The transcription factor GA-binding protein (GABP) is composed of
two subunits,
and ß1. GABP
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.
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RESULTS
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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
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. 1A
). Subsequent gel
mobility-shift assays showed that GST-GABP
bound to the
sequence nt -116 to -80 only in the presence of GST-GABPß1 (Fig. 1B
).

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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 ( , ß1, or and
ß1) as described in Materials and Methods.
Arrows indicate probe-protein complexes.
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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
and GABPß1 (Fig. 2A
). The same complex of GABP was also
observed with crude nuclear extracts prepared from FRTL-5 cells (Fig. 2B
). To confirm the presence of GABP
and GABPß1 in FRT and FRTL-5
cells, we subjected crude nuclear extracts from these cells to
immunoblot analysis. Antibodies to GABP
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 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.
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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. 3
, and Fig. 4
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 13) 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. 4 .
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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.
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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. 5A
, 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. 5B
). We further examined the effect of
methylation on GABP binding to the TSHR gene promoter by DNase I
footprint analysis (Fig. 5C
). Nucleotides -116 to -86 of the TSHR
gene were protected by GST-GABP
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
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 and GST-GABPß1 (1 µg
each). The protected region is indicated by the solid
bar showing the 5'- and 3'-positions.
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Effect of Methylation on TSHR Gene Transcription
Finally, we investigated the effect of methylation on TSHR gene
transcription by chloramphenicol acetyltransferase (CAT) assay (Fig. 6
). FRTL-5 cells were transfected with
mock-methylated or HpaII-methylated plasmids in the absence
or presence of GABP
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 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.
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DISCUSSION
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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
1020 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-
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.
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MATERIALS AND METHODS
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Cell Culture
FRTL-5 cells were kindly provided by Dr. L. D. Kohn (NIH,
Bethesda, MD) and maintained in Coons modified Hams 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
Coons modified Hams 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 Dulbeccos 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
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 [
-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
suppliers protocol and then end-labeled. Labeled DNA fragments
(30,000 cpm) were incubated with recombinant mouse GABP
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 [
-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
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
(pCR3-
), 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).
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ACKNOWLEDGMENTS
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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
and GABPß1 cDNAs and
anti-GABP antiserum.
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FOOTNOTES
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Address requests for reprints to: Toshimasa Onaya, M.D., Ph.D., Professor and Chairman, Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Yamanashi 40938, 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.
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