Analysis of Differentiation-Induced Expression Mechanisms of Thyrotropin Receptor Gene in Adipocytes
Hiroki Shimura,
Asako Miyazaki,
Kazutaka Haraguchi,
Toyoshi Endo and
Toshimasa Onaya
The Third Department of Internal Medicine Yamanashi Medical
University Yamanashi 4093898, Japan
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ABSTRACT
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Rat adipose tissue, as well as differentiated
3T3-L1 cells, has been shown to express TSH receptor (TSHR) mRNA in
amounts approaching those in the thyroid. We investigated the molecular
mechanisms of TSHR gene expression in adipose cells. Primer extension
and cloned cDNA sequences showed that transcription of the TSHR gene in
rat adipose tissue was from multiple start sites clustered between -89
to -68 bp and almost identical to those in FRTL-5 thyroid cells. By
transient expression analysis, we localized, between -146 and -90 bp,
a positive regulatory element, the activity of which was markedly
increased after the differentiation of 3T3-L1 cells.
Deoxyribonuclease I protection showed that nuclear extracts from
differentiated 3T3-L1 cells strongly protected two sequences, from
-146 to -127 bp, including a cAMP response element-like
sequence and from -112 to -106 bp containing a putative
Ets-binding sequence. In differentiated 3T3-L1 cells, disruption
or deletion of either sequence was found to result in the loss of
enhancer activity, suggesting both elements may synergistically
activate the TSHR promoter. Electrophoretic mobility shift analysis
revealed the induction of new protein/DNA complexes formed either with
the cAMP response element-like site or with putative Ets elements after
the differentiation into adipocytes. In contrast, nuclear proteins,
whose binding to DNA was diminished after the differentiation of 3T3-L1
cells, were found to interact with the site contiguous to the 5'-end of
the putative Ets-binding sequence. Mutations of this binding site,
which reduced the protein/DNA complex formation, increased TSHR
promoter activity in undifferentiated cells. These observations
suggested that differentiation-induced diminution of suppressor
interactions may allow the enhancers to synergistically activate the
transcription of TSHR gene in adipocytes.
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INTRODUCTION
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Autoantibodies to the TSH receptor (TSHR), which are
present in patients with Graves disease, increase thyroidal cAMP
levels and cause hyperfunctioning of the thyroid gland (1). In
addition, Graves disease patients with a high titer of
thyroid-stimulating antibodies often exhibit exophthalmos and pretibial
dermopathy (2). Although TSH has been thought to act solely in the
control of thyroid function, extrathyroidal TSH binding has also been
documented (3, 4, 5, 6, 7, 8, 9, 10, 11), suggesting that expression of TSHR in these cells
may account for the extrathyroidal manifestations of Graves
disease.
Although several extrathyroidal cells, such as lymphocytes and
fibroblasts, have been shown to express TSHR mRNA detectable only by
RT-PCR (12, 13, 14, 15), functional TSHR has been detected in adipocytes (3, 16, 17, 18) and may be involved in the regulation of adipose function. We
have recently shown that rat adipose tissue (19), as well as cultured
rat preadipocytes (20), express amounts of TSHR mRNA similar to that
detected in the thyroid gland. The full-length TSHR cDNA, which we
isolated from rat fat cells (19), differed by only one amino acid from
the rat thyroid TSHR cDNA. Moreover, when transfected into Chinese
hamster ovary (CHO) cells, rat adipocyte TSHR cDNA was functionally
indistinguishable from thyroid TSHR cDNA (19). These observations
suggest that the presence of functional TSHR in adipocytes is related
to the extrathyroidal manifestations of Graves disease.
A suitable model for the study of adipocytes is 3T3-L1 cells, a
fibroblast-like cell line derived from the Swiss mouse embryo, which,
upon reaching confluence, can be induced to differentiate into
adipocytes by the hormonal pulse with insulin,
isobutylmethylxanthine, and dexamethasone (21, 22). In addition,
we recently showed that this differentiation was accompanied by the
expression of TSHR mRNA (23). This cultured cell system is therefore
useful for studying the regulation of TSHR gene expression in
adipocytes.
Our recent report (24) showed that, in 3T3-L1 adipocytes, TSH induces
cAMP-mediated down-regulation of TSHR gene transcription. The
mechanism of TSHR mRNA regulation in 3T3-L1 cells, however, was
different from that in thyroid cells. For example, in the former,
TSH-induced down-regulation of TSHR mRNA is evident within 1 h and
peaks within 4 h; the transient increase in TSHR mRNA detected in
the rat thyroid cell line, FRTL-5, is not observed in 3T3-L1
adipocytes. While the down-regulation of TSHR gene expression in FRTL-5
cells was found to be cycloheximide sensitive, that in 3T3-L1 cells is
cycloheximide resistant. Furthermore, while insulin or serum was found
to be required for TSH-induced regulation of TSHR mRNA in FRTL-5 cells,
neither was required for down-regulation in 3T3-L1 cells.
The 5'-flanking region of the rat TSHR gene has recently been cloned
(25), and a minimal promoter region, -220 to -39 bp, exhibiting
thyroid-specific expression and TSH/cAMP regulation, has been
identified (26, 27, 28, 29, 30). The region between -220 and -177 bp was shown to
act as a thyroid-specific enhancer (28, 29, 30). Within this region, a
regulatory element from -189 to -175 bp was observed to bind thyroid
transcription factor-1 (TTF-1) (28, 31, 32), and single-strand
DNA-binding proteins (SSBPs) were found to interact with an element
contiguous with the 5'-end of the TTF-1 element (30, 33). TTF-1 and the
SSBPs have been observed to function jointly in the regulation of TSHR
gene expression in thyroid cells (28, 30, 32, 33). The region between
-220 and -192 bp was found to act as an insulin-response
element, the activity of which appears to be thyroid specific
(29). A sequence between -139 and -132 bp, TGAGGTCA, which is
homologous to a consensus cAMP-response element (CRE), TGACGTCA, was
found to function as a constitutive enhancer and to be responsible for
efficient expression of this gene (26, 27, 28).
Our previous findings, that adipose cells do not express TTF-1 and that
hormonal regulation of TSHR gene expression in adipose cells is
different from that in thyroid cells (24), led us to hypothesize that
the TSHR gene expression in adipocytes may be regulated by a different
set of transcription factors from that in thyroid cells, or may be
directed by an alternative promoter. We have therefore dissected the
5'-flanking region of the rat TSHR gene The present study shows that
the TSHR mRNAs in rat adipocytes are transcribed from almost identical
transcriptional start sites to those used in thyroid cells. Using
3T3-L1 adipocytes, we identify an element responsible for the TSHR gene
expression in adipocytes. This element includes the cAMP response
element (CRE)-like site and Ets-binding motifs and differs from the
thyroid-specific enhancer element containing TTF-1-binding sequence. We
further demonstrate that repressing factors whose binding to DNA are
diminished in differentiated 3T3-L1 adipocytes interact within the
fat-specific element.
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RESULTS
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Determination of Transcriptional Initiation Sites in Rat Adipose
Tissue
We performed primer extension to identify the transcriptional
initiation sites in the 5'-flanking region of the TSHR gene in rat
adipose tissue. The extension products observed with rat adipose tissue
RNA were identical to those detected in RNA from FRTL-5 thyroid cells
(Fig. 1A
). The major sites of
transcriptional initiation mapped at -83, -72, -69, and -68 bp
(arrows in Fig. 1
, A and B), which almost concurred with
those in FRTL-5 cells identified in a previous report (25).

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Figure 1. Primer Extension and Anchored PCR Analyses of the
Transcriptional Initiation Sites of the Rat TSHR Gene in Rat Fat Tissue
A, The transcriptional initiation sites were determined by primer
extension analysis. As described in Materials and
Methods, the end-labeled primer was hybridized to 50 µg total
RNAs from rat epididymal fat tissue, FRTL-5, rat liver, and yeast tRNA
as control. The primer was extended by reverse transcriptase, and the
products were analyzed on PAGE. A sequencing ladder, using the same
primer, was run in parallel. Major transcriptional initiation sites are
indicated by arrows (in A and B). B, Dots
indicate the 5'-end of the TSHR cDNA clones obtained by the anchored
PCR. Nucleotide numbering is relative to the translational initiation
site, which is designated 1. In addition, elements important for the
TSHR gene expression in thyroid cells are indicated. Thus, the
insulin-response element (29 ) and TTF-1-binding sequence (28 )
determined in FRTL-5 thyroid cells are indicated by single or double
underlines, respectively. The dashed line indicates the
SSBP-1-binding site (30 33 ). Horizontal arrows indicate
the tandem repeat sequences in the repressive element that appear to be
active ubiquitously (27 43 ). The CRE-like sequence is
boxed.
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When we also assayed transcriptional initiation sites by anchored PCR,
we detected the 5'-end of clones at -110 (one clone), -106 (one
clone), -91 (one clone), -86 (one clone), -83 (two clones), -81
(two clones), -79 (one clone), -72 (one clone), -68 (two clones),
-64 (one clone), -62 (one clone), -45 (one clone), and -34 bp (one
clone) (Fig. 1B
). None of these clones contained alternative splicing
in the 5'-untranslated region. Data from anchored PCR and primer
extension experiments indicate that TSHR mRNAs in adipose tissue are
transcribed from multiple sites almost identical to those in thyroid
cells.
Identification of Regulatory Elements in the TSHR Promoter Required
for Its Expression in Adipose Cells
Using electroporation, we transfected chimeric constructs of 5'-
deletion mutants of the TSHR promoter (Fig. 2A
), ligated to the chloramphenicol
acetyltransferase (CAT) reporter gene, into 3T3-L1 cells before and
after the induction of differentiation. In differentiated 3T3-L1 cells
(adipocytes), the full-length plasmid, pTRCAT5'-1707 (5.3 ±
1.4%), expressed significantly higher CAT activity than the shortest
construct, pTRCAT5'-90 (0.8 ± 0.2%). In undifferentiated 3T3-L1
cells (preadipocytes), however, there was no significant difference in
CAT activity between pTRCAT5'-1707 and 5'-90. The differentiation of
3T3-L1 cells into adipocytes resulted in a 4.3-fold increase of CAT
activity expressed by pTRCAT5'-1707.

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Figure 2. CAT Activities of Chimeric Constructs Containing
5'-Deletion of the 5'-Flanking Region of the TSHR in 3T3-L1 Cells
before and after Differentiation
Regulatory elements of the TSHR promoter determined in FRTL-5 thyroid
cells are diagrammatically presented in panel A. 3T3-L1 cells before
(preadipocytes, open bars) or after (adipocytes,
solid bars) were cotransfected with the noted plasmids
and pCH110 by electroporation. Conversion rates were normalized to
ß-galactosidase activities and are presented relative to the activity
expressed by the pSV2CAT positive control. A statistically significant
(P < 0.05) increase induced by differentiation is
noted by an asterisk (*). The significant
(P < 0.05) effect of 5'-deletion from
pTRCAT5'-1707 on the CAT activity in the undifferentiated or
differentiated cells is denoted by an open circle ( )
or a solid circle (), respectively.
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When we compared the activity of 5'-deletion mutants that lacked the
sequences from -1707 to -1191 (5'-1190), -908 (5'-907), -639
(5'-638), and -420 (5'-419) bp in 3T3-L1 adipocytes, we observed no
significant changes in CAT activity (Fig. 2B
). The deletion mutant,
pTRCAT5'-190 (9.8 ± 0.3%), however, exhibited significantly
(P < 0.05) higher CAT activity than either
pTRCAT5'-1707 (5.3 ± 1.4%) or 5'-220 (6 .5 ± 1.1%)
(Fig. 2B
). While deletion of the sequences from -190 to -178 bp,
containing the TTF-1- and SSBP-1-binding sites determined in thyroid
cells, and from -146 to -91 bp, containing the CRE-like element
between -139 and -132 bp (Fig. 2A
), significantly (P
< 0.01) decreased CAT activity, deletion of the sequence from -177 to
-147 bp, in which the TSHR suppressor element binding protein-1
(TSEP-1) interacted (Fig. 2A
), increased CAT activity 22-fold (Fig. 2B
). These data suggest that the regions of the TSHR gene between -190
to -178 bp and between -146 to -91 bp constitute positive regulatory
elements, whereas sequences between -220 to -191 bp and between -177
to -147 bp constitute suppressor elements.
In 3T3-L1 preadipocytes, only the plasmid pTRCAT5'-146 exhibited
significantly higher CAT activity than did pTRCAT5'-1707 (Fig. 2B
).
The CAT activity of pTRCAT5'-146, however, was 7.5-fold higher in
3T3-L1 adipocytes than in preadipocytes while no significant difference
in adipocyte and preadipocyte activity was observed with 5'-90
construct (Fig. 2B
), i.e. relative CAT activities were
0.8 ± 0.2% vs. 0.5 ± 0.1%, respectively,
suggesting that the region between -146 and -91 bp is a major
positive regulatory element in 3T3-L1 adipocytes.
To identify nuclear factor-interacting sequences in the major positive
regulatory element, we performed deoxyribonuclease I (DNase I)
protection analysis with nuclear extracts from 3T3-L1 preadipocytes and
adipocytes, as well as with extracts of FRTL-5 thyroid cells (Fig. 3
). In all three nuclear extracts, the
region from nucleotides -146 to -127 bp, centered on the CRE-like
element, was protected. In addition, nuclear extracts of 3T3-L1
adipocytes and FRTL-5 cells demonstrated protection of the region
between -112 to -106 bp on the coding strand, which is located
downstream of the CRE.

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Figure 3. DNase I Protection Analysis of the Positive
Regulatory Element in the TSHR Gene
Genomic fragment spanning -220 to -50 bp was labeled at 3'-end of the
coding strand with [ -32P]dATP and Klenow fragment.
Lane 1 is A+G ladder determined by the Maxam and Gilbert sequence
reaction (57 ); lane 2 is control digestion pattern in the absence of
added nuclear extract. Other lanes contain the probe preincubated with
30 µg of nuclear extracts from 3T3-L1 cells before (preadipocytes) or
after (adipocytes) differentiation, or from FRTL-5 cells. The
open bar diagrammatically denotes the region where all
three extracts protected; the solid bar indicates the
sequence protected by the extracts from differentiated 3T3-L1 and
FRTL-5 cells.
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Activation of the TSHR Promoter by the CRE-Like Site and the
Element Downstream of the CRE
To analyze the role of the protected regions in TSHR promoter
activity, we introduced mutations into the pTRCAT5'-146 construct
(Fig. 4
). In differentiated 3T3-L1 cells,
while mutation of the CRE-like element, TGAGGTCA, to that of the CRE
consensus sequence, TGACGTCA (pTRCAT5'-146CRE), did not alter promoter
activity, mutation to a nonpalindromic sequence, CGAGGACA, disrupting
the dyad symmetry (pTRCAT5'-146NP), significantly decreased promoter
activity. In addition, deletion of the CRE-like site, pTRCAT5'-131, led
to loss of promoter activity. Mutation of the downstream sequence
(pTRCAT5'-146DSM1) also showed reduced promoter activity, almost
identical to that of pTRCAT5'-146NP. The downstream sequence contains
the complementary sequence of the consensus DNA motif for the Ets
family, GGAA (Figs. 4A
and 5A
). In addition, this sequence, TTCC, also
exists between -100 and -97 bp (Fig. 5A
).

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Figure 4. Effect of Mutations in the Region Protected by the
Nuclear Extract from Differentiated 3T3-L1 Cells on the CAT Activity
The mutated plasmid constructs are noted in panel A. The open
and solid bars denote the region protected by the extract from
differentiated 3T3-L1 cells (see Fig. 3 ). The CRE-like sequence and the
Ets-binding motif are boxed. The differentiated cells
were cotransfected with the TSHR CAT mutants and pCH110, conversion
rates were normalized to ß-galactosidase activities, and CAT activity
was presented relative to pSV2CAT positive control. The significance
(P < 0.05) of the effect of the mutation or
deletion of pTRCAT5'-146 on the CAT activity is denoted by an
asterisk.
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Figure 5. CAT Activity in the Differentiated 3T3-L1 Cells
Transfected with Chimeric Plasmids Containing One, Two, or Three Copies
of the CRE-Like Sequence (CRE, open bars), the
Downstream Sequence (DS, hatched bars) or the CRE Plus
the Downstream Sequence (CRE+DS, solid bars) Ligated to
the 3'- End of an SV40 Promoter-driven CAT Gene
Panel A depicts the sequences of the coding strands of the ligated
oligonucleotides. The CRE-like element and putative Ets-binding sites
are indicated by double or single
underlines, respectively. In panel B, the arrows
depict the number of copies and direction of each construct; an
right arrow denotes 5' to 3'. As in Figs. 2 and 4 , the
differentiated 3T3-L1 cells were cotransfected with plasmid pCH110, and
conversion rates were normalized to ß-galactosidase activities. The
CAT activities are presented relative to that of the pCAT-promoter
control with no CRE-like or Ets sites. An asterisk (*)
or a solid circle () denotes a statistically
significant increase relative to pCAT-promoter-CRE or -DS construct,
respectively.
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To confirm the synergism between the CRE-like site and the
downstream sequence, we tandemly ligated oligonucleotides containing
the CRE-like site alone (CRE), two putative Ets-binding sequences (DS),
or the CRE site plus two Ets sites (CRE+DS) to the downstream of SV40
promoter-driven CAT gene (Fig. 5A
). CAT activity was measured after
transient transfection into differentiated 3T3-L1 cells (Fig. 5B
). We
found that the SV40 promoter was not activated when up to three copies
of either oligonucleotide containing the CRE-like site or the Ets sites
were inserted. In contrast, the presence of both the CRE and the
downstream sequence significantly enhanced the SV40 promoter
activity.
Interactions of Nuclear Proteins with the Protected Elements
Gel mobility shift analyses were performed to characterize the
protein-DNA interactions involving the positive regulatory elements of
the TSHR gene in 3T3-L1 cells. When a double-stranded oligonucleotide
encompassing the sequence from -146 to -114 (CRE) was used as a probe
(Fig. 6A
), we observed multiple
protein-DNA complexes in nuclear extracts from 3T3-L1 preadipocytes,
similar to those detected in FRTL-5 extracts. In contrast, nuclear
extracts from 3T3-L1 adipocytes formed a specific protein/DNA complex
not observed in the other cell (Fig. 6
, A and B, solid
arrow) and exhibited less of the slowly migrating complexes (Fig. 6A
, dashed arrows). These complexes, however, were not
formed with the oligonucleotide probe containing the nonpalindromic
mutation (CRENP). Competition analysis (Fig. 6B
) showed that the
formation of adipocyte-specific protein-DNA complex was self-competed
by incubating with unlabeled oligonucleotide, as well as by an
unlabeled, double-stranded oligonucleotide containing the CRE consensus
sequence but with a surrounding sequence different from that of the
TSHR promoter (somatostatin CRE, 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3',
Promega, Madison, WI). Formation of this complex, however, was not
competed by incubation with an unlabeled oligonucleotide spanning the
region from -127 to -93 bp (DS1) and lacking the CRE-like sequence or
with unlabeled CRENP. These results indicated that the differentiation
of 3T3-L1 cells into adipocytes induced a new protein interacting with
the CRE-like octamer sequence. The slowly migrating complexes (Fig. 6C
, dashed arrows), formation of which were decreased after the
differentiation, were shown to be formed with activating transcription
factor-2 (ATF-2) since these complexes were abolished and supershifted
(Fig. 6C
, arrowhead) by antiserum to ATF-2. Inhibition of
complex formation by antiserum to CRE-binding protein (CREB) was
evident only in protein-DNA complexes formed with nuclear extracts from
FRTL-5 cells (Fig. 6C
). The adipocyte-specific protein-DNA complex,
however, was not altered by antiserum to CREB or ATF-2 (Fig. 6C
).

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Figure 6. Ability of the CRE-Like Element to Form Protein-DNA
Complexes with Nuclear Extract with 3T3-L1 Cells before and after
Differentiation
A, Radiolabeled, double-stranded oligonucleotide spanning -146 to
-114 bp with (CRENP) or without (CRE) the nonpalindromic mutation (see
Fig. 4 ) was incubated with nuclear extracts from 3T3-L1 cells before
(PA) and after (A) differentiation, as well as from FRTL-5 cells (F)
maintained in medium without TSH for 7 days. Dashed
arrows depict the protein-DNA complexes diminished by the
induction of differentiation of 3T3-L1 cells. A solid
arrow denotes the protein-DNA complexes induced by
differentiation. B, The double-stranded oligonucleotide CRE was
radiolabeled and incubated with nuclear extracts from differentiated
3T3-L1 cells (A) and unlabeled oligonucleotides, CRE (self competition,
a 250-fold excess over probe in molar amount), CRENP (250-fold), DS1
(-127 to -93 bp, 250-fold), or S-CRE (50-fold), which contains the
CRE and its surrounding sequence of the somatostatin gene. C, The
radiolabeled oligonucleotide CRE was incubated with nuclear extracts
from 3T3-L1 cells before (PA) and after (A) differentiation or FRTL-5
cells (F) in the presence of an antiserum against CREB or ATF-2 or a
control normal rabbit IgG (NRIgG). An arrowhead denotes
a supershifted complex formed by antiserum to ATF-2. Protein-DNA
complexes inhibited by antiserum to CREB are indicated with
bars.
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When we performed gel mobility shift analysis using an oligonucleotide
probe spanning the region from -118 to -80 bp, including the
protected element containing the putative Ets-binding site and the
additional Ets site, we found that nuclear extracts from 3T3-L1
preadipocytes formed a faint protein/DNA complex (Fig. 7A
, complex B). Formation of this complex
was increased (Fig. 7A
, lane 2), and an additional complex (Fig. 7A
, complex A) became detectable by the differentiation of 3T3-L1 cells to
adipocytes. Nuclear extracts from FRTL-5 and Buffalo rat liver cells
(BRL 3A) also formed greater amounts of complexes than did the
preadipocyte extracts. These protein-DNA complexes were shown to be
specific, since they could be self-competed by unlabeled
oligonucleotide, but not by an unlabeled oligonucleotide spanning the
region from -146 to -114 bp (Fig. 7B
). Nuclear extracts from FRTL-5
cells also formed an additional complex (complex C) exhibiting the
highest mobility. This complex, however, appeared to be nonspecific
since it was not self-competed (data not shown).

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Figure 7. Ability of the Putative Ets Site to Form
Protein-DNA Complexes with Nuclear Extract with 3T3-L1, FRTL-5, and BRL
Cells
A, Radiolabeled, double-stranded oligonucleotide spanning the region
from -118 to -80 bp (DS2) with or without mutations was incubated
with nuclear extracts from 3T3-L1 cells before (PA) and after (A)
differentiation, as well as from FRTL-5 (F), or BRL (B) cells. Mutants
are denoted in the lower panel. Putative Ets-binding
sites are boxed, and the black bar
indicates the sequence protected by nuclear extracts from
differentiated 3T3-L1 cells and FRTL-5 cells. B, The double-stranded
oligonucleotide, DS2, was radiolabeled and incubated with nuclear
extracts from differentiated 3T3-L1 cells and a 250-fold excess of
unlabeled DS2 or CRE as a competitor. C, Increasing amounts of
unlabeled, double-stranded oligonucleotide were incubated with nuclear
extracts from 3T3-L1 cells after differentiation and the radiolabeled
oligonucleotide probe DS2. The data were subjected to quantitative
densitometry and plotted as percent of B complex in the absence of
competitor. The amount of each unlabeled oligonucleotide is presented
in fold excess over probe.
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To identify the site in the promoter region involved in the formation
of differentiation-induced protein-DNA complexes, we introduced
mutations into the protected sequence (DS2DSM1) (e.g.
pTRCAT5'-146DSM1 in Fig. 4A
). We observed decreased formation of these
protein/DNA complexes (Fig. 7A
, lanes 5- 8), a result consistent with
the decreased CAT activity of pTRCAT5'-146DSM1 in 3T3-L1 adipocytes.
Since this mutant, DS2DSM1, retained detectable binding activity,
we introduced additional mutations into another putative Ets-binding
element (DS2DSM2). Disruption of both Ets sites resulted in complete
loss of the binding activity (Fig. 7A
, lanes 912). In competition
analysis (Fig. 7C
), significant inhibition of the B complex formation
was already evident at a 32-fold excess of unlabeled homologous
oligonucleotide (DS2), but not by both mutants at the same amount.
Higher amounts of oligonucleotide (
500-fold excess over probe) were
required for the 50% inhibition of the complex formation by unlabeled
DS2DSM2 in comparison with DS2DSM1 (120-fold). These results suggested
that the putative Ets-binding sites in the downstream sequence of the
CRE-like site interacted with the differentiation-induced activating
factors. However, nuclear extracts from BRL 3A liver cells, expressing
no TSHR gene, formed protein-DNA complexes similar to those by extracts
from 3T3-L1 adipocytes (Fig. 7A
). We therefore pursued an interaction
of another nuclear factor within the positive regulatory element.
Interaction of Differentiation-Reduced Nuclear Proteins with TSHR
Promoter
To analyze the region between the CRE-like site and the putative
Ets sites, we performed gel mobility shift analyses using the
oligonucleotide, DS1, spanning the region from -127 to -93 bp, as a
probe. While nuclear extracts from 3T3-L1 preadipocytes formed multiple
protein/DNA complexes, two of these complexes were diminished in
nuclear extracts from 3T3-L1 adipocytes (Fig. 8A
). The proteins forming these complexes
appeared to be ubiquitously expressed, since they were also detected in
nuclear extracts from FRTL-5 and BRL 3A cells (Fig. 8A
). These
complexes were also found to be specifically formed with
double-stranded DNA since formation of these complexes was
self-competed, but not by double-stranded unlabeled oligonucleotide
containing somatostatin CRE (S-CRE, Fig. 8B
). In addition, neither
unlabeled coding nor noncoding strand of DS1 oligonucleotide competed
the formation of these complexes (Fig. 8B
). All extracts also formed
two minor complexes (Fig. 8
, B and C complexes). While the B complex
was nonspecific, since it was not reduced by competition with a
250-fold excess of unlabeled oligonucleotide (Fig. 8B
), formation of
the C complex was competed out not only by unlabeled double-stranded
oligonucleotide, but also by its coding or noncoding strand.

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Figure 8. Ability of the Sequence between -127 to -93 bp to
Form Protein-DNA Complexes with Nuclear Extract with 3T3-L1, FRTL-5,
and BRL Cells
A, Radiolabeled, double-stranded oligonucleotide between -127 to -93
bp (DS2) was incubated with nuclear extracts from 3T3-L1 cells before
(PA) and after (A) differentiation, as well as from FRTL-5 (F), or BRL
(B) cells. B, The double-stranded oligonucleotide, DS1, was
radiolabeled and incubated with nuclear extracts from undifferentiated
3T3-L1 cells and unlabeled double- or single-stranded DS1 as a
competitor. A double-stranded oligonucleotide containing a consensus
CRE site in somatostatin gene (S-CRE) was also used as a nonspecific
competitor. The amount of each unlabeled oligonucleotide present in
each assay, in fold excess over probe, is noted.
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To define the binding site of the proteins forming the A complexes, the
G residues on both strands of the oligonucleotide in contact with
protein were determined by methylation interference (Fig. 9A
). While methylation of G residues from
-117 to -110 bp on the noncoding strand interfered with formation of
the major A complexes (Fig. 9C
), methylation of G residues on the
coding strand had no effect (data not shown). This result is supported
by DNAase I protection analysis using a probe labeled on the noncoding
strand (Fig. 9B
). Nuclear extracts from 3T3-L1 preadipocytes and FRTL-5
cells incompletely protected the region between -120 and -110 bp, in
which extracts from adipocytes showed no protection.

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Figure 9. Identification of the Sequence Interacting with
Differentiation-Reduced Nuclear Proteins by Methylation Interference
and DNase I Protection Analyses
In panel A, the dimethyl sulfate-modified, double-stranded DS2
oligonucleotide 32P-labeled in the coding strand was
incubated with 25 µg nuclear extract from undifferentiated 3T3-L1
cells. Protein-DNA complexes and free DNA probe were separated on a
preparative native gel as described in Materials and
Methods. Free DNA and the upper complex of the A complexes
shown in Fig. 8A were eluted from the gel and cleaved at the modified
residues. The cleavage products were resolved by electrophoresis
through a 12% denaturing gel. Open circles define the
bases whose modification reduces the proportion of DNA in the bound
fraction; X defines bases whose methylation does not alter the protein
binding. In panel B, genomic fragment spanning -220 to -50 bp was
labeled at the 3'-end of the noncoding strand. Lane 1 is A+G ladder
determined by the Maxam and Gilbert sequence reaction (57 ); lane 2 is
the control digestion pattern in the absence of added nuclear extract.
Other lanes contain the probe preincubated with 30 µg of nuclear
extracts from 3T3-L1 cells before (Preadipocytes) or after (Adipocytes)
differentiation, or from FRTL-5 cells. The black bar
diagrammatically denotes the region where all three extracts protected;
the hatched bar indicates the sequence incompletely
protected by the extracts from undifferentiated 3T3-L1 and FRTL-5
cells. In panel C, results of methylation interference and DNase
protection analyses are summarized. The CRE-like site and the putative
Ets-binding element are boxed.
|
|
Based on the results of methylation interference analyses, we again
introduced mutations into both the TSHR promoter-CAT chimeric
construct and the oligonucleotide probe and performed gel mobility
shift analysis (Fig. 10A
). Mutations in
the oligonucleotide probe (DS1DSM3) almost abolished the formation of
both the major and minor bands of the A complexes (Fig. 10B
), thus
suggesting that the proteins constituting the major and minor A
complexes interact with the same sequence. When these constructs were
transiently transfected into 3T3-L1 cells, we observed activation of
the TSHR promoter, an effect more evident in preadipocytes (5.8-fold)
than in adipocytes (1.9-fold) (Fig. 10C
). This observation is
consistent with the difference in the activity of A complex formations
between 3T3-L1 preadipocytes and adipocytes (Fig. 8A
). In sum, these
results indicate that the nuclear factors in which binding is decreased
after the differentiation of 3T3-L1 cells act as repressive factors on
the TSHR promoter.

View larger version (22K):
[in this window]
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|
Figure 10. Effect of a Mutation in the Binding Site of
Differentiation-Reduced Nuclear Proteins on an Ability of the Complex
Formation and on the CAT Activity
The sequences in panel A depict the mutation (DS1DSM3) and the
wild-type (DS1) sequence. Open circles indicate the
bases whose methylation interferes with the protein binding. Putative
Ets sites are boxed. In panel B, each oligonucleotide
was used as radiolabeled probe and incubated with nuclear extracts from
undifferentiated 3T3-L1 cells. In panel C, 3T3-L1 cells before
(Preadipocytes) and after (Adipocytes) the induction of differentiation
were transfected with the TSHR-CAT plasmids with or without the
mutation. The CAT activities are normalized with ß-galactosidase
activities expressed by pCH110 cotransfected and are presented relative
to that of positive control plasmid, pSV2CAT, or as the ratio of
activity of the mutant vs. the wild type.
|
|
 |
DISCUSSION
|
---|
We have shown previously that the regulation of TSHR gene
expression in 3T3-L1 adipocytes is distinct from that in FRTL-5 thyroid
cells (24). We were therefore somewhat surprised to find that the gene
is initiated at almost identical sites in adipose and thyroid cells.
The rat TSHR promoter has been shown to exhibit features of a
house-keeping gene, including the absence of a TATA box, a high G+C
content, and the presence of multiple transcriptional initiation sites
(25). At typical TATA-less promoters, binding of SP1 to a GC-rich
element is required for expression (34, 35, 36). In addition, a
transcription factor IID (TFIID) complex containing TATA
box-binding protein is thought to be essential for transcription even
at a promoter lacking a TATA box, and SP1 bound to a GC can recruit a
tethering factor and TFIID to lie along the DNA next to the GC box (34, 35). In some TATA-less genes, the SP1-interacting GC box has been found
to act as a start site selector at about the same distance, 3653 bp
(36). It has been shown, however, that the minimal promoter region of
the TSHR gene is not protected by purified SP1 protein in DNAase I
protection analysis (26). Nevertheless, the common element activated in
both adipocytes and thyroid cells may be important for the initiation
of gene transcription. Since the major start sites are 4459 bp
downstream from the protected sequence centered on the CRE, CREBs may
be a candidate for the initiation of transcription of the gene.
In 3T3-L1 cells, deletion analysis of the 5'-flanking region of the
TSHR gene indicated that a 56-bp region between -146 and -90 bp was a
positive regulatory element whose activity was induced by the induction
of differentiation. Within this 56-bp region, we found that nuclear
proteins from differentiated 3T3-L1 cells protected two elements. The
upstream element between -146 and -127 bp contains a CRE-like element
that was necessary for gene expression in thyroid cells (26, 27, 28). The
downstream element spanning the region between -112 to -106 bp
includes a putative Ets family-binding site. Both of these elements
were found to be indispensable for the enhancer activity in adipocytes,
suggesting that the differentiation-induced nuclear factors interact
with these sequences, thus contributing to the promoter activity in
adipocytes. In fact, we found that differentiation of 3T3-L1 cells
induced the formation of a complex between the upstream element and an
as-yet-unidentified protein. Competition and mutation analyses,
however, suggested that it was a member of the CREB family. The
downstream element was also observed to interact with nuclear proteins,
the binding of which was increased by the induction of differentiation.
This downstream element or the CRE-like site, however, cannot by itself
activate the TSHR promoter, since either deletions or mutations of each
sequence resulted in loss of promoter activity.
The synergism between the CRE site and putative Ets-binding elements in
activating the TSHR promoter has been observed in other genes (37, 38, 39, 40).
Members of the Ets family often show functional synergism with other
transcription factors to achieve efficient activation of target genes
through an Ets-binding site and adjacent DNA sequences (37, 38, 39, 40, 41, 42). For
example, in the gene encoding mitochondrial uncoupling protein,
Ets motifs have been identified as brown adipocyte regulatory elements
(39). Synergistic cooperation of the brown adipocyte regulatory
elements and the CRE is essential for the expression of the uncoupling
protein gene in brown fat cells (39).
Although differentiation-dependent expression of the TSHR gene
may be due to synergistic activation by the CRE-like and Ets sites, a
third element may also be involved. This possibility was suggested by
lines of evidence indicating that the complexes with the downstream
sequence, including Ets sites, were formed by nuclear extracts of BRL
liver cells, which did not express the TSHR, and that nuclear proteins
from BRL cells and the CRE-like site have been shown to form multiple
complexes other than the differentiation-induced complex (26, 27),
whereas the construct containing both elements, pTRCAT5'-146, had
slight activity in BRL cells (26, 27). We have identified nuclear
factors with diminished binding to DNA only in differentiated 3T3-L1
cells. These nuclear proteins appear to suppress the promoter activity
of the TSHR gene. The high activity of the complex formations in FRTL-5
and BRL cell nuclear extracts may, therefore, account for the weak
activity of pTRCAT5'-146 in these cell lines (27, 28). Methylation
interference analysis showed that these factors bind to a sequence
contiguous to the 5'-end of the Ets-binding element, thus suggesting
that binding of these factors may interfere with the binding of Ets
family members to the TSHR promoter.
Transient expression analysis of 5'-deletion mutants revealed that the
region between -177 and -146 bp acts as a repressive element in both
differentiated and undifferentiated 3T3-L1 cells. This sequence
contains the binding site of TSHR suppressor element-binding protein-1
(TSEP-1), a member of the Y-box family (27, 43), which binds to the
coding strand of this region in a sequence-specific manner and
suppresses the TSHR gene expression in thyroid cells (43). The TSEP-1
mRNA is also detected in both 3T3-L1 cells before and after
differentiation (data not shown), suggesting that TSEP-1 represses the
TSHR promoter activity in 3T3-L1 cells and that this repression may
overcome the weak, but significant, enhancing activity of the region
between -146 and -90 bp observed in undifferentiated 3T3-L1
cells.
The sequence between -190 and -177 bp was found to exhibit enhancing
activity only in differentiated 3T3-L1 cells. In FRTL-5 cells, this
region has been shown to interact with both TTF-1 and the SSBP-1 (30, 33). TTF-1 mRNA, however, was not detected in 3T3-L1 cells before or
after differentiation (24). The interactions of nuclear proteins,
including SSBP-1, to this element in differentiated 3T3-L1 cells are
under current investigation.
Accumulating evidence has revealed that the differentiation of
preadipocytes into adipocytes is regulated by at least three
transcription factors, CCAAT/enhancer binding protein
(C/EBP
)
(44), adipocyte determination- and differentiation-dependent factor 1
(ADD1) (45), and peroxisome proliferator-activated receptor
2
(PPAR
2) (46, 47), all of which appear to be important in regulating
the expression of adipose-specific genes (45, 48, 49). The enhancer
elements in the TSHR promoter, however, contain neither the CCAAT box
nor the E-box motif, the binding sites of C/EBP
and ADD1,
respectively. The third transcription factor, PPAR
2, has been found
to form a heterodimeric complex with RXR
(50), which then binds to a
sequence known as DR-1 [direct repeat with one nucleotide spacer (46, 49, 50)]. Interestingly, the CRE-like site in the TSHR promoter
contains a half-site of the DR-1 sequence, AGGTCA. Although we were
unable to detect binding of the PPAR
2/RXR
heterodimer, produced
in the reticulocyte lysate system, to an oligonucleotide probe
containing the CRE-like site (data not shown), these adipogenic
transcription factors may indirectly regulate the expression of the
TSHR gene in adipocytes.
 |
MATERIALS AND METHODS
|
---|
Primer Extension
The 5'-end of the rat TSHR gene transcript was determined as
previously described (25), using a 24-bp oligonucleotide primer
complementary to 1235 bp of the rat TSHR cDNA
(5'-AGCAGAGTGAGCTGGAGCAGGGAC-3') (25) end-labeled with
[
-32P]ATP and T4 polynucleotide kinase. The primer
(3 x 105 cpm) was hybridized with 50 µg total RNA
or yeast tRNA at 30 C overnight and extended with SuperScript II
reverse transcriptase (GIBCO BRL, Rockville, MD). Resulting products
were analyzed on an 8% polyacrylamide-7M urea gel in parallel with a
sequencing reaction generated with the extension primer.
Anchored PCR
Anchored PCR (5' rapid amplification of cDNA ends) was
used to clone the 5'-end of the TSHR cDNA. Total RNA (1 µg) from rat
epididymal fat tissue was used to synthesize the first-strand cDNA
using SuperScript II reverse transcriptase (GIBCO BRL) and 2.5 pmol of
primer (5'-AAATTGTAGAAAGAATGTGGC-3'; complementary to 279299 bp of
the TSHR cDNA) (51). After first-strand cDNA synthesis, the original
mRNA template was destroyed with ribonuclease H (RNase H), and the
first-strand product was purified. An anchor sequence was then added to
the 3'-end of the cDNA using terminal deoxynucleotidyl transferase
(GIBCO BRL) and dCTP. Tailed cDNA was amplified by PCR using a nested
primer containing dUMP residues for uracil DNA glycosylase cloning (52)
[5'-CAUCAUCAUCAUTCGATGAGCTTCAGAGT-CTGG-3'; complementary to
162182 bp of the rat TSHR cDNA (51)], and a deoxyinosine-containing
anchor primer
(5'-CUACUACUACUAGGCCACGCGTCGACTACGGGIIGG-GIIGGGIIG-3').
The amplified products were cloned into pAMP1 plasmid (GIBCO BRL) by
the uracil DNA glycolase cloning method (52), and clones containing the
amplified DNA were sequenced.
Cell Culture
3T3-L1 cells (CCL 92.1; ATCC, Rockville, MD) were grown in
high-glucose DMEM and supplemented with 10% calf serum. Confluent
3T3-L1 preadipocytes were induced to differentiate into adipocytes as
previously described (23, 24). Briefly, 1 day after confluence, cells
were treated with media containing 10% FBS, 10 µg/ml insulin, 0.2
µg/ml dexamethasone, and 0.5 mM isobutylmethylxanthine.
After 3 days, this medium was replaced by media supplemented with 10
µg/ml insulin and 10% FBS, and 2 days later, the media were replaced
with media containing 10% FBS. Cells were used for studies 9 or 10
days after induction of differentiation.
FRTL-5 rat thyroid cells (CRL 8305 from ATCC) were grown in Coons
modified Hams F-12 medium supplemented with 5% calf serum and a
mixture of six hormones containing bovine TSH (10 mU/ml), insulin (10
µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml),
glycyl-L-histidyl-L-lysine acetate (10
ng/ml), and somatostatin (10 ng/ml) (53). Buffalo rat liver cells (BRL
3A; ATCC No. CRL 1442) were grown in Hams F-12 supplemented with 5%
FBS.
Plasmid Construction
Chimeric constructs containing 5'-flanking region of the rat
TSHR gene and chloramphenicol acetyltransferase (CAT) gene,
pTRCAT5'-1707, 5'-1190, 5'-907, 5'-638, 5'-419, 5'-220, 5'-190, 5'-177,
5'-146, 5'-90, and a plasmid for positive control, pSV2CAT, were kindly
provided by Dr. Leonard D. Kohn (Metabolic Diseases Branch,
NIDDK, NIH, Bethesda, MD 20892). Mutations of promoter sequences were
generated by PCR using forward primers that had the mutated sequence
and the ON-8L primer described previously as a reverse primer (25).
Amplified fragments were cloned into plasmid p8CAT and sequenced to
ensure nucleotide fidelity.
To generate pCAT-promoter-DS or -CRE+DS plasmids, both sense and
antisense strand oligonucleotides containing the sequence from -118 to
-80 or from -146 to -80 bp, respectively, as well as the
XbaI recognition site on their 5'-end were synthesized,
annealed, and inserted into the XbaI site of the
pCAT-promoter plasmid (Promega). The inserts were sequenced to confirm
copy number and direction. pCAT-promoter-CRE plasmids were kindly
provided by Dr. Leonard D. Kohn.
All plasmid preparations were purified by CsCl gradient centrifugation
(54).
Transient Expression Analysis
3T3-L1 cells before (preadipocytes) and after (adipocytes)
differentiation were transfected by electroporation (Gene Pulser,
Bio-Rad, Richmond, CA). When undifferentiated 3T3-L1 cells were
transfected, cells being grown to 80% confluency in medium with 10%
calf serum (3T3-L1 preadipocytes) were used. After the initiation of
differentiation, 3T3-L1 cells on the 9th or 10th day (3T3-L1
adipocytes) were transfected. Cells were harvested, washed, and
suspended at 5 x 106 cells/ml in 0.8 ml PBS. Either
50 µg pTRCAT5'-1707, equivalent molar amounts of the deletion
mutants, or 5 µg pSV2CAT (positive control) were added with 5 µg
pCH110 (ß-galactosidase expression plasmid; Pharmacia Biotech,
Uppsala, Sweden). Cells were pulsed (300 V; capacitance, 960
µfarads), plated on one (adipocytes) or two (preadipocytes)
10-mm culture dishes, and cultured for 72 h. ß-Galactosidase was
measured as described (55). After ß-galactosidase assay, cell
extracts were heated at 65 C for 10 min for CAT assays (56). CAT
activities were normalized to ß-galactosidase activities and then
presented as the activities relative to that expressed by pSV2CAT,
which remained in quantitatively measurable range of the assay.
Nuclear Extracts
Nuclear extracts were prepared using a procedure previously
described (26, 27, 28, 29, 30) with minor modifications. 3T3-L1 extracts were
either from cells after being grown to 80% confluency in medium with
10% calf serum (preadipocytes) or from cells on the 9th or 10th day
after initiation of differentiation. FRTL-5 cells maintained in 5H
medium (-TSH) for 7 days after near-confluency in 6H medium was
achieved. BRL cells were grown until near-confluency in Coons
modified Hams F-12 supplemented with 5% FBS. Cells were harvested,
washed with PBS, and after centrifugation at 500 x g,
suspended in 5 pellet volumes of 0.3 M sucrose and 3%
Tween 40 in Buffer A [10 mM HEPES-KOH, pH 7.9, containing
10 mM KCl, 1.5 mM MgCl2, 0.1
mM EGTA, 0.5 mM dithiothreitol (DTT), 0.5
mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml
leupeptin, and 2 µg/ml pepstatin A]. After freezing, thawing, and
gently homogenizing, nuclei were isolated by centrifuging at
25,000 x g on a 1 M sucrose cushion
containing 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%
glycerol, 0.5 mM DTT, 0.5 mM PMSF, 2 µg/ml
leupeptin, and 2 µg/ml pepstatin A]; after centrifugation at
100,000 x g for 1 h, the supernatant was dialyzed
in Buffer C [20 mM HEPES-KOH, pH 7.9, 100 mM
KCl, 0.1 mM EDTA, 20% glycerol, 0.5 mM DTT,
0.5 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml pepstatin
A].
DNase I Protection Analysis
Genomic fragment between -220 and -50 bp was synthesized by
PCR and subcloned into the p8CAT vector as described previously
(25, 26, 27, 28, 29, 30). The plasmid was cut with either HindIII or
BamHI, end labeled with [
-32P]dATP and
Klenow fragment, and recut with either BamHI or
HindIII. The probe was purified on an 8% native
polyacrylamide gel. Initial incubations were for 15 min at room
temperature in 25 mM HEPES-KOH, pH 7.6, containing 5
mM MgCl2, 34 mM KCl, 1 µg
poly(dI-dC), and 30 µg nuclear extracts. Incubations continued 20
more min in the presence of the probes (50,000 cpm) and in a reaction
volume of 20 µl. DNA probes were digested with 0.5 U DNase I
(Promega) for 5 min on ice, and the reaction was terminated with 80
µl stopping solution (20 mM Tris HCl, pH 8.0, 250
mM NaCl, 20 mM EDTA, 0.5% SDS, 10 µg
proteinase K, and 4 µg sonicated calf thymus DNA). After incubation
at 37 C for 15 min, the digested products were phenol extracted,
ethanol precipitated, and separated on a 8% sequencing gel. The Maxam
and Gilbert A+G sequencing reaction (57) was used to locate the
footprinted regions.
Gel Mobility Shift Analyses
Gel mobility shift analyses were performed as described
previously, with minor modifications (24, 26, 27, 28, 29, 30). Synthesized
double-stranded oligonucleotides were end labeled with
[
-32P]ATP and T4 polynucleotide kinase and then
purified on an 8% native polyacrylamide gel; 2.5 µg nuclear extract
were incubated in a 20 µl reaction volume for 20 min at room
temperature, with or without unlabeled competitor oligonucleotides as
noted in individual experiments, and in the following buffer: 10
mM Tris HCl, pH 7.6, 50 mM KCl, 5
mM MgCl2, 1 mM DTT, 1
mM EDTA, 12.5% glycerol, 0.1% Triton X-100, and 1 µg
poly(dI-dC). Labeled probe, 50,000 cpm (
0.5 ng DNA), was added and
incubated an additional 20 min at room temperature. DNA-protein
complexes were separated on 5% native polyacrylamide gels. In
experiments using antiserum to CREB or ATF-2 (Santa Cruz Biotechnology,
Santa Cruz, CA), the antiserum or normal rabbit IgG was added to
nuclear extracts in the same buffer subsequent to addition of the
labeled probe and then incubated for 40 min at room temperature.
Methylation Interference Assays
Methylation interference assays were performed as previously
described (30). Single-stranded oligonucleotide spanning the region
from -146 to -80 bp was end-labeled with [
-32P]ATP
and T4 polynucleotide kinase and then purified on an 8% native
polyacrylamide gel. To obtain double-stranded probes that had the
coding or noncoding strand labeled, end-labeled single-stranded
oligonucleotides were annealed with their cold complementary strand and
then purified on an 8% native polyacrylamide gel. The double-stranded
probes were modified with dimethyl sulfate as described by Maxam and
Gilbert (57) for 20 min on ice. For the preparative mobility shift,
5 x 105 cpm of modified oligonucleotides were
incubated with 10 µg poly(dI-dC) and 25 µg nuclear extract. The
undried gel was exposed to an imaging plate of a BAS2000 image analyzer
(Fuji Film Co., Tokyo, Japan) for 10 min, and the regions corresponding
to the protein-DNA complex and unbound probe in the gel were excised,
eluted, and then precipitated. Base elimination and strand scission
reactions at guanines were performed (57). The samples were then
purified by butanol extractions and analyzed on a 12% sequencing
gel.
Other Procedures
Protein concentration was measured using a Bio-Rad kit;
recrystallized BSA was the standard. All experiments were repeated at
least three times with different batches of cells. Where noted, values
are the mean ± SE of these experiments; significance
(P < 0.05) between experimental values was determined
by the Students t test.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Toshimasa Onaya M.D., Ph.D., Professor and Chairman, The Third Department of Internal Medicine, Yamanashi Medical University, 1110 Tamaho, Yamanashi 409-3898, Japan. E-mail: onayat{at}res.yamanashi-med.ac.jp
Received for publication February 11, 1998.
Revision received June 15, 1998.
Accepted for publication June 16, 1998.
 |
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