Tannic acid, which comprises polyphenolic
compounds from tea leaves, suppresses the glucocorticoid-induced gene
expression of mouse mammary tumor virus (MMTV) integrated into 34I
cells. To investigate whether this suppression is due to promoter
responsiveness to tannic acid, we performed chloramphenicol
acetyltransferase analysis transfecting a MMTV promoter containing a
chloramphenicol acetyltransferase expression vector into mouse
fibroblast L929 cells. Deletion analysis of the promoter region
revealed that a 50-base pair (bp) region located downstream of the TATA
element is responsible for the suppressive effect of tannic acid. The tannic acid-sensitive suppressibility was introduced into a thymidine kinase promoter by inserting the 50-bp region into the region on the
5'-upstream side of the promoter. Detailed point mutation analyses
revealed that two elements, a 13-bp element and an ACTG motif in the
50-bp region, contribute to tannic acid sensitivity and promoter
repressibility, respectively. Interestingly, this repressive ACTG motif
is found in the human immunodeficiency virus promoter, the activity of
which is also suppressed by tannic acid (Uchiumi, F., Maruta, H.,
Inoue, J., Yamamoto, T., and Tanuma, S. (1996) Biochem. Biophys.
Res. Commun. 220, 411-417). Furthermore, electrophoretic
mobility shift analysis revealed that a protein factor(s) in nuclear
extracts from L929 cells binds to the 50-bp region in a
sequence-specific manner and that the amount of DNA-protein complex is
increased by tannic acid treatment. Moreover, the negative regulatory
sequence ACTG and the tannic acid-sensitive 13-bp element in this
region were shown to be responsible for the formation of the
DNA-protein complex by electrophoretic mobility shift analysis and
footprint analyses. These findings suggest that the suppressive effect
of tannic acid on MMTV gene expression is mediated by a protein
factor(s) that binds to the negative regulatory element containing the
common ACTG motif in a cooperative manner with the tannic
acid-sensitive 13-bp element.
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INTRODUCTION |
The MMTV long terminal repeat
(LTR)1 has been used as a
model system for studying the regulation of steroid-induced
transcription and the conformational effect of chromatin structure on
transcription (1). The chromatin structure of the MMTV LTR has been
well characterized, and it has been shown that the LTR acquires a
phased array of six nucleosomes (1, 2). Binding motifs for
hormone-responsive elements and other elements, such as the nuclear
factor I (NF-I) and octamer binding factor binding motifs, are located
in the MMTV promoter region (3-5). Both the NF-I and octamer binding factor motifs are known to be positive cis-regulatory
sequences for MMTV transcription of transiently introduced templates
(4). However, when the motifs are stably introduced into chromatin, they do not function unless the chromatin structure of MMTV LTR is
altered by hormone (glucocorticoid) action (4, 6, 7). These
observations suggest that integrated MMTV gene expression is controlled
by both transcription factors and chromatin structural changes in the
LTR.
We previously reported that the glucocorticoid-induced gene expression
of MMTV is sensitive to tannin and related compounds (8-10). Here, we
have investigated a tannic acid-responsive region in the MMTV promoter
by transient transfections of the MMTV promoter containing
chloramphenicol acetyltransferase (CAT) expression plasmids into the
mouse fibroblastic cell line L929. A 50-bp region located downstream of
the TATA element was found to be responsible for the suppressive effect
of tannic acid. The introduction of various mutations in the 50-bp
region suggested the presence of two separate elements, a tannic
acid-sensitive 13-bp element and a negative regulatory ACTG motif.
Furthermore, gel mobility shift analysis showed that a protein
factor(s) that binds specifically to the 50-bp region is induced into
L929 cell nuclei by tannic acid treatment. The results of gel shift
competition analysis using mutated-oligonucleotides suggested that the
ACTG motif and the 13-bp element play important roles in the formation
of the DNA-protein complex. The specific binding of the nuclear
factor(s) to the ACTG motif and the 13-bp element was confirmed by
footprint analysis. These results suggest that the signal produced by
tannic acid treatment is transduced to the negative regulatory motif under the control of the tannic acid-sensitive element. Previously, we
found a tannic acid-responsive 30-bp element in the human
immunodeficiency virus (HIV) promoter (11). A comparison of the
sequences of the 50-bp element in the MMTV promoter and the 30-bp
element in the HIV promoter revealed the presence of a common ACTG
motif. Thus, our results may provide an important clue to controlling the regulation of retroviral gene expression through the tannic acid-sensitive element along with the ACTG motif by using tannic acid
related compounds.
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EXPERIMENTAL PROCEDURES |
Materials--
Tannic acid and dexamethasone were purchased from
Sigma. Acetyl CoA was from Wako Chemical Co. (Japan).
[14C]Chloramphenicol and [
-32P]ATP were
from NEN Life Science Products. Restriction enzymes and other DNA
modifying enzymes were from Takara (Japan). Poly(dI-dC) was from
Amersham Pharmacia Biotech.
Cell Cultures--
L929 is a mouse fibroblastic cell line (12).
The 34I cell line is derived from a C3H mouse mammary carcinoma kindly
provided from Dr. Gordon Hager (13, 14). These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum, 2 mM L-glutamine, and
antibiotics (100 IU/ml of penicillin and 100 µg/ml of
streptomycin).
Northern Blot Analysis--
Total cellular RNAs were extracted
from cultured cells by the guanidine isothiocyanate cesium chloride
method. Five micrograms of total RNA was fractionated in a
formaldehyde-containing 1.0% agarose gel and transferred to a nylon
membrane (Pall Inc.) Hybridization of this filter with a
32P-labeled 2.0-kilobase pair
EcoRI-BglII fragment covering the env
region of the MMTV gene was carried out as described previously. (8,
14). The filter was washed with 0.05× SSC buffer containing 10 mM EDTA and 0.1% SDS at 90 °C for 30 min and hybridized
with a 32P-labeled 1.9-kilobase pair BamHI
fragment covering the human
-actin coding sequence.
Plasmid Constructs--
The pMMTVCAT plasmid was constructed by
inserting the 1.3-kilobase HindIII-XhoI fragment
of the LTR region (from
1175 to +140 (15)) of the MMTV gene into the
SacI site of pUC00CAT (16). The pTKCAT (pBLCAT2 (17))
plasmid contains a herpes simplex virus thymidine kinase (TK) promoter.
These constructs carry the bacterial CAT gene that is expressed in
eukaryotic cells under the control of each promoter.
pMMTVCAT was treated with the restriction enzyme SacI, and
the large fragment was excised. This fragment, which lacks nucleotides
1175 to
109 of the MMTV promoter region, was blunt ended with T4
DNA polymerase and self-ligated. The resultant plasmid was designated
pMMTV
1CAT. Similarly, pMMTV
2CAT, from which nucleotides +90 to
+140 of the MMTV LTR were deleted, was made by excising the 50-bp
PpuMI/SmaI fragment from the pMMTVCAT construct.
The pM50TKCAT and pCMTKCAT plasmids contain a 50-bp sequence of
the nucleotide from +90 to +140 (M50 sequence;
5'-CCCCCCCGCAGATCCGGTCACCCTCAGGTCGGCCGACTGCGGCAGACGGATCA-3') and three
tandem repeats of seven nucleotides (CM sequence;
5'-C(ACTGCGG)3A-3'), respectively, at the BamHI
site of pTKCAT in the correct orientation. The CAT reporter plasmids,
pM50m4TKCAT, pM50m6TKCAT, and pM50m62TKCAT, carry mutations
on the ACTG motif of the M50 sequence of pM50TKCAT. Similarly,
pM50mG0TKCAT and its derivatives have mutations on the tannic
acid-responsive sequence of the M50 sequence of pM50TKCAT. The mutated
sequences are indicated in Fig. 6B. The mutated nucleotide
on the M50 sequence in pM50mTTKCAT is
5'-CCCCCCCGCAGATCTGGTCACCCTCAGGTCGGCCGACTGCGGCAGACGGATCA-3'.
The pCM2TKCAT and pMC2TKCAT plasmids
contain two tandem repeats of the CM sequence at the BamHI
site of pTKCAT in the correct and reverse orientations, respectively.
pCMm5TKCAT and pCMm6TKCAT are plasmids with mutations introduced on the
ACTG motifs of pCM2TKCAT, as illustrated in Fig.
9A. These plasmids were used for transfection assays.
Transfection Assay--
Plasmid DNAs were transfected into L929
cells by the DEAE-dextran method (16). Adherent cells (2 × 106) were treated with 0.4 ml of TBS (25 mM
Tris-HCl (pH 7.4), 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM
CaCl2, and 0.5 mM MgCl2) containing
4 µg of the reporter plasmid, and 500 µg/ml of DEAE-dextran for 30 min at room temperature. The cells were then washed with TBS to remove
unadsorbed DNA and cultivated for another 48 h in Dulbecco's
modified Eagle's medium-10% fetal calf serum. Next, dexamethasone and
tannic acid were added to the culture medium. After 16 h of
incubation, the cells were collected and used for the preparation of
CAT samples. CAT assays and thin-layer chromatography were performed as
described previously (11, 16). Briefly, cell extracts containing 100 µg of protein were incubated with [14C]chloramphenicol
and acetyl coenzyme A at 37 °C for 24 h. CAT activities of the
reaction products were examined by thin-layer chromatography and
quantified with a Fuji BAS 2000 image analyzer system (Fuji Film,
Tokyo, Japan).
DNase I Footprint Analysis--
DNA-protein binding reactions
and DNase I treatments were performed as described elsewhere (18).
Briefly, a double-stranded DNA probe was prepared by annealing a
32P 5'-end-labeled M50 sense oligonucleotide
(5'-GATCCCCCCCCGCAGATCCGGTCACCCTCAGGTCGGCCGACTGCGGCAGACGGATCA-3') with
a nonlabeled M50 antisense oligonucleotide
(5'-GATCTGATCCGTCTGCCGCAGTCGGCCGACCTGAGGGTGACCGGATCTGCGGGGGGG-3'). About 0.2 ng of the probe (20,000 cpm) was incubated with 48 µg of protein (nuclear extract and bovine serum albumin) and poly(dI-dC) (0, 4, or 8 µg) in binding buffer (25 mM HEPES-KOH (pH
7.9), 50 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA,
and 10% (v/v) glycerol) (50 µl) for 30 min at room temperature.
Then, a
volume of a solution containing 0.175 U/µl of
DNase I and 80 mM MgCl2 was added, and the
samples were incubated for 1 min at room temperature. The enzyme
reaction was stopped by the addition of 3 volumes of stop buffer (0.1 M Tris-HCl (pH 8.0), 0.5 M NaCl, 1% sodium
sarkosyl, 10 mM EDTA, and 25 µg/ml calf thymus DNA). The
partially digested DNAs were extracted by phenol/chloroform and
precipitated with 80% ethanol. The recovered samples were dried and
analyzed in 8 M urea-10% polyacrylamide sequence gel. An
A+G ladder was prepared by a chemical sequencing reaction (19).
Electrophoretic Mobility Shift Assay (EMSA)--
DNA binding
reactions and EMSAs were performed by a modification of a procedure
described previously (16). Briefly, two complimentary synthetic
oligonucleotides were annealed (see below) and labeled with
[
-32P]ATP with T4 polynucleotide kinase. Samples of
about 0.1 ng of 32P-labeled oligonucleotides (10,000 cpm)
were incubated with 1 µg of nuclear extracts from L929 cells and 1 µg of poly(dI-dC) in a solution (20 µl) of 25 mM
HEPES-KOH (pH 7.9), 50 mM KCl, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and 10% (v/v) glycerol (binding buffer) for 30 min at room temperature. Excess (0.8-100-fold in moles) unlabeled
oligonucleotide was added to the incubation mixtures in competition
assays. DNA-protein complexes were analyzed in 4% native
polyacrylamide gels with buffer containing 6.7 mM Tris-HCl
(pH 7.5), 3.3 mM sodium acetate, and 1 mM
EDTA.
The oligonucleotide DNAs used were as follows.
M50 (oligonucleotide containing nucleotides +90 to +140 of the MMTV
promoter) has the following sequence.
M50m1, M50m4,
M50m6, M50mG2, M50mG4, and M50mG5 are the mutated DNA fragments of M50
as illustrated in Fig. 9B.
CM (oligonucleotide containing three tandem repeats of the
ACTGCGG motif in the tannic acid-responsive element of the MMTV promoter) has the following sequence.
CMm1 to CMm6 are mutated DNA fragments of CM as illustrated in
Fig. 9A.
DNA fragment CH (oligonucleotide containing three tandem repeats of the
ACTGTTG motif in the tannic acid-responsive element of the HIV promoter
(11)) has the following sequence.
OC (oligonucleotide containing two tandem repeats of the
silencer ACCCTCTCT motif in the human osteocalcin promoter (20)) has
the following sequence.
HES-1 (oligonucleotide containing the HES-1 binding motif (21))
has the following sequence.
TL (oligonucleotide containing three tandem repeats of the
telomeric sequence, TTAGGG motif (22)) has the following sequence.
 |
RESULTS |
Suppression of MMTV Gene Expression by Tannic Acid--
Tannin
related polyphenolic compounds, especially oligomeric ellagitannins,
are potent and specific inhibitors of poly(ADP-ribose) glycohydrolase (8, 9). Because de-poly(ADP-ribosyl)ation of
chromosomal proteins has been suggested to be involved in the initiation of MMTV transcription (8-10), we examined the effect of
tannic acid on glucocorticoid-induced MMTV gene expression in 34I
cells.
Dexamethasone, a synthetic glucocorticoid, induced dramatically induced
both the 35S and 24S (spliced form) RNAs of MMTV transcripts (Fig.
1A, top panel; compare
lane 2 with lane 1). The induction of MMTV
transcription by dexamethasone was potently suppressed by pretreatment
with tannic acid in a dose-dependent manner (Fig. 1A,
top panel, lanes 2-4). To determine the specific suppression of
MMTV expression, we performed Northern blot analysis in the same filter
using
-actin cDNA as a labeled probe (Fig. 1A, middle panel). The relative levels of both the 35S and 24S transcripts compared against 18S and 28S rRNA levels were reproducibly suppressed by the tannic acid treatment (Fig. 1B). In contrast, the
relative level of the
-actin transcript changed little (Fig.
1B). These results indicate that glucocorticoid-induced MMTV
gene expression is specifically lowered or suppressed by tannic acid
treatment. Although tannic acid is a mixture of gallotannins (tri-,
tetra-, and penta-O-galloyl-
-D-glucose) and
ellagitannins (23), the above result is consistent with our previous
findings that oligomeric ellagitannins suppress glucocorticoid-induced
MMTV gene expression in 34I cells (8, 10).

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Fig. 1.
Effect of tannic acid on MMTV gene
expression. A, total RNAs were prepared from 34I cells
treated with 100 (lane 3) or 200 µg/ml (lane 4)
tannic acid or without tannic acid (lanes 1 and
2) for 1 h, and then 0 (lane 1) or 200 nM (lanes 2-4) dexamethasone was added for 30 min. The total RNAs were then subjected to Northern blot analysis with
a DNA probe for the MMTV gene as described in "Experimental
Procedures" (top panel). After autoradiography, the filter
was reprobed with a -actin cDNA, and a similar experiment was
performed (middle panel). The ethidium bromide-stained gel
after electrophoresis is shown (bottom panel). B,
the signal intensities of the 35S (columns 1, 4, 7, and
10) and 24S (columns 2, 5, 8, and 11)
MMTV transcripts and -actin mRNA (columns 3, 6, 9,
and 12) were quantified and normalized with the signal
intensities of the 18S plus 28S rRNAs. Histograms show the relative RNA
levels of 34I cells treated with 100 (columns 7-9) or 200 (columns 10-12) µg/ml tannic acid for 1 h and then
treated with 200 nM dexamethasone (columns
4-12) for 30 min compared with non-drug-treated 34I cells
(columns 1-3). Results are shown as means and S.E. of three
independent experiments.
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Inhibitory Effect of Tannic Acid on MMTV Promoter
Activity--
Previously, we reported that the HIV promoter is
negatively responsive to tannic acid (11). To examine whether the MMTV promoter is also responsive to tannic acid, the 1315-bp LTR region of
the MMTV gene was inserted upstream of the bacterial CAT gene of
reporter plasmid pUC00CAT. The resulting plasmid, pMMTVCAT, was
transfected into L929 cells, which were then treated with dexamethasone
and tannic acid, and CAT activity was examined (Fig. 2A). The CAT activity of the
dexamethasone-treated cells was about 12-fold greater than that of
control cells (Fig. 2A, compare lanes 4-6 with
lanes 1-3). This dexamethasone-induced MMTV promoter activity was decreased to 40% by the addition of tannic acid at a
final concentration of 100 µg/ml (Fig. 2A, lanes 7-9).
Because tannic acid at this concentration has essentially no effect on cell viability or protein content, further analyses were performed under these experimental conditions.

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Fig. 2.
Effect of tannic acid on glucocorticoid
induced MMTV promoter activity in L929 cells. A, the
pMMTVCAT plasmid was transfected into L929 cells in triplicate. After
48 h of transfection, the cells were incubated with (lanes
4-9) or without (lanes 1-3) 200 nM
dexamethasone in the absence (lanes 1-6) or presence
(lanes 7-9) of 100 µg/ml tannic acid. The CAT activity of
the reaction products was determined by thin-layer chromatography.
Transacetylation in each lane was quantified, and relative activities
were compared with the mean CAT activities of dexamethasone-treated
cells (lanes 4-6). B, experiments similar to
those described in A were performed by transfecting the
pMMTV 1CAT plasmid into L929 cells in duplicate. After 48 h of
transfection, the cells were incubated with (lanes 3, 4, 7,
and 8) or without (lanes 1, 2, 5, and
6) 200 nM dexamethasone in the absence
(lanes 1-4) or presence (lanes 5-8) of 100 µg/ml tannic acid.
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To investigate whether the tannic acid-responsive element(s) is located
in the MMTV promoter region, we constructed an MMTV promoter-deleted
plasmid, pMMTV
1CAT, from which nucleotides
1175 to
109 of the
MMTV promoter region were deleted. As shown in Fig. 2, the CAT activity
of cells transfected with pMMTV
1CAT was not induced by
dexamethasone. However, we noticed that the CAT activities of both
dexamethasone-treated and untreated cells were markedly lowered by
tannic acid treatment (Fig. 2B, compare lanes 5 and 6 with lanes 1 and 2, or
lanes 7 and 8 with lanes 3 and
4). Furthermore, the negative effect of tannic acid on the CAT activity of pMMTV
1CAT-transfected cells was apparent and reproducible, although the CAT activity was low (Fig.
3A). We therefore consider
that there is a tannic acid-responsive element(s) located within the
region comprising nucleotides
109 to +140 of the MMTV promoter.

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Fig. 3.
Location of the tannic acid negatively
responsive sequence in the MMTV promoter. A, CAT reporter
plasmids pMMTVCAT, pMMTV 1CAT, and pMMTV 2CAT were transfected into
L929 cells. After 48 h of transfection, the cells were treated
with 200 nM dexamethasone in the absence (open
columns) or presence (gray shaded columns) of 100 µg/ml tannic acid. CAT assays were performed and the activities were
quantified as absolute percentage of transacetylation. Results are
shown as mean and S.E. of six independent experiments. GRE,
glucocorticoid-responsive element; OTF, octamer binding
factor. B, sequence comparison of the 50-bp MMTV element
with the 30-bp HIV element. Related motifs in the MMTV and HIV elements
are underlined.
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Identification of a Tannic Acid-responsive Region in the MMTV
Promoter--
To determine the presence of a negative tannic
acid-responsive element(s) within the region comprising nucleotides
109 to +140 of the MMTV promoter, we constructed another MMTV
promoter-deleted CAT expression plasmid, pMMTV
2CAT, from which
nucleotides +90 to +140 were deleted. The deletion of these 50 bp
caused a reduction in the CAT activity of dexamethasone-treated cells
of about 20% (Fig. 3A, compare columns 1 and
5), suggesting that this 50-bp region functions as a
positive element in the absence of tannic acid treatment. Thus, we
speculate that this is why this 50-bp sequence has not been identified
as a negative element within the MMTV LTR. However, the
dexamethasone-induced CAT activity of cells transfected with
pMMTV
2CAT was not suppressed by tannic acid treatment as effectively
as that of cells transfected with pMMTVCAT or pMMTV
1CAT (Fig.
3A, compare lane 1 with lane 2,
lane 3 with lane 4, and lane 5 with
lane 6). These results suggest that the 50-bp region
(nucleotides +90 to +140) located downstream of the transcriptional
start site contains a negative responsive element(s) to tannic acid
treatment.
Previously, we investigated negative element(s) in the HIV promoter and
found that the 12-O-tetradecanoylphorbol-13-acetate-induced promoter activity of Jurkat cells is suppressed by tannic acid treatment (11). The 30-bp sequence located just upstream of the two
NF-
B motifs was determined to be the negative element responsive to
tannic acid. A comparison between the 50-bp MMTV and the 30-bp HIV
tannic acid-responsive elements revealed a consensus nucleotide
sequence of 5'-ACTG-3' (Fig. 3B). We therefore expected that
this ACTG motif is involved in a the negative response to tannic acid
treatment.
Determination of the Binding Sequence for Nuclear Factor(s) in the
50-bp Region--
To examine whether any DNA binding nuclear factor(s)
interacts with the 50-bp region including the ACTG motif, we performed DNase I footprint analysis (Fig. 4). The
amount of undigested probe increased when nuclear extracts of L929
cells were added to the binding reaction. Most of the probe was
protected in the presence of nuclear extracts when poly(dI-dC) was
absent from in the reaction mixture (Fig. 4A, lanes 4, 7,
and 10). Because 8 µg of poly(dI-dC) was enough to prevent
the nonspecific binding of nuclear protein(s) to the probe (Fig.
4A, compare lanes 5, 8, and 11 with
lanes 6, 9, and 12, respectively), we decided to quantify the relative signal intensities of regions I
(5'-CAGACGGAT-3'), II (5'-CTCAGGTCGGCCGACTGCGG-3'), and III
(5'-CGGTCACC-3') (Fig. 4A) against that of the
undigested probe in the presence of equal amounts of poly(dI-dC). As
shown in Fig. 4B, the relative signal intensity of region II
clearly decreased upon the addition of L929 cell nuclear extracts,
whereas the signal intensities of regions I and III were essentially
unaffected. These results indicate that the nuclear factor(s) binds
specifically to region II. The relatively broad sequence in this region
may include both tannic acid-sensitive and tannic acid-negative
response elements.

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Fig. 4.
Footprint analysis of the nuclear protein
binding region within the 50-bp element. A, DNase I
footprint analysis of the 50-bp element. The M50 sense strand
oligonucleotide was 5'-end-labeled with [ -32P]ATP with
T4 polynucleotide kinase. An equal amount of nonlabeled antisense
oligonucleotide was annealed with the sense oligonucleotide to generate
a DNase I footprint probe. The binding reaction was performed by
incubating the probe (2 × 104 cpm: approximately 0.2 ng) for 30 min at room temperature with 0 (lanes 1-3), 12 (lanes 4-6), 24 (lanes 7-9), or 48 (lanes
10-12) µg of nuclear proteins obtained from tannic acid-treated
L929 cells. The total amount of protein contained in each binding
reaction was brought to 48 µg by adding appropriate amounts of bovine
serum albumin. The amounts of poly(dI-dC) were 0 (lanes 1, 4, 7, and 10), 4 (lanes 2, 5, 8, and
11), or 8 (lanes 3, 6, 9, and 12)
µg. After DNase I treatment, DNA fragments were recovered, and equal
amounts of radioactivity (3 × 103 cpm) were subjected
to 8 M urea-10% polyacrylamide gel electrophoresis. Sizes
of the bands were determined by comparison with the adjacent
oligonucleotide sequence generated by Maxam and Gilbert (19) sequencing
of the 32P-end labeled M50 sense oligonucleotide
(Lane M). B, quantitative analysis of the DNA
fragments after electrophoresis. Signal intensities of regions I-III
and the undigested probe in lanes 3, 6, 9, and 12 in A were quantified with a Fuji BAS 2000 image analyzer
system. The ratio of the signal intensity of the region to that of the
undigested probe was calculated. Results show relative values compared
with the value calculated from lane 3. Open
circles, region I; closed circles, region II;
closed squares, region III.
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The ACTG Motif Is a Negative Response Element--
To determine
the repressive effect of the ACTG motif, we prepared ACTG
motif-containing pTKCAT plasmids (pCMTKCAT, pCM2TKCAT, and
pMC2TKCAT; see "Experimental Procedures"). In all
cases, a reduction in CAT activity was observed in the presence or
absence of tannic acid (Fig. 5 and data
not shown). This repressive effect increased in relation to the number
of ACTG motifs (CM fragments) inserted upstream of the TK promoter. Six
tandem arrays of CM fragments in either orientation were enough to
repress the TK promoter activity by about 80%. Thus, the ACTG motif is
repressive by itself independent of orientation. We further confirmed
the repressive effect of the ACTG motif by using point mutants of pCM2TKCAT (Fig. 5). The CAT activities in pCMm5TKCAT- and
pCMm6TKCAT-transfected cells increased by 2.5-3.5-fold over that of
pCM2TKCAT-transfected cells. It is estimated that a single
ACTG motif confers about 30% repression, whereas the mutated motifs in
CMm5 and CMm6 confer about 10% repression. These results are
consistent with those of gel shift competition analyses, showing that
the CMm5 and CMm6 probes have about one-third the competition ability
of the wild type CM probe (see below, Fig. 9A). This
suggests that the decrease in repressibility seen for the point mutants
is due to lower binding affinities of the nuclear factor(s) in L929
cells for the mutated ACTG motifs.

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Fig. 5.
The ACTG motif by itself has a negative
regulatory effect on the TK promoter. L929 cells were transfected
using pTKCAT, pCMTKCAT, pCM2TKCAT, pMC2TKCAT,
pCMm5TKCAT, and pCMm6TKCAT as reporter plasmids. After 48 h of
transfection, the cells were treated with tannic acid (100 µg/ml) for
16 h. The histograms show CAT activities relative to those of
cells transfected with the pTKCAT plasmid.
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A 13-bp Sequence Is Involved in Tannic Acid
Responsiveness--
The above observations show that the ACTG motif in
the 50-bp region confers negative promoter activity in the presence of tannic acid. Because the motif by itself does not possess tannic acid
sensitivity, we suspected that another sequence in region II indicated
by footprint analysis may regulate negative responsiveness under the
control of tannic acid. To investigate responsiveness in detail, we
introduced various point mutations into the region II of the pM50TKCAT
plasmid. A single nucleotide substitution introduced in the ACTG motif
(pM50m4TKCAT, pM50m6TKCAT, or pM50m62TKCAT) resulted in
less tannic acid-negative responsiveness (Fig. 6A). On the other hand, a
point mutation introduced into region III (Fig. 4) of the 50-bp region
(pM50mTTKCAT) did not affect responsiveness. Surprisingly, a single
nucleotide point mutation within the 13-bp sequence immediately
upstream of the ACTG motif (pM50mG0TKCAT) disrupted the tannic
acid-negative responsiveness of pM50TKCAT completely. Thus, the tannic
acid-responsive sequence is located in this 13-bp sequence.

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Fig. 6.
MMTV 50-bp sequence contains a tannic
acid-responsive region and a negative regulatory region that includes
an ACTG motif. A, reporter plasmids pTKCAT and pM50TKCAT and
its derivatives carrying various mutations in the M50 region were
transfected into L929 cells. After 48 h of transfection, the cells
were treated with or without 100 µg/ml tannic acid for 16 h, and
CAT activity was measured as described under "Experimental
Procedures." The relative CAT activities of the tannic acid-treated
and untreated cells are illustrated in shaded and open
columns, respectively. Results are shown as average with S.E. of
at least three independent experiments. The bracket
represents the region II indicated in Fig. 4A. The ACTG
motif (black box) and the tannic acid-responsive sequence
(hatched box) or the sequence with mutations (open
box) in the M50 fragments are illustrated schematically to the
left. B, similar experiments were performed using
pM50mG0TKCAT variants as reporter plasmids. Their mutants are
illustrated to the left. The relative CAT activities of the
tannic acid-treated and the untreated cells are illustrated in
shaded and open columns, respectively. Results
are shown as average with S.E. of three independent experiments.
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To confirm the tannic acid responsiveness, we introduced other point
mutations into the 13-bp sequence (5'-CTCAGGTCGGCCG-3'). As shown in
Fig. 6B, all mutations disrupted the tannic acid
responsiveness of the M50 fragment, indicating that the 13-bp sequence
is responsible for the tannic acid sensitivity. Because partial
disruption of sensitivity was observed in ACTG motif-mutated reporter
plasmids (Fig. 6A), there may be a binding factor(s) that
associates with the two separate elements, the 13-bp sequence and the
ACTG motif. In other words, the two elements may crosstalk to respond
negatively to tannic acid treatment.
Tannic Acid Induces the Binding Activity of a Nuclear Factor(s)
that Interacts with the 50-bp Region--
The above results suggest
that the 50-bp region of the MMTV promoter is responsible for tannic
acid sensitivity and the negative response ability. Moreover, footprint
analysis showed a DNA sequence (region II) responsible for the binding
of a nuclear factor(s) present in tannic acid-treated L929 cells.
Therefore, we examined in detail whether any nuclear factor(s) binds
sequence specifically to the 50-bp region of the MMTV promoter. Nuclear
extracts were prepared from L929 cells treated with dexamethasone,
tannic acid, or both. The nuclear extracts were incubated with a
synthetic 50-bp double-stranded DNA probe, M50, end-labeled with
32P. As shown in Fig.
7A (compare lanes 1 and 4), the complex of M50 and nuclear protein(s) from L929
cells treated with both tannic acid and dexamethasone is clearly
observable in EMSA. The binding activity of nuclear protein(s) to M50
increased by 3-4-fold, although this increase did not appear when the
cells were treated with dexamethasone alone (Fig. 7A,
lane 2). We further tested whether the CM probe can form a
DNA-protein complex with L929 cell nuclear proteins (Fig. 7A,
lane 5-8). The reaction profile of the retarded bands of the
32P-labeled CM protein complex was essentially identical to
that of the 32P-labeled M50 protein complex. Therefore, the
shifted bands are thought to be due to a tannic acid-inducible protein
factor(s) present in L929 nuclear extracts.

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Fig. 7.
Induction of MMTV 50-bp tannic
acid-responsive element binding activity in L929 cells. A,
the double-stranded oligonucleotide probes M50 (lanes 1-4)
and CM (lanes 5-8) were 5'-end-labeled with
[ -32P]ATP using T4 polynucleotide kinase. L929 cells
were treated with (lanes 2, 4, 6, and 8) or
without (lanes 1, 3, 5, and 7) 200 nM
dexamethasone in the presence (lanes 3, 4, 7, and
8) or absence (lanes 1, 2, 5, and 6)
of 100 µg/ml tannic acid, and nuclear extracts were prepared. The
labeled probe (approximately 10 fmol) was incubated in buffer with 2 µg of poly(dI-dC) and 1 µg of nuclear protein extract for 30 min at
room temperature. The reaction mixture was electrophoresed in a 4%
nondenaturing polyacrylamide gel. A band showing a shift in mobility is
indicated by the arrowhead. B, nuclear extracts
were prepared from L929 cells treated with 100 µg/ml tannic acid for
0, 0.5, 1, 2, 4, 8, 16, or 24 h (lanes 1-8,
respectively) or prepared from cells treated with 0.05% ethanol for 0, 8, or 24 h (lanes 9-11, respectively). EMSA was
performed using 1 µg of nuclear protein extract with a
32P-end labeled M50 probe and 2 µg of poly(dI-dC). A band
showing a shift in mobility is indicated by the
arrowhead.
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To examine the induction profile more precisely, we performed EMSA
using the 32P-labeled M50 probe and nuclear extracts from
L929 cells treated with tannic acid (100 µg/ml) for 0-24 h. Although
a small amount of the 32P-M50 protein complex was detected
in the reaction mixture of nuclear extracts from untreated cells, the
amount of shifted 32P-M50 probe was estimated to be about
1% of the total (Fig. 7B, lane 1). The binding activity of
the nuclear extracts began to increase after 2 h of tannic acid
treatment and reached a plateau after a further 6 h of treatment.
The binding activity was not increased by treatment with 0.05% ethanol
used as the solvent for tannic acid (Fig. 7B, lanes 9-11).
Therefore, we conclude that the binding activity of nuclear factor(s)
to the 50-bp region (region II) is induced by tannic acid
treatment.
Sequence-specific Binding of the Nuclear Factor(s) to Tannic Acid
Response Elements in the 50-bp Region--
To examine the sequence
specificity of M50-protein complex formation, we performed competition
analyses using double-stranded DNA probes M50, CM, TL, OC, and HES-1
(see "Experimental Procedures") as competitors. As shown in Fig.
8A, both M50 and CM, which
contain the ACTG motif, competed in a dose-dependent manner
with the formation of the 32P-M50 protein complex. On the
other hand, the TL probe, which has been shown to bind to the large
fragment of replication factor C (20), did not inhibit complex
formation. Similarly, no competition was observed for the other
double-stranded DNA probes, OC and HES-1 fragments, which have been
reported to be suppressive element motifs (Fig. 8B). These
results suggest that a nuclear factor(s) from tannic acid-treated L929
cells recognizes and binds to the 50-bp region. However, the CM probe
is less effective than the M50 probe in binding (Fig. 7) and
competition (Fig. 8) analyses. These results indicate that both the
ACTG motif and the 13-bp sequence in the 50-bp region are involved in
the specific binding of a nuclear factor(s).

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Fig. 8.
Sequence specific binding of a nuclear
factor(s) to probes containing the ACTG nucleotide motif of the MMTV
50-bp element. A, competition analysis of
32P-M50 protein complex formation. The competitors used for
EMSA were M50 (lanes 2-4), TL (lanes 5-7), and
CM (lanes 8-10). Binding of the 32P-labeled M50
probe (10 fmol) and 1 µg of nuclear extract from L929 cells treated
with 100 µg/ml tannic acid for 4 h and 2 µg of poly(dI-dC) was
examined as described in the legend to Fig. 7A. The amounts
of competitor DNA were 100 fmol (lanes 2, 5, and
8), 300 fmol (lanes 3, 6, and 9), or 1 pmol (lanes 4, 7, and 10). Lane 1 shows a binding assay without competitor. B, an experiment
similar to that described in A was done using the
double-stranded DNA probes M50 (closed circles), CM
(closed triangles), TL (open circles), OC
(open squares), and HES-1 (open triangles) as
competitors. The radioactivities of the shifted band in each assay
mixture formed by incubating 32P-labeled M50 with the
nuclear extract of L929 cells treated with 100 µg/ml tannic acid for
4 h were quantified with a Fuji BAS 2000 image analyzer system.
Results represent the averages of two independent experiments.
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To confirm the requirement of the negative ACTG motif for M50 protein
complex formation, we performed similar gel shift competition analyses
using various mutated CM probes as competitors. As summarized in Fig.
9A, the CM probe containing
the ACTG motif competed in the formation of M50-protein complex by
about 30%. CM mutant probes showed only 5-6% (CMm2 and CMm3), 10%
(CMm4, CMm5, and CMm6), and 20% (CMm1) reductions in the complex
formation. Taking into account the data depicted in Fig. 5, these
results suggest that the four-nucleotide consensus sequence 5'-ACTG-3'
is responsible for at least the formation of the M50-protein complex,
as well as the repressive ability.

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Fig. 9.
Mutation analysis of the ACTG motif and the
13-bp element for M50-protein complex formation. A, gel
shift competition analysis using the M50 DNA fragment as a
32P-labeled probe was performed. Experimental conditions
were similar to those described in the legend to Fig. 8. Competitor DNA
fragments and their mutated nucleotides are shown (left).
Hyphens indicate nucleotides identical to those in the CM
probe. Competitors (300 fmol) were added to the binding reaction
mixtures. Histograms show the relative complex formation of
32P-M50 with protein(s) compared with the reaction without
competitors. Results are shown as average with S.E. from three
independent experiments. B, a similar experiment was
performed using M50 probes containing point mutations as competitor
DNAs (300 fmol) The mutations are illustrated to the left.
Histograms show the relative complex formation of 32P-M50
with protein(s) compared with the reaction without competitors. The
results are shown as the average of two independent experiments.
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Because not only the ACTG motif but also the 13-bp sequence are
responsible for the negative response ability with tannic acid
sensitivity (Fig. 6), we further examined the role of the two elements
in the binding of nuclear factor(s) by the gel shift competition
analysis using various mutated M50 probes as competitors (Fig.
9B). All mutations that disrupted the tannic acid
sensitivity showed almost the same level of competition as the M50
competitor probe in M50-protein complex formation. These results
suggest that both independent elements, the 13-bp and the ACTG motif, are required for the binding of the nuclear factor(s) and that the two
elements act cooperatively through the binding protein(s).
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DISCUSSION |
So far, several negative regulatory elements have been identified
in MMTV LTR (24), all located upstream of the transcription start site.
Recently, Dudley and colleagues (25) found a protein (special AT-rich
sequence-binding protein 1) that binds specifically to the
promoter-proximal negative element. In this study, we characterized the
suppressive effect of tannic acid on MMTV promoter activity and found a
50-bp tannic acid negatively responsive element located at downstream
of the other negative regulatory elements. The consensus sequence for
repressiveness was determined to be 5'-ACTG-3' by comparing the 50-bp
region of MMTV LTR with the 30-bp region in HIV LTR previously
identified as a tannic acid-responsive negative element (11). This ACTG
motif is able to confer a repressive effect on the TK promoter activity
in CAT assay. EMSAs show the importance of the ACTG motif for the
specific binding of a nuclear protein factor(s). Point mutations
introduced into the ACTG motif resulted in decreases in both the
specific binding of the nuclear factor(s) and repression in the
transient CAT assay. Furthermore, point mutations in the 50-bp region
of CAT constructs revealed a tannic acid-sensitive 13-bp sequence just
upstream of the ACTG motif. The binding of the protein factor(s) to
this 13-bp sequence was supported by footprint analysis. The 50-bp
binding activity of the protein factor(s) from L929 cell nuclear
extracts was found to be inducible by tannic acid treatment. This is
the first evidence that MMTV LTR contains a novel tannic
acid-responsive negative regulatory element (containing the ACTG motif
plus the 13-bp sequence) downstream of the previously identified
negative regulatory elements (24, 25).
MMTV LTR has been well characterized as a steroid-inducible promoter
(1). It locates a hormone-responsive region that contains four hormone
receptor binding elements and confers responsiveness to steroid hormone
action (26). Furthermore, the chromatin structure of integrated MMTV
LTR has been shown to be organized into an array of at least six
positioned nucleosomes (2, 15). Recently, the importance of chromatin
remodeling by the glucocorticoid receptor in the transcription of the
stably integrated MMTV gene was clearly demonstrated (27). The LTR
region also locates other protein factor binding-sites, such as the
NF-I binding site (28), AP-2 element (29), and two octamer motifs (5,
30). Associations between the protein factors and these transcriptional
elements and the subsequent alterations in chromatin structure by the
interactions between the factors the chromatin constructing proteins
are thought to play important roles in chromosome-integrated MMTV gene
expression (4-7, 15, 31).
MMTV transcription is also known to be regulated by modifications in
chromosomal proteins, such as acetylation (32, 33) and
poly(ADP-ribosyl)ation (10, 14). Hyperacetylation of histones induced
by histone deacetylase specific inhibitors has been shown to increase
chromosome-integrated MMTV and HIV-1 gene expression (32, 33). Histone
hyperacetylation catalyzed by histone acetyltransferases (34) is
believed to lead to allosteric changes in nucleosomal conformation that
destabilize chromatin structure (35, 36). Thus, the hyperacetylation of
histones is thought to cause transcriptional activation. Furthermore,
several transcriptional factors, including p300/CBP and
p300/CBP-associated factor, have recently been shown to possess histone
acetyltransferase activity in mammalian cells (37, 38). Previously, we
reported that the induction of the MMTV mRNA is associated with a
loss of poly(ADP-ribose) from the HMG 14 and HMG 17 proteins (14).
Moreover, we observed that oenotein B, a specific inhibitor of
poly(ADP-ribose) glycohydrolase, suppresses glucocorticoid-induced MMTV
transcription in 34I cells (10). In this case, suppression is
accompanied by the inhibition of glucocorticoid-induced endogenous
de-poly(ADP-ribosyl)ation of the HMG 14 and HMG 17 proteins, which are
known transcriptional activators (39, 40). Our present data clearly
show that tannic acid treatment induces the ability of L929 cell
nuclear extracts to bind to the 50-bp region of the MMTV LTR.
Interestingly, the 50-bp region partly overlaps with the boundary of
the Nuc-A position (2, 41). The specific protein factor(s) that binds
to the tannic acid-responsive sequence containing the ACTG motif and the 13-bp sequence might interact with poly(ADP-ribosyl)ated HMG proteins and/or histones to affect their nucleosomal assembly systems
and thereby suppress MMTV transcription. Further investigations are
needed to confirm this hypothesis and to elucidate the role of the
tannic acid-responsive DNA binding factor(s) that negatively regulates
MMTV gene expression.
The integrated HIV promoter has been shown to contain a periodic array
of nucleosomes, like the MMTV promoter (42). Transcriptional regulatory
elements such as NF-
B, Sp-1, and TATA motifs are located in the HIV
LTR (43-45). A recent study has shown that several changes in the
local chromatin structure of the HIV promoter induced by NF-
B play
important roles in regulating transcription from the integrated HIV
provirus (46). The HS4 element in the HIV promoter has been shown to be
associated with the 3' terminus of the U5 region and to contain a
cluster of potential binding sites for the AP-1, AP-3-like, DBF-1, and
Sp-1 proteins (47). In addition, the nucleotide sequence of this
element has been shown to be required for the control of transcription
at the chromatin-organized HIV promoter (48). Like that of the MMTV
promoter, the chromatin structure the of integrated HIV promoter is
affected by specific transcription factors (49, 50). It is noteworthy
that the negative ACTG motif found in the MMTV promoter is also present in the HIV LTR. As indicated in Fig. 6, the role of this motif by
itself may be to repress the inherent activities of the proviral promoters in the absence of inducers, such as glucocorticoids and tumor
necrosis factor. The factor(s) that binds to the ACTG motif that is
present in both the MMTV and HIV promoters may negatively regulate MMTV
and HIV gene expression by interacting with other transcription factors
or chromatin-associated proteins to alter the nucleosomal conformations
to transcriptionally inactive states. To clarify the molecular
mechanism by which the ACTG motif represses MMTV and HIV gene
expression in tannic acid-treated cells, molecular cloning of cDNAs
that encode the MMTV 50-bp and HIV 30-bp element (or ACTG motif)
binding protein(s) is required. Our present data support the proposal
of Smith and Hager (51) that consideration of chromatin structural
changes in LTR is important in understanding transcriptional regulation
in vivo. We believe that the characterization of viral LTR
binding proteins and their interactions with chromosomal proteins may
not only clarify transcription regulatory mechanisms but also
contribute to the development of therapies for retrovirus-associated diseases.