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Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, Texas 77843-4466
Address all correspondence and requests for reprints to: Dr. Stephen H. Safe, Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and Sp1 proteins were
required for hormone-induced trans-activation that
involved ER/Sp1 binding to the G-rich site in which only Sp1 protein
bound DNA. Both proteins also interacted in Drosophila
cells in functional assays, confirming the transcriptional activation
of TS-involved ER/Sp1, and this adds to the increasing number of
genes that are activated through this pathway in breast cancer cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and have been used extensively as a model
for investigating the effects of estrogens, mitogenic growth factors,
and antiestrogens on cell proliferation and gene expression (reviewed
in Refs. 1, 2, 3, 4, 5, 6). Several studies demonstrate that 17ß-estradiol
(E2), insulin-like growth factor I (IGF-I),
transforming growth factor-, epidermal growth factor, and insulin
induce the growth of MCF-7 and other ER-positive breast cancer cell
lines, and in some studies combined treatment with
E2 plus mitogenic polypeptides gave more than
additive cell proliferation (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Differences in responsiveness to
E2/mitogenic polypeptides are frequently
reported, and these may be due in part to serum factors and variable
expression of wild-type and truncated forms of ER in MCF-7 cells
(18, 19, 20, 21).
Hormone-induced proliferation of MCF-7 cells is accompanied by
increased DNA synthesis and [3H]thymidine
uptake, and several enzymes and/or genes associated with purine and
pyrimidine synthesis are also up-regulated (22, 23, 24, 25, 26, 27, 28). For example,
dihyrofolate reductase, thymidylate synthase (TS), and thymidine kinase
responses are increased after treatment of MCF-7 cells with
E2; however, the magnitude of these responses to
and effects of antiestrogens were variable (26, 27). For example,
E2 caused a slight increase in TS activity, and
both tamoxifen and 4'-hydroxytamoxifen also increased activity, but
only at some concentrations. We have been investigating the regulation
of E2-induced gene expression, and analysis of
messenger RNAs (mRNAs) from MCF-7 cells by suppressive subtractive
hybridization identified TS as 1 of at least 45 genes up-regulated by
E2. This induction response was confirmed by
Northern blot analysis. Analysis of the TS gene promoter has shown that
a downstream GC-rich (-150 to -142) Sp1 protein-binding site is
required for E2-mediated transcriptional
activation of the TS gene. ER/Sp1 action through binding G/GC-rich
motifs has now been characterized in several gene promoters, including
c-fos, heat shock protein 27, retinoic acid receptor 1,
adenosine deaminase, cathepsin D, bcl-2, and IGF-binding
protein 4 (IGFBP-4) (29, 30, 31, 32, 33, 34, 35, 36), and the results of this study indicate
that TS is induced via a comparable pathway in MCF-7 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was prepared in this laboratory (37).
Dimethylsulfoxide (Me2SO) was used as solvent for
E2 and the antiestrogens. 4'-Hydroxytamoxifen was
purchased from Sigma; ICI 182,780 was provided by Alan
Wakeling (Astra USA, Inc.-Zeneca Pharmaceuticals, Macclesfield, UK). ß-Galactosidase activity
in cotransfection studies was determined using an assay kit purchased
from Invitrogen (Carlsbad, CA). All other chemicals and
biochemicals were the highest quality available from commercial
sources. Oligonucleotides derived from the TS gene promoter and a consensus Sp1 oligonucleotide were synthesized by the Gene Technologies Laboratory, Texas A & M University (College Station, TX). Structures of these oligonucleotides (sense strands) are summarized below. The putative Sp1 binding sites are underlined, and mutations incorporated in the mutant oligonucleotides are denoted by an asterisk: TS4 (-229/-140), 5'-AGC TTG CCA CAC CCG TGG CTC CTG CGT TTC CCC CTG GCG CAC GCT CTC TAG AGC GGG GGC CGC GAC CCC GCC GAG CAG GAA GAG GCG GAG CG-3'; TS4m1 (-229/-140), 5'-AGC TTG A1A1T1 A1T1T1 CCG TGG CTC CTG CGT TTC CCC CTG GCG CAC GCT CTC TAG AGC GGG GGC CGC CGC GAC CCC GCC GAG CAG GAA GAG GCG GAG CG-3'; TS4m2 (-229/-140), 5'-AGC TTG CCA CAC CCG TGG CTC CTG CGT TTC CCC CTG GCG CAC GCT CTC TAG AGC GGG GGC CGC CGC GAC CCC GCC GAG CAG GAA GAT1 C1T1T1 A1AG CG-3'; TS4m3 (-229/-140), 5'-AGC TTG CCA CAC CCG TGG CTC CTA1 G1A1T C1TC CCC CTG GCG CAC GCT CTC TAG AGC GGG GGC CGC GAC CCC GCC GAG CAG GAA GAG GCG GAG CG-3'; TSSp1 (-145/-124), 5'-AGC TTC AGG AAG AGG CGG AGC GCG GGA G-3'; TSSp1m (-145/-124), 5'-AGC TTC AGG AAG AT1C1 T1T1A1 AGC GCG GGA G-3'; and Sp1 (consensus), 5'-AGC TTA TTC GAT CGG GGC GGG GCG AGC G-3'.
Cloning
The inserts encoding the wild-type hER were removed by digesting
the appropriate plasmids with EcoRI. The inserts were then
religated into pcDNA3-Neo (Invitrogen, Carlsbad, CA),
which had been linearized with EcoRI and treated with CIAP
(29, 30, 31, 32). The ligation products were transformed into DH5a cells, and
clones were verified by sequencing. Efficiencies for in
vitro transcription/translation of these genes were periodically
determined using the rabbit reticulocyte lysate system and
[35S]methione, followed by SDS-PAGE and
quantitation of radiolabeled proteins by densitometry. Results showed
that levels of immunoreactive wild-type and variant ER proteins were
not significantly different. The pBLTATA-CAT plasmid was made by
digesting the pBLCAT2 vector with BamHI and XhoI
to remove the thymidine kinase promoter; the double stranded E1B
oligonucleotide containing complementary 5'-overhangs was then inserted
into the corresponding sites. TS4, TS4m1, TS4m2, and TS4m3
oligonucleotides were cloned into the pBLTATA-CAT vector at the
HindIII and BamHI sites to give the pTS4, pTS4m1,
pTS4m2, and pTS4m3 constructs, respectively. pTS1, pTS2, and pTS3
constructs contained -441 to +28, -229 to +28, and -140 to +28 TS
gene promoter inserts (37).
Transient transfection assay
MCF-7 cells were transfected using the calcium phosphate method
with 10 µg TS gene promoter-derived constructs and 5 µg wild-type
or variant ER expression plasmids; in the absence of cotransfected
wild-type ER, no hormone-responsiveness was observed, and this was due
to overexpression of the TS promoter-derived constructs.
ß-Galactosidase-lacZ plasmid (5.0 µg) obtained from
Invitrogen was cotransfected in studies determining
differences in basal CAT activities with constructs containing TS gene
promoter inserts; activities are corrected for transfection
efficiencies. Previous studies have shown the requirement for
cotransfection of hER expression plasmid using other
E2-responsive constructs in MCF-7 cells (29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43). pcDNA3-Neo (Invitrogen) was used as an empty
vector (control) and was also added in some experiments to maintain
uniform levels of added DNA. Transfection efficiency was high, and no
additional shock was required. After 18 h, medium was changed, and
cells were treated with Me2SO (0.2% total
volume), E2, 4'-hydroxytamoxifen, ICI 182,780, or
their combinations in Me2SO for 44 h. Cells
were then washed with PBS and scraped from the plates. Cell lysates
were prepared in 0.15 ml 0.25 M Tris-HCl (pH 7.5)
by three freeze-thaw-sonication cycles (3 min each). Protein
concentrations were determined using BSA as a standard, and analysis of
CAT activity in cell lysates used a constant amount of protein from
each treatment group. Lysates were incubated at 56 C for 7 min to
remove endogenous deacetylase activity. CAT activity was determined by
incubating aliquots of the cell lysates with 0.2 mCi
D-threo-[dichloroacetyl-1-14C]chloramphenicol
and 4 mM acetylcoenzyme A. Acetylated products
were visualized and quantitated using a Instant Imager system (Packard
Instruments Co., Downers Grove, IL). CAT activity was calculated as a
fraction of that observed in cells treated with
Me2SO alone (arbitrarily set at 100), and results
are expressed as the mean ± SD. At least
three separate experiments were carried out for each treatment
group.
Schneider cell maintenance and transfection
Schneider SL-2 cells were obtained from American Type Culture Collection and were grown at room temperature in T-150
flasks in Schneider’s medium (Life Technologies, Inc.,
Gaithersburg, MD) supplemented with 5% FCS (heat inactivated at 56 C
for 30 min) and 0.5 x antibiotic/antimycotic solution. Three
milliliters of cell suspension per plate were pipetted onto 60-mm
plates, and after incubation for 24 h at room temperature, cells
were transfected with 0.5 ml transfection cocktail containing 5 µg
pTS4 report plasmid, 1 µg ß-galactosidase, 250 µl 2 x
HBSS, and 25 µl 2.5 M CaCl2, with
different amounts of pPac/Sp1 or pPac/hER plasmids. The vector pPac
was used to make the total amount of plasmid DNA 7 µg/incubation.
After incubation for 20 h at room temperature, cells transfected
with ER were treated with 10-8 M
E2 or received only the solvent carrier
(Me2SO) for 44 h and were harvested by
scraping. CAT activities were determined and normalized to
ß-galactosidase activity as described above.
Ribonuclease protection assay
Cells were plated into 100-mm petri dishes and cultured in 5%
stripped serum for 24 h, then synchronized for 2 days and treated
with Me2SO (control) and 1 nM
E2 at various time points. A solution of RNAzol B
(Tel-Test, Inc., Friendswood, TX) was added, cells were
scraped from the plates, and total RNA was extracted. Using a T7
promoter sequence appended to 5' of reverse PCR primers, the PCR
products of TS and ß-actin complementary DNAs (cDNAs) were in
vitro transcribed to [-32P]UTP-labeled
complementary RNAs (cRNAs) by a T7 RNA polymerase using the standard
protocol, as described in the assay kit (Ambion, Inc.
Austin, TX). The 251- and 306-nucleotide riboprobes were complementary
to the coding region from 469–720 of TS mRNA and from 144–450 of
ß-actin mRNA. The ribonuclease protection assay was performed
following the standard protocol supplied with the assay kit
(Ambion, Inc., Austin, TX). Briefly, total RNA (30 µg)
was incubated for 15 min at 68 C with 80,000 cpm gel-purified
[-32P]UTP-labeled cRNAs in 10 µl
hybridization buffer. After hybridization, the samples were digested
with ribonuclease A/T1 in 100 µl RNase digestion buffer for 30 min at
37 C. The digestion reaction was terminated by the addition of 150 µl
inactivation/precipitation mix. The mixture was precipitated then
denatured and electrophoresed on a 5% polyacrylamide gel containing 8
M urea. The gel was dried and exposed to a x-ray
film for 24 h. Levels of protected cRNA probe were standardized
relative to protected ß-actin cRNA probe in the same sample, and band
intensities were determined on Betagen Betascope 603 blot analyzer
(Intelligenetics, Inc., Mountain View, CA) or
autoradiography using X-Omat film (Eastman Kodak Co.,
Rochester, NY). Quantitation of band intensities used a Zero-D software
package (Molecular Dynamics, Inc., Sunnyvale, CA) and a
JX-330 scanner (Sharp Electronics, Mahwah, NJ).
Electrophoretic mobility shift assays with pure protein
Gel electromobility shift assays were performed using
recombinant Sp1 protein and different amounts of ER protein.
E2 was added to the reaction at a final
concentration of 20 nM and then incubated on ice for 15
min. Sp1 protein and 32P-labeled oligonucleotides
were then added to the reaction mixtures in the presence of 1 µg
poly[deoxyinosinic-cytidylic acid] and incubated for 15 min at
25 C. In competition experiments, different amounts of unlabeled
oligonucleotides were also included in the incubation mixture. Aliquots
of these mixtures were loaded onto a 4% polyacrylamide gel
(acrylamide-bisacrylamide ratio, 30:0.8) and run at 110 V in 0.09
M Tris-0.09 M borate-2 mM EDTA (pH
8.0). 32P-Labeled DNA and DNA-protein bands were
visualized by autoradiography and quantitated by densitometry using the
Molecular Dynamics, Inc. Zero-D software package and a
Sharp JX-330 scanner. For some of these studies, relative band
intensities are presented as the mean ± SD for three
separate experiments.
Electrophoretic mobility shift assays with nuclear extracts
Synthetic oligonucleotides were synthesized, purified, annealed,
and 32P labeled, and DNA binding was measured
using a gel retardation assay as previously described (34). Nuclear
extracts were incubated in HEGD buffer (25 mM HEPES, 1.5
mM EDTA, 10% glycerol, and 1.0 mM
dithiothreitol, pH 7.6) with poly[d(I-C)] (200 ng) for 15 min at 20
C. The mixture was then incubated for an additional 15 min (20 C) after
the addition of 32P-labeled DNA. Aliquots from
reaction mixtures were loaded into a 5% polyacrylamide gel
(acrylamide-bisacrylamide, 30:0.8) and electrophoresed at 110 V in 0.9
M Tris-borate and 2 mM EDTA, pH 8.0. The gel
was dried, and protein-DNA interactions were determined, quantitated by
scanning on an Instant Imager system, and visualized by autoradiography
as described above.
In vitro SssI footprinting
This is a highly sensitive assay for studying protein-DNA
interactions and depends on SssI methylation of CpG sites
(36, 44, 45). Fifty micrograms of pTS1 containing the -441 to +28
insert from the TS gene promoter were restricted with XhoI
and diluted (10 ng/µl), and 1 µl was incubated with different
concentrations of human recombinant ER, Sp1, and both proteins
simultaneously. Binding reactions were carried out in 1 x NS
binding buffer (0.02 M HEPES, 0.1
M KCl, 0.005 M
MgCl2, 0.004 mM EDTA, 5%
glycerol, and 50 mM 5-adenosylmethionine)
in a volume of 25 µl. The binding reactions were incubated on ice for
5 min and then equilibrated to room temperature for 20 min. One
microliter of purified SssI (New England Biolabs, Inc., Beverly, MA) was added to the equilibrated reactions and
incubated at 30 C for 15 min and then at 75 C for 15 min; 10 µl
freshly made deamination denaturation buffer (0.9
N sodium hydroxide, 25 mM
EDTA, and 0.2 mg/ml sheared salmon sperm DNA) were added. After
incubation for 5 min at 98 C, 200 µl of a saturated solution of
sodium metabisulfite were added, and the samples were processed as
described previously (44, 45). The primers used to amplify from the
purified deaminated plasmid DNA were: WB1, 5'-CAC CAA CTA ACA AAA AAA
AAA TAT ACT AC-3'; and WB2, 5'-TTT TAA AAT GTT TTT TAT GAT GTT ATT
G-3'.
Statistics
Results are expressed as the mean ± SD for
three separate determinations for each data point. Statistical
significance was determined using ANOVA and Scheffe’s
post-hoc test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Basal and
E2-induced CAT activities were investigated in
transient transfection studies in MCF-7 cells using a series of
constructs containing promoter inserts from the TS gene (37). Basal CAT
activities in MCF-7 cells transfected with pTS1, pTS2, and pTS3 were
comparable (Fig. 1B), and these data contrasted with the results of
previous studies using HeLa cells (37), which showed that the -229 to
-140 region of the gene promoter was primarily responsible for high
basal activity that was decreased by upstream negative regulatory
elements (-441 to -229).
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(5 µg) and treated
with Me2SO or 10 nM
E2. In the absence of cotransfected ER,
induction by E2 was not observed due to
insufficient endogenous ER in transfected cells that overexpress the
TS constructs. With the exception of plasmids that contain one or more
copies of palindromic estrogen-responsive elements, most constructs
containing promoter inserts from E2-responsive
genes also require cotransfection with wild-type ER in MCF-7 cells
for trans-activation by E2 (29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43). The results (Fig. 1B
) show that E2 did
not induce CAT activity in MCF-7 cells transfected with pTS1; however,
after removal of the upstream -441 to -230 sequence, the resulting
pTS2 construct was E2 responsive; hormone
inducibility was subsequently lost in pTS3, indicating that elements
required for ER action were located in the -229 to -140 region of
the TS gene promoter.
The construct pTS4 contains the -229 to -140 region of the TS gene
promoter linked to a CAT reporter gene, and E2
induced CAT activity in MCF-7 cells transiently transfected with pTS4.
Previous studies demonstrated that a CACCC box and a nonconsensus Sp1
binding motif (GAGGCGGA) were important for basal activity of
constructs containing TS gene promoter inserts (37), and we have
investigated the roles of both sites in E2
responsiveness using constructs mutated in the CACCC box (pTS4m1) and
the downstream Sp1 binding site (pTS4m2). A third construct, pTS4m3,
was mutated within a negative regulatory sequence (-212 to -202)
identified in transfection studies in HeLa cells. The results of
transient transfection studies showed that pTS4m1 and pTS4m3 were
E2 responsive (>2.5-fold induction), indicating
that the downstream G-rich site was required for ER action. Mutation
of the negative regulatory sequence did not affect
E2 responsiveness; however, basal activity was
higher. The results in Fig. 2B show that
in MCF-7 cells transfected with pTS4 plus pEBG (empty vector),
E2 induced CAT activity; in contrast, when pEBG
was replaced by a plasmid expressing the dominant negative form of Sp1
(pEBG-Sp1), hormone-induced CAT activity was lost. These data confirm
that Sp1 protein is required for ER/Sp1 action at the G-rich site in
the TS gene promoter. The results in Fig. 2C summarize the effects of
E2, 4'-hydroxytamoxifen, ICI 182,780, and their
combination on CAT activity in MCF-7 cells transfected with pTS4 and
ER expression plasmid. ICI 182,780 alone was inactive and inhibited
E2-induced CAT activity, whereas
4'-hydroxytamoxifen was an ER agonist, and a previous report also
showed that TS activity was induced by 4'-hydroxytamoxifen in MCF-7
cells (26).
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and Sp1 with TSSp1 oligonucleotide (-145 to
-124). show that
nuclear extracts from MCF-7 cells treated with DMSO (control) and 10
nM E2 bound to
[32P]TSSp1 (contains the -145 to -124 region
of the TS gene promoter) to give a major broad high mol wt retarded
band (see arrow, lanes 1 and 2), and several lower mol wt
bands. Treatment with E2 increased the intensity
of the retarded band (lane 2). Competition with excess unlabeled
consensus Sp1 and TSSp1 oligonucleotides significantly decreased the
intensity of the high mol wt band. Incubation of MCF-7 cell nuclear
extracts with 32P-labeled consensus Sp1 (lane
1) and TSSp1 (lane 2) oligonucleotides gave a similar pattern of
retarded bands, including the high mol wt complex (Fig. 3B). The
intensity of the [32P]TSSp1-protein retarded
band was decreased after competition with excess unlabeled consensus
Sp1 and TSSp1 oligonucleotides (lanes 3–6), but not that of the TSSp1m
oligonucleotide that is mutated in the G-rich Sp1 protein-binding site.
The high mol wt retarded band formed after incubation of MCF-7 cell
nuclear extracts with 32P-labeled consensus Sp1,
and TSSp1 oligonucleotides (Fig. 3C, lanes 1 and 4, respectively) were
supershifted after coincubation with Sp1 antibody (lanes 2 and 5), but
not nonspecific goat IgG (lanes 3 and 6).
32P-Labeled mutant TSSp1 did not form the high
mol wt retarded band (lane 7) after incubation with nuclear
extracts.
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proteins were also determined in gel mobility shift
assays (Fig. 4). Both radiolabeled
consensus Sp1 and TSSp1 oligonucleotides bound Sp1 protein (Fig. 4A, lanes 1–7), whereas mutation of the G-rich site in TSSp1 resulted in
the loss of Sp1-DNA complex formation (lanes 8 and 9). Moreover, the
intensity of the [32P]TSSp1-Sp1 retarded band
(Fig. 4B, lane 2) was decreased after competition with excess unlabeled
consensus Sp1 (lanes 3 and 4) and TSSp1 (lanes 5–7), but not TSSp1m
(lane 8) oligonucleotides. Thus, results with both nuclear extracts and
recombinant human Sp1 protein suggest that the G-rich site (-150 to
-142) in the TS gene promoter binds with Sp1 protein, and these
results complement previous studies with nuclear extracts from HeLa
cells (37). Incubation of [32P]TSSp1 with
recombinant human ER alone did not give a retarded band (Fig. 4C, lane 6); however, coincubation of recombinant ER plus Sp1 proteins
resulted in significantly enhanced Sp1-DNA binding (lanes 3–5)
compared with binding studies using Sp1 protein alone (lane 2).
Thus, ER enhanced Sp1-DNA interactions, but did not form a
supershifted band. Similar results were reported in previous studies
using Sp1 and ER proteins and G/GC-rich oligonucleotides derived
from other E2-responsive gene promoters in which
ER enhanced the on-rate of Sp1-DNA complex formation (29, 30, 31, 32, 33, 34, 35, 36).
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and Sp1 proteins and their
combination were further investigated using a highly sensitive DNA
footprinting assay (44, 45). Yeast SssI methyltransferase
methylates CpG sites, and this technique has been adapted to
investigate in vitro interactions of ER and Sp1 proteins
interacting with the G-rich E2-responsive region
of the TS gene promoter. Incubation with ER alone (Fig. 5, lanes 2 and 3) did not significantly
alter methylation patterns compared with the control (lane 1);
increasing concentrations of Sp1 protein (20–80 ng; lanes 4–6) did
not alter the footprint in the -150 to -142 regions (
);
however, methylation of upstream G-rich sequences (1) was decreased,
indicating some enhanced Sp1 binding, but only at the highest
concentration of Sp1 protein. In contrast, after coincubation of ER
(100 or 400 fmol) with the lowest concentration of Sp1 protein (20 ng),
there was an enhanced footprint in the -150 to -142 region as well as
other upstream (1) and downstream G-rich sites. Thus, only the
combination of ER plus Sp1 proteins footprinted the
E2-responsnive G-rich sequence (-150 to -142)
in the TS gene promoter.
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and Sp1 interactions in Drosophila Schneider SL2
cells, and these cells have been extensively
used to study functional interactions of proteins with various
promoters. The results in Fig. 6A
demonstrate that basal reporter gene activity in SL-2 cells transfected
with pTS4 is increased after cotransfection with Sp1 expression
plasmid. In cells cotransfected with pTS4, Sp1 (100 ng) and different
concentrations of ER expression plasmids (plus 10 nM
E2), there was significant
trans-activation using 20 ng ER expression plasmid,
whereas in the absence of Sp1 protein, CAT activity was similar to that
in the solvent (Me2SO) control (data not shown).
The enhanced ER-Sp1 interaction at G-rich sites in SL2 cells was
also observed in a comparable study using a G-rich construct containing
a bcl-2 gene promoter insert (36).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have used suppressive subtractive hybridization (55) with polyadenylated mRNA from MCF-7 cells to identify E2-regulated genes (56), and TS was 1 of over 40 genes up-regulated by E2. Northern analysis confirmed a more than 60% increase in steady state mRNA levels, and up to a 2-fold increase has been observed in some individual experiments (data not shown). Horie and Takeishi (37) previously analyzed the TS gene promoter in HeLa cells, and their studies showed that basal activity was dependent on sequences that both increased and decreased activity. In HeLa cells, transient transfection of constructs containing an upstream negative regulatory sequence (-342 to -269) exhibited low basal activity; however, results obtained for pTS1 and pTS2 suggest that this upstream sequence does not significantly affect the basal activity of TS constructs in MCF-7 cells. In contrast, E2 responsiveness was observed with pTS2, but not pTS1 or pTS3, suggesting that 1) the -441 to -229 region contains elements that inhibit transcriptional activation by E2; and 2) the -229 to -140 region is required for induction by E2. Although the major focus of this research was to characterize the E2-responsive region of the promoter, future studies will investigate those elements and proteins in the -441 to -229 region that block estrogen action.
Basal activity of the -229 to -140 region in HeLa cells also is
regulated by a CACCC box (-228 to -212), a negative regulator
sequence (-212 to -202), and a nonclassical G-rich Sp1-binding site
(-150 to -142), and mutations of the latter two sites result in
increased and decreased basal activities, respectively, whereas
mutation of the CACCC box only slightly decreased activity (37).
Mutational analysis of the activating sites in the TS promoter followed
by transfection in MCF-7 cells also confirmed the relative importance
of the downstream G-rich site for basal activity in this cell line, and
mutation of the negative regulator sequence (pTS4m3) increased basal
activity in MCF-7 and HeLa cells (37). Moreover, hormone activation of
pTS4, pTS4m1, pTSm2, and pTSm3 showed that only the G-rich site was
required for ER-mediated trans-activation, and this
response was inhibited by ICI 182,780, whereas 4'-hydroxytamoxifen was
an ER agonist.
Physical and functional interactions of Sp1 with other nuclear proteins
have previously been characterized. For example, Sp1 and
E2F1 interact in vitro, and
E2F1 enhances Sp1-mediated trans-activation in promoter
constructs containing only GC-rich sites (57, 58). Previous studies
have demonstrated that ER and Sp1 physically interact, and ER
preferentially binds to the C-terminal DNA-binding domain of Sp1
protein (29), the region that is also required for binding other
transcription factors including E2F1 (57, 58). The results
summarized in Figs. 3 and 4 demonstrate that Sp1 protein bound the
downstream G-rich sequence (TSSp1, -145 to -124), and ER protein
enhanced Sp1-DNA binding but did not supershift the band. These
observations were consistent with previous reports on ER/Sp1
interactions with other G/GC-rich site from the c-fos,
adenosine deaminase, retinoic acid receptor 1, IGFBP-4, cathepsin D,
bcl-2, and E2F1 genes, where ER also enhanced the
on-rate of Sp1-DNA binding without forming a supershifted ternary
complex (29, 30, 31, 32, 33, 34, 35, 36). The failure to observe a supershifted ER/Sp1-DNA
complex is somewhat surprising in view of the known physical and
functional interactions of ER and Sp1 proteins (29, 30, 31, 32, 33, 34, 35, 36); however,
comparable observations have been reported for other interacting
nuclear transcription factors. For example, sterol regulatory
element-binding protein and cyclin D1 also enhanced binding of Sp1 and
ER to their cognate enhancer elements (59, 60), and the human T cell
lymphotropic virus type 1 Tax protein activates the transcription of
several nuclear proteins by enhancing their DNA binding without forming
a supershifted complex (61, 62, 63, 64, 65, 66). We have also used a highly sensitive
in vitro footprinting assay in which yeast SssI
methyltransferase methylates CpG sites, and this can be carried out
under a variety of incubation conditions. The results show that neither
Sp1 nor ER protein alone footprinted the
E2-responsive G-rich region of the TS gene
promoter; however, in combination, a significant footprint was
observed. Thus, the in vitro footprint complements gel
mobility shift data to demonstrate convergent interactions of ER and
Sp1 proteins at the -150 to -142 region of the TS gene promoter.
The results of this study in MCF-7 cells demonstrate that transiently
transfected ER expression plasmid is required for hormone-mediated
trans-activation of TS through a G-rich site that binds
endogenous Sp1 protein. ER/Sp1 action was further confirmed in MCF-7
cells transiently transfected with ER, pTS4, and a dominant negative
expression plasmid (pEBG-Sp) for Sp1, confirming a role for Sp1 protein
in this transcriptional factor complex. Moreover, functional
interactions of ER and Sp1 proteins were also observed in Schneider
SL-2 cells, where ER clearly enhanced Sp1-dependent
trans-activation.
In summary, this study shows that transcriptional activation of TS by
E2 in MCF-7 cells involves ER/Sp1 interactions
at a G-rich promoter sequence. This mode of ER action is important
for regulating an increasing number of
E2-responsive genes, including E2F1,
bcl-2, cathepsin D, IGFBP-4, retinoic acid retinoic acid
receptor 1, c-fos, and adenosine deaminase (29, 30, 31, 32, 33, 34, 35, 36).
Functional and/or physical interactions of Sp1 or Sp1-like proteins
have been characterized for other members of the nuclear receptor
superfamily, including chicken ovalbumin upstream promoter transcriptor
factor (COUP-TF), progesterone receptor, and steroidogenic
factor-1 (67, 68, 69, 70, 71, 72), suggesting that Sp1 may play an increasingly
important role in the cell-specific functions of nuclear receptor
transcription factors.
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Footnotes |
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2 Sid Kyle Professor of Toxicology.
Received October 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 gene expression: role of estrogen
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), but the high
concentration of Sp1 protein (lane 6) decreased methylation at upstream
G-rich sites (*). Incubation with ER