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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 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-fos protooncogene expression is induced by E2 alone or in combination with insulin and other mitogenic polypeptides (15, 16, 17, 18, 19, 20). A 2.25-kilobase human c-fos gene promoter sequence linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene was E2 responsive in transient transfection assays in HeLa cells, and hormone inducibility was localized to a 240-bp region (-1300 to -1060) (27). Sequence analysis identified an imperfect palindromic estrogen-reponsive element (ERE) within this region (5'-CGGCAGCGTGACC-3') that bound the ER in gel mobility shift assays. However, insertion of a single copy of this sequence in the promoter-reporter construct did not result in the induction of reporter gene activity by E2 in HeLa cells, and hormone-induced trans-activation was observed only with a promoter containing multiple (n = 3) EREs (27).
Initial studies in this laboratory showed that E2 induced CAT activity in MCF-7 cells transiently transfected with a construct containing 1.4 kilobases from the c-fos gene promoter linked to a CAT reporter gene. Induction was not observed using a construct containing the imperfect palindromic ERE (-1212 to -1200), and this paralleled results obtained in HeLa cells (27). A recent study in this laboratory showed that ER and Sp1 physically interact, and E2-induced trans-activation was observed using constructs containing only a GC-rich oligonucleotide insert (28). The results were in contrast to studies with constructs containing cathepsin D gene promoter inserts in which E2 responsiveness was associated with the formation of an ER/Sp1 complex that bound to an Sp1(N)23ERE half-site motif (29, 30). A GC-rich motif was identified downstream from the imperfect palindromic ERE in the fos gene promoter at -1168 to -1161, and the E2 responsiveness of the -1220 to -1155 region was investigated using promoter-TATA-CAT constructs in transient transfection assays in MCF-7 cells. E2 induced trans-activation of fos gene promoter-reporter constructs that do not contain the imperfect palindromic ERE (-1212 to -1200), and the enhancer element required for induction by E2 was the GC-rich sequence (5'-GGGGCGTGG-3') that bound the nuclear protein Sp1 and formed a transcriptionally active ER/Sp1 protein complex. Hormone-induced trans-activation did not require the ER DNA-binding domain, suggesting that ER acts directly on the Sp1 protein and increases transcriptional activity through enhanced binding of Sp1 to its cognate GC-rich site.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oligonucleotides derived from the c-fos protooncogene promoter and a consensus Sp1 oligonucleotide were synthesized by the Gene Technologies Laboratory, Texas A&M University (College Station, TX). The structures of these oligonucleotides are summarized below, and the putative GC-rich Sp1 and ERE sites are underlined. Mutations incorporated in the mutant oligonucleotides are denoted by an asterisk: fos1 (-1220/-1155), sense strand, 5'-AGC TTG GCT GAG CCG GCA GCG TGA CCC CGG CTG TCC TAC GCA GCA GGG CAG GAG ATT GGG GGG CGT GGC ACG-3'; fos2 (-1220/-1171), sense strand, 5'-AGC TTG GCT GAG CCG GCA GCG TGA CCC CGG CTG TCC TAC GCA GCA GGG CAG GAG AG-3'; fos3 (-1222/-1197), sense strand, 5'-AGC TTG GCT GAG CCG GCA GCG TGA CCC CGG CG-3'; fos4 (-1175/-1153), sense strand, 5'-AGC TTG GAG ATT GGG GGG CGT GGC ACA CG-3'; fos1-m1 (-1220/-1155), sense strand, 5'-AGC TTG GCT GAG CCG GCA GCG TGA CCC CGG CTG TCC TAC GCA GCA GGG CAG GAG ATT GGG T1T1A1 A1GT GGC ACG-3' (mutation of Sp1 site); fos1-m3 (-1220/-1155), sense strand, 5'-AGC TTG GCT GAG CCA1 T1A1T1 GCG TA1G1 A1C1C CGG CTG TCC TAC GCA GCA T1A1G1 A1T1G GAG ATT GGG GGG CGT GGC ACG - 3' (mutation of three ERE half-sites); and Sp1 (consensus), sense strand, 5'-AGC TTA TTC GAT CGG GGC GGG GCG AGC G-3'.
Cloning
The inserts encoding the wild-type hER, HE11, HE15, and HE19 ER
deletion variants were removed by digesting the appropriate plasmids
with EcoRI. The inserts were then religated into pcDNA3-neo
(Invitrogen, Carlsbad, CA) that had been linearized with
EcoRI and treated with calf intestinal alkaline phosphatase.
The ligation products were transformed into DH5a cells, and clones were
verified by sequencing. The in vitro translation efficiency
for these plasmids was determined by 35S labeling
experiments followed by separation of radiolabeled wild-type and
variant ER by electrophoresis. The levels of wild-type and variant ER
protein expressed were comparable based on radiolabeled band
intensities (corrected for methionine content in the individual
proteins). 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 (28)
containing complementary 5'-overhangs was then inserted into the
corresponding sites. The fos1, fos2, fos3, fos4, and fos1.m3
oligonucleotides were cloned into the pBLTATA-CAT vector at the
HindIII and BamHI sites to give the pF1, pF2,
pF3, pF4, and pF1.m3 constructs, respectively, as previously described
(28).
Transient transfection assay
MCF-7 and MDA-MB-231 cells were transfected using the calcium
phosphate method with 10 µg c-fos gene promoter-derived
constructs and 1 µg (MCF-7 cells) or 5 µg (MDA-MB-231 cells)
wild-type or variant ER expression plasmids; this was due to
overexpression of these constructs. Previous studies have also shown
the requirement for cotransfection of hER expression plasmid using
other E2-responsive constructs (27, 28, 29, 30).
E2 responsiveness was observed only after
cotransfection with ER expression plasmids. 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, the medium was changed, and the cells were treated with DMSO
(0.2% total volume), E2, 4'-hydroxy-tamoxifen,
ICI 164,384, or their combinations in DMSO 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 for 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 acetyl coenzyme A. Acetylation was allowed to
proceed to less than 20–25% completion (linear range), and acetylated
metabolites were analyzed by TLC. After TLC, acetylated products were
visualized and quantitated using a Betagen Betascope 603 blot analyzer
(Intelligenetics, Mountain View, CA). CAT activity was calculated as a
fraction of that observed in cells treated with DMSO alone (arbitrarily
set at 100), and results are expressed as the mean ±
SD. The experiments were carried out at least in
triplicate.
Electrophoretic mobility shift assays
Pure Sp1 protein was purchased from Promega (Madison, WI).
Expression plasmids for wild-type ER, HE11, HE15, and HE19 were used to
in vitro transcribe and translate the corresponding proteins
in a rabbit reticulocyte lysate kit (Promega). Gel electromobility
shift assays were performed using Sp1 protein and in vitro
translated proteins in 1 x binding buffer (20 mM
HEPES, 5% glycerol, 100 mM potassium chloride, 5
mM magnesium chloride, 0.5 mM dithiothreitol,
and 1 mM EDTA in a final volume of 25 µl).
E2 was added to the reaction at a final
concentration of 20 nM and then incubated on ice for 15
min. Sp1 and 32P-labeled oligonucleotides were then added
to the reaction mixtures in the presence of 1 µg poly(dI-dC) and
incubated for 15 min at 25 C. In competition experiments, different
amounts of unlabeled oligonucleotides were also included in the
incubation mixtures. 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 8.3). 32P-Labeled DNA and
DNA-protein bands were visualized by autoradiography and quantitated by
densitometry using the Molecular Dynamics Zero-D software package
(Molecular Dynamics, Sunnyvale, CA) and a Sharp JX-330 scanner (Mahwah,
NJ).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Results
of transient transfection studies using pF1-m2 (containing the
-1220/-1155 insert mutated in the perfect ERE half-site) also showed
that this construct was E2 responsive (data not
shown). These results suggested that the -1171 to -1153 region of the
promoter containing a GC-rich site (GGGGCGTGG) was required for
E2 responsiveness, and in MCF-7 cells transiently
transfected with pF4, E2 caused a 3.2-fold
increase in CAT activity (Fig. 1B); similar results were obtained using
pF1 (5.5-fold induction) and pF1-m3 (4.7-fold induction) in which the
three ERE half-sites were mutated. The results (Fig. 1) indicate that
E2 responsiveness of pF1 is associated with the
GC-rich region of the c-fos gene promoter (-1220 to
-1155).
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, A and B). Sp1
protein bound to [32P]Sp1 (containing a consensus Sp1
sequence) to form a retarded band (Fig. 2A, lane 2) that exhibited less
mobility than Sp1 complexes with fos gene promoter-derived
oligonucleotides; [32P]fos1 and
[32P]fos1-m3 also bound Sp1 to form retarded bands (lanes
3, 4, and 6), whereas [32P]fos1-m1 with mutations in the
GC-rich site did not form a complex with Sp1 (lane 5). Formation of the
retarded band associated with the [32P]fos1-m3 complex
(Fig. 2B, lane 1) was competitively inhibited after coincubation with a
400-fold excess of unlabeled Sp1 (lane 2) or fos1-m3 (lane 3)
oligonucleotides, whereas competition with the fos1-m1 oligonucleotide,
which is mutated in the GC-rich site, did not decrease formation of the
retarded band (lane 4). These results demonstrate that Sp1 protein
interacts with the same GC-rich sequence (-1175 to -1153) required
for E2 responsiveness in transient transfection
assays (Fig. 1
). [32P]fos1-m3 bound Sp1 to form a
specifically bound retarded band (Fig. 3B, lanes 2–4), and coincubation of Sp1
(3 ng) (lane 6) with different concentrations of recombinant ER
(lanes 7–9) resulted in a concentration-dependent increase in the
intensity of the retarded band. Previous studies using a consensus
[32P]Sp1 oligonucleotide also showed that wild-type ER or
HE11 enhanced [32P]Sp1-Sp1 (protein) binding without
forming a ternary complex (28). Similar results were observed for the
Sp1-[32P]fos1-m3 complex (Fig. 3B, lane 2) in which
coincubation with wild-type hER caused a 2-fold increase in the
intensity of the Sp1-[32P]fos1-m3 retarded band, and
increased retarded band intensity was also observed after coincubation
with HE11 (lane 6), an ER variant in which the DNA-binding domain has
been deleted. In contrast, coincubation with HE15 (containing AF-1) or
HE19 (containing AF-2; lanes 8 and 10, respectively) did not enhance
formation of the retarded band. Similar results were previously
reported after coincubation of Sp1 plus full-length or variant ER
protein with consensus [32P]Sp1 oligonucleotide (28).
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). The pure antiestrogen ICI 164,384
did not induce CAT activity in MCF-7 cells transiently transfected with
pF1 (Fig. 4A, lane 3); however, in cells cotreated with
E2 plus ICI 164,384, the hormone-induced response
was decreased by 79% (lane 4). In contrast, 1 µM
4'-hydroxytamoxifen did not exhibit antiestrogenic activity (lane 6),
but was active as an ER agonist and induced CAT activity (4.2-fold;
lane 5). Similar results were obtained in MCF-7 cells transiently
transfected with pF1-m3, which contains the -1220 to -1155 region of
the fos promoter in which the GC-rich site is intact and all
three ERE sites are mutated (Fig. 4B). E2 and
4'-hydroxytamoxifen induced CAT activity (2.6- and 2.1-fold,
respectively; lanes 2 and 5), whereas ICI 164,384 was not an ER agonist
(lane 3). In cells cotreated with both antiestrogens (lanes 4 and
6), only ICI 164,384 inhibited E2-induced CAT
activity. The results summarized in Figs. 5 and 6
show that CAT activity is induced by E2 in
MCF-7 and MDA-MB-231 cell cotransfected with pF1 or pF1-m3 and hER or
HE11 expression plasmids. Induction was not observed in cells
cotransfected with HE15 or HE19 expression plasmids or with a control
vector (pcDNA3-neo) that does not contain an
E2-responsive promoter insert.
4'-Hydroxytamoxifen also induced CAT activity in MDA-MB-231 cells
transiently cotransfected with pF1 and full-length ER expression
plasmid (Fig. 7); in contrast, CAT
activity was not induced in cells cotransfected with variant HE11,
HE15, or HE19 ER expression plasmids. These results demonstrate that
E2 activates HE11/Sp1 or ER/Sp1 complexes (Figs. 5 and 6), whereas 4'-hydroxytamoxifen activates only the ER/Sp1 complex
(Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An Sp1-like motif was identified (-1168 to -1161) 3' to the
palindromic ERE in the fos gene promoter, and the results of
transient transfection studies using a construct (pF1) that contained
the -1220 to -1155 region of the fos gene promoter showed
that E2 induced CAT activity (Fig. 1). In
contrast, Weisz and Rosales (27) previously reported that a construct
containing the imperfect palindromic ERE (-1212 to -1200) was not
E2 responsive. This confirmed that downstream
sequences (-1200 to -1155) were required for E2
responsiveness, and the identities of these elements were further
investigated using a series of wild-type (pF1-pF4) and mutant
(pF1·m3) constructs containing fos gene promoter
sequences.
The fos gene promoter sequence inserted in pF1 (-1220 to
-1155) contains two ERE half-sites that include the imperfect
palindromic ERE, a third ERE half-site, and a GC-rich sequence (-1168
to -1161) that is a putative binding site for the nuclear Sp1 protein.
Previous studies have shown that Sp1(N)xERE half-site
motifs within the cathepsin D, heat shock protein-27, c-myc,
creatine kinase B, and retinoic acid receptor-
gene promoters bind
ER/Sp1 complexes and confer E2 inducibility on
their corresponding promoter-reporter constructs (30, 31, 33, 34, 35). The
-1120 to -1155 region of the fos gene promoter insert in
pF1 contains three potential Sp1(N)xERE half-site
motifs; however, incubation of [32P]fos1 with
nuclear extracts from MCF-7 cells did not give an ER/Sp1-retarded band
(data not shown). Therefore, the functionality of fos gene
promoter sequences was further investigated. The results illustrated in
Fig. 1 show that pF1, which contains both the GC-rich and ERE
sequences, was E2 responsive, and deletion of the
GC-rich region (pF2 and pF3) within the promoter resulted in the loss
of hormone-induced trans-activation. These data showed that
the GC-rich site was necessary for E2
responsiveness, but did not exclude a role for a functional
Sp1(N)xERE half-site. However, transient transfection
studies using a plasmid containing mutations in the three ERE
half-sites (pF1-m3) or a construct containing the GC-rich site alone
(pF4; -1175 to -1153) demonstrated that the GC elements were both
necessary and sufficient for E2-induced
trans-activation.
The E2-responsive -1175 to -1153 region of the c-fos protooncogene promoter contains a potential nonconsensus Sp1 element (5'-GGGGCGTGG-3'), and the result show that, like [32P]Sp1, both wild-type [32P]fos1, and mutant [32P]fos1-m3 oligonucleotides bind Sp1 protein to form retarded bands that are competitively decreased only after coincubation with unlabeled oligonucleotides containing intact Sp1-binding sites. Miltenberger and co-workers (36) have previously identified a GGGGCGTGG sequence in the multidrug-resistant gene promoter that also binds Sp1 protein and plays an important role in basal expression of this gene. A comparison of the results obtained with pF1, pF2, pF3, and pF4 indicates that basal CAT activity was markedly decreased using plasmids that did not contain the GC-rich site (i.e. pF2 and pF3), suggesting that Sp1 binding may also be required for optimal basal expression of c-fos.
Recent studies in this laboratory have shown that GC-rich sites that
bind the Sp1 transcription factor mediated
E2-induced trans-activation (28). For
example, E2 induced CAT activity in transient
transfection studies in MCF-7 cells (cotransfected with hER) using a
plasmid containing a consensus Sp1 oligonucleotide insert. Moreover, in
gel mobility shift assays using the same consensus
[32P]Sp1 oligonucleotide, coincubation with ER increased
the overall rate and levels of Sp1-[32P]Sp1 complex
formation, and in parallel studies using wild-type and ER variant
(HE11, HE15, and HE19) expression plasmids, only hER and HE11 enhanced
Sp1-[32P]Sp1 binding (28). The results illustrated in
Fig. 3 using the [32P]fos1-m3 oligonucleotide demonstrate
that wild-type hER and HE11 (DNA-binding domain deficient) also
enhanced (>2-fold) formation of the Sp1-[32P]fos1-m3
complex, whereas HE15 and HE19, which express AF1 and AF2,
respectively, did not enhance retarded band formation. Although ER and
Sp1 physically interact (28), a ternary ER-Sp1-DNA complex was not
observed in this study or a previous report (28). The observation that
a protein (i.e. ER homodimer) enhanced the binding of
another protein (i.e. Sp1) to its cognate sequence without
forming a ternary complex in a gel mobility shift assay has been
observed in other studies showing that human T cell leukemia virus
type-1 Tax, sterol regulatory element-binding protein, and cyclin D1
enhanced bZIP, Sp1 and ER binding to their respective DNA enhancer
sequences (37, 38, 39).
c-fos proto-oncogene expression is induced by both
E2 and 4'-hydroxytamoxifen in MCF-7 cells,
whereas ICI 164,384 exhibits ER antagonist activity (19, 40). The
activities of these ligands as ER agonists/antagonists were comparable
in MCF-7 cells cotransfected with hER and pF1 or pF1-m3, thus
confirming that ER/Sp1 binding to the GC-rich motif (-1168 to -1161)
plays an important role in E2-mediated
trans-activation of this gene. Transcriptionally active
ER-protein complexes have also been observed with the AP-1 Fos-Jun
complex, and the effects of ER-AP-1 trans-activation through
AP-1 sites are dependent on the cell and promoter context and on
the ligand (i.e. estrogens vs. antiestrogens)
(24, 25, 26). For example, ER binds to Jun (but not Fos),
E2 activates AP-1-dependent activity in transient
transfection assays, and trans-activation is observed with
both wild-type ER and HE11 in both MCF-7 and Ishikawa (endometrial)
cell lines. The results summarized in Figs. 5 and 6 demonstrate that
E2-mediated induction of CAT activity in MCF-7 or
MDA-MBA-231 breast cancer cells transfected with pF1 or pF1-m3 is also
dependent on cotransfection with wild-type hER or HE11. In contrast,
tamoxifen-induced CAT activity in MDA-MB-231 cells transfected with pF1
was observed only in cells cotransfected with wild-type hER. Thus,
important ligand-dependent (E2 vs.
tamoxifen) differences were observed for ER/Sp1-mediated
trans-activation. These data also illustrate important
ligand-dependent differences in trans-activation by ER/Sp1
and ER-AP-1 complexes, as ER-AP-1 was not activated by tamoxifen in
human breast cancer cells (26). Interestingly, Weisz and Rosales (27)
previously reported [32P]fos3 binds both the ER and AP-1
transcription factors in a gel mobility shift assay. In contrast, the
corresponding plasmid containing the fos3 (-1222 to -1997) insert was
not E2 responsive in transient transfection
assays in HeLa cells (cotransfected with wild-type ER). Thus, the AP-1
site in the c-fos gene promoter was not responsive to
E2 in HeLa cells or breast cancer cells, and
these results further emphasize the importance of both promoter
and cellular context for hormone-induced activation (26).
In summary, the results of this study show that the induction of fos protooncogene expression by E2 in MCF-7 cells is regulated through interaction of an ER/Sp1 protein complex binding to a GC-rich (Sp1) region in the promoter (-1168 to -1161), and this represents the first example of an E2-responsive gene regulated by this complex interacting with a GC-rich motif. These data are in contrast to a report by Weisz and Rosales (27), who identified an imperfect palindromic ERE that was required for E2-induced trans-activation in HeLa cells. The results of preliminary studies in this laboratory using HeLa cells were not in conflict with the previous report (27), and the 5'-GGGGCGTGG-3' sequence was not required for E2 responsiveness in HeLa cells (data not shown). Thus, genomic Sp1-binding sites may play an important role in the E2 responsiveness of some genes and also influence cell- and promoter/gene-specific differences in hormone-induced trans-activation (41). Current research in this laboratory is focused on studying functional Sp1 sites in other E2-responsive genes that play a role in ER-mediated trans-activation in diverse mammalian cell lines.
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Footnotes |
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2 Sid Kyle Professor of Toxicology.
Received September 19, 1997.
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