Functional Synergy between the Transcription Factor Sp1 and the Estrogen Receptor
W. Porter,
B. Saville,
D. Hoivik and
S. Safe
Department of Veterinary Physiology and Pharmacology, Texas A &
M University, College Station, Texas 77843-4466
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ABSTRACT
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A GC-rich oligonucleotide containing an estrogen
responsive element (ERE) half-site from the heat shock protein 27 (Hsp
27) gene promoter (-105 to -84) [i.e.
GGGCGGG(N)10GGTCA;
Sp1(N)10ERE] forms a complex with the Sp1 and
estrogen receptor (ER) proteins. Moreover, promoter-reporter constructs
containing this sequence (-108 to -84 or -108 to +23) are also
estrogen-responsive. Mutation of the ERE half-site in the Hsp
27-derived oligonucleotides did not result in loss of estrogen
responsiveness in transient transfection studies, suggesting that
estrogen inducibility was mediated through the Sp1-DNA motif. Gel
mobility shift assays using 32P-labeled wild
type and ERE mutant Sp1(N)10ERE and consensus
Sp1 oligonucleotides showed that Sp1 protein formed a DNA-protein
complex with all three nucleotides, and the intensities of retarded
bands were enhanced by coincubation with wild type ER and 11C-ER, which
does not contain the DNA-binding domain. ER mutants in which N-terminal
(19C-ER) and C-terminal (15C-ER) regions were deleted did not enhance
Sp1-DNA binding or hormone-induced transactivation of GC-rich
promoter-reporter constructs in ER-negative MDA-MB-231 cells, whereas
both wild type and 11C-ER restored inducibility. Immunoprecipitation
studies also confirmed that the Sp1 and ER proteins physically
interact. The interaction of the Sp1 and ER proteins and the resulting
enhanced Sp1-DNA binding is observed in the presence or absence of
estrogen (hormone-independent), whereas transactivation of
promoter-reporter constructs is estrogen-dependent. Thus, the results
illustrate a new estrogen-dependent transactivation pathway that
involves ER-protein interactions and is ERE-independent.
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INTRODUCTION
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Modulation of genetic and cellular responses at the level of
transcription by physiological or environmental stimuli may involve
synergistic or antagonistic interactions of transcription factors with
target gene promoter sequences (1, 2, 3, 4, 5, 6, 7, 8, 9). Transactivation through nuclear
hormone receptors are regulated by lipophilic hormones, which include
steroids, retinoids, thyroid hormones, and vitamin D3 (2).
Structural characterization of nuclear receptors has shown that these
proteins are comprised of six domains (10). The DNA-binding domain, C,
is involved in binding and recognizes specific DNA sequences referred
to as hormone response elements. The E domain comprises a large
structural component of the protein, which contains ligand-binding and
dimerization motifs, and the F domain plays a role in transactivation.
The A/B and E domains, located in the N-terminal and ligand-binding
regions of the receptor, contain activator function 1 (AF1) and AF2,
respectively. In combination, the two domains associate and
subsequently synergize to activate transcription (11).
The molecular mechanism by which nuclear receptors modulate gene
transcription has, in its simplest form, been recognized for some time
(12). For example, 17ß-estradiol (E2) modulates
transcription by binding with the estrogen receptor (ER); the liganded
ER homodimerizes and binds to its cognate sequence, the estrogen
responsive element (ERE), in the promoter of a target gene, resulting
in either transcriptional activation or repression in a cellular and
promoter-specific manner (13, 14, 15). This selectivity by steroid hormone
receptors can be the result of ligand modification, such as differences
in pharmacokinetics and metabolism (16), or changes in the receptor
through splice variants (17, 18), or covalent modifications (2, 19, 20, 21, 22, 23). In the context of complex promoters, EREs are generally found
in multiple copies or encased among binding motifs for other
transcription factors. For example, analysis of 5'-promoter regions in
the c-myc and rat creatine kinase B (CKB) genes
identified estrogen-responsive regions that did not contain classic
palindromic EREs (1, 24). Dubik and Shiu (1) pointed out that both of
these promoter sequences contained Sp1 and ERE-half sites, and it was
suggested that transactivation of the c-myc and
CKB genes may be due to interactions between ER and Sp1
complexes, which are stabilized by interactions with an
Sp1(N)xERE half-site DNA-binding motif. Research in this
laboratory has identified a functional Sp1(N)23ERE
half-site in the noncoding strand of the cathepsin D gene (25, 26).
Using the Sp1(N)23ERE sequence in both gel mobility shift
and functional transient transfection studies in MCF-7 human breast
cancer cells and HeLa cells, it was shown that an ER/Sp1 complex binds
to the Sp1(N)23ERE oligonucleotide, and E2
induces reporter gene activity using a cathepsin D Sp1/ERE-CAT
construct. Although the results clearly demonstrated that a DNA-bound
ER/Sp1 complex was formed, the nature of this interaction and the
involvement of other proteins was not determined. Subsequent
studies have also demonstrated that Sp1(N)xERE motifs also
play a role in estrogen-regulated retinoic acid receptor
(RAR
)
and heat shock protein 27 (Hsp 27) gene expression in human breast
cancer cells (27, 28). Thus, the cooperative interactions of Sp1 and ER
proteins play a role in regulation of at least five estrogen-inducible
genes, including c-myc, CKB, cathepsin D, RAR
,
and Hsp 27, and requires the presence of Sp1 and ERE half-site motifs
in which there is considerable variability in the ERE half-site
sequences, their orientation, and the number of intervening nucleotides
between Sp1 and ERE DNA-binding sites. This study reports that estrogen
induces reporter gene activity in MCF-7 cells transiently transfected
with a human ER expression plasmid and constructs containing GC-rich
Sp1-binding sequences linked to a bacterial chloramphenicol acetyl
transferase (CAT) reporter gene. In gel mobility shift assays, ER
enhanced Sp1 binding to 32P-labeled oligonucleotides
containing GC-rich binding sites, and, in the absence of DNA,
[35S]ER and Sp1 proteins could be coimmunoprecipated with
Sp1 antibodies. Thus, the functional synergy between ER and Sp1 is
associated with protein-protein interactions and represents an
estrogen-induced transactivation pathway that does not require ER-DNA
binding.
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RESULTS
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Previous studies have identified an Sp1(N)10ERE motif
in the 5'-promoter region of the Hsp 27 gene that confers estrogen
inducibility on promoter-reporter constructs (28). Mutation of both
Sp1- and ERE-binding sites resulted in loss of estrogen inducibility
and failure to form an ER/Sp1 complex in gel mobility shift assays
(28). The results in Fig. 1
compare the
induction of CAT activity by E2 in MCF-7 cells transiently
transfected with wild type Hsp-CATs Sp1/ERE (lanes 1 and 2) and
Hsp-CAT
Sp1/ERE (lanes 7 and 8) plasmids and with the corresponding
mutant plasmids Hsp-CATs Sp1/ERE and Hsp-CAT
Sp1/ERE plasmids, which contain mutations in the ERE
half-sites. The results show that E2 induced CAT activity
using both wild type (lanes 1, 2, 7, and 8) and ERE mutant (lanes 3, 4,
9, and 10) plasmids, suggesting that hormone responsiveness of these
constructs did not require an intact ERE half-site. Moreover,
E2 induced CAT activity in cells transfected with the
Sp1-CAT(Hsp) plasmid, which contains only the Sp1 oligonucleotide
insert from the Hsp 27 gene promoter. The role of Sp1 DNA-binding sites
on E2-responsiveness was confirmed in MCF-7 cells
transiently cotransfected with a construct containing an consensus Sp1
element (Sp1-TATA-CAT) and human ER (hER) expression plasmids.
E2 caused a concentration-dependent induction of CAT
activity (1.4- to 10.6-fold) (Fig. 2
, lanes 1 through 4), and the effects were similar to those observed for
wild-type Hsp-CATs Sp1/ERE and Hsp-CAT
Sp1/ERE plasmids and
constructs containing mutations in their ERE half-sites (Fig. 1
).

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Figure 1. Role of Sp1 Motifs on E2-Induced CAT
Activity in MCF-7 Cells Transiently Transfected with Plasmids
Containing Wild Type or Mutant Oligonucleotide Inserts from the Hsp 27
Gene Promoter
The cells were transiently cotransfected with Hsp-CATs Sp1/ERE (lanes 1
and 2), Hsp-CATs Sp1/ERE (lanes 3 and 4), Sp1-CAT(Hsp)
(lanes 5 and 6), Hsp-CAT Sp1/ERE (lanes 7 and 8), Hsp-CAT
Sp1/ERE plasmids (lanes 9 and 10), and hER. The
transient transfection and CAT assays were performed as described in
Materials and Methods. Cells were treated with
Me2SO (lanes 1, 3, 5, 7, and 9) or 10 nM
E2 (lanes 2, 4, 6, 8, and 10). The relative intensities of
lanes 2, 4, 6, 8, and 10, when compared with control (arbitrarily set
at 100) (lane 1, 100 ± 13; lane 3, 100 ± 21; lane 5,
100 ± 2; lane 7, 100 ± 10; and lane 9, 100 ± 21) were
1013 ± 13, 1263 ± 189, 1166 ± 46, 251 ± 21 and
391 ± 17, respectively (means ± SD for three
determinations).
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Figure 2. Dose-Dependent Effects of E2 on
Consensus Sp1-TATA-CAT in MCF-7 Cells
MCF-7 cells were transiently cotransfected with hER and
Sp1-TATA-CAT. The transient transfection and CAT assays were performed
as described in Materials and Methods. Cells were
treated with Me2SO (lane 1) or 10-10,
10-9, and 10-8 M E2
(lanes 2 through 4, respectively). The relative intensities of lanes 2
through 4, when compared with control (arbitrarily set at 100) (lane 1,
100 ± 12), were 136 ± 13, 269 ± 19, and 1057 ±
79, respectively (means ± SD for three
determinations).
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The effects of ER on Sp1 binding to a [32P]Hsp 27-Sp1/ERE
short oligonucleotide were determined in gel mobility shift assays.
Increasing amounts of Sp1 protein (0, 10, 20, and 40 ng) caused a
dose-dependent increase in an Sp1-oligonucleotide retarded band (Fig. 3
, lanes 1 through 4). In the presence of
in vitro translated ER, the dose-dependent increase in
retarded band formation by the same concentration gradient of Sp1
protein was further enhanced (Fig. 3
, lanes 5 through 8). The intensity
of the ER-enhanced retarded band (lane 8) was significantly decreased
by competition with a 100-fold excess of unlabeled Sp1 oligonucleotide
(lane 10), but not by a 100-fold excess of wild type or mutant ERE
oligonucleotides (lanes 9 and 10, respectively). Intensity of the
retarded band was not decreased in competition with 100-fold excess
wild type ERE (lane 9). The results summarized in Fig. 4
also demonstrate that Sp1 binding to
the mutated [32P]Hsp 27-Sp1/ERE short
oligonucleotide (lanes 1 through 4) is enhanced by the ER (lanes 5
through 8), and the retarded band intensity (lane 8) is decreased by
unlabeled Sp1 (lane 10) but not by mutant ERE oligonucleotides (lane
9). The pattern of enhanced Sp1-DNA binding by ER using GC-rich sites
in the Hsp 27 promoter was also observed using a consensus
[32P]Sp1 oligonucleotide (Fig. 5A
). The intensity of the bound
Sp1-[32P]Sp1 band (lanes 2 through 4) was enhanced after
incubation with ER (lanes 6 through 8), decreased by competition with
100-fold excess unlabeled Sp1 oligonucleotide (lane 9), but not
affected by unlabeled mutant ERE (lane 10). The time-dependent
formation of the Sp1-DNA retarded band was determined in the presence
or absence of ER (Fig. 5B
). The results show that binding was maximal
after 10 min, and the overall on rate was increased 2.0- to 3.2-fold
over the 15-min incubation period. In contrast, the time-dependent loss
of the Sp1-DNA complex was independent of ER, and the off rate for
decomposition of the complex was comparable in the presence or absence
of ER (data not shown). In all of the protein-DNA-binding studies
(Figs. 3 through 5

) enhanced Sp1-DNA binding by ER was
hormone-independent, since comparable results were obtained in the
presence or absence of 10-8 M E2
(data not shown).

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Figure 3. Enhanced Binding of Sp1 to Wild Type
[32P]Hsp 27-Sp1/ERE Short Oligonucleotide by in
Vitro Translated ER
In vitro translated proteins were obtained as described
in Materials and Methods. In vitro
translated ER (3 µl) or unprogrammed rabbit reticulocyte lysate (3
µl, UPL) was incubated with 2 x 10-8 M
E2 on ice for 15 min. Increasing amounts of Sp1 (0, 10, 20,
and 40 ng) were added to the preincubated proteins and then subjected
to gel electrophoretic mobility gel shift assay using
[32P]Hsp 27-Sp1/ERE short oligonucleotide. The retarded
Sp1 bands (see arrow) were visualized by autoradiography
and quantitated by densitometry using a Molecular Dynamics Zero-D
software package and a Sharp JX-330 scanner. The intensity values of
lanes 2 through 4 and 6 through 11 relative to the control bands
(arbitrarily set at 100) (lane 1, 100 ± 29 and lane 5, 100
± 7) were 90 ± 3, 127 ± 3, and 367 ± 11 (lanes 2
through 4, respectively) and 213 ± 5, 436 ± 12, 627 ±
10, 649 ± 9, 289 ± 8, and 534 ± 1 (lanes 6 through
11, respectively) (means ± SD for three
determinations). Similar results were seen with Baculovirus expressed
ER (data not shown).
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Figure 4. Enhanced Binding of Sp1 to the Mutant
[32P]Hsp 27-Sp1/ERE Short Oligonucleotide
by in Vitro Translated ER
In vitro translated proteins were obtained as described
in Materials and Methods. In vitro
translated ER (3 µl) or unprogrammed rabbit reticulocyte lysate (3
µl, UPL) was incubated with 2 x 10-8 M
E2 on ice for 15 min. Increasing amounts of Sp1 (0, 10, 20,
and 40 ng) were added to the preincubated proteins and then subjected
to gel electrophoretic mobility gel shift assay using mutant
[32P]Hsp 27-Sp1/ERE short oligonucleotide.
The retarded Sp1 bands (see arrow) were visualized by
autoradiography and quantitated by densitometry. The intensity values
of lanes 2 through 4 and 6 through 10 relative to the control bands
(arbitrarily set at 100) (lane 1, 100 ± 3; and lane 5, 100
± 2) were 124 ± 1, 160 ± 2, and 310 ± 2 (lanes 2
through 4, respectively) and 211 ± 1, 389 ± 7, 918 ±
6, 917 ± 3, and 266 ± 5 (lanes 6 through 10, respectively)
(means ± SD for three determinations).
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Figure 5. Enhanced Binding of Sp1 to [32P]Sp1
Oligonucleotide by ER
A, Enhanced binding of Sp1 to a consensus [32P]Sp1
oligonucleotide by in vitro translated ER. In
vitro translated proteins were obtained as described in
Materials and Methods. In vitro
translated ER (3 µl) or unprogrammed rabbit reticulocyte lysate (3
µl, UPL) was incubated with 2 x 10-8 M
E2 on ice for 15 min. Increasing amounts of Sp1 (0, 10, 20,
and 40 ng) were added to the preincubated proteins and then subjected
to gel electrophoretic mobility gel shift assay using a consensus
[32P]Sp1 oligonucleotide. The retarded Sp1 bands (see
arrow) were visualized by autoradiography and
quantitated by densitometry. The intensity values of lanes 2 through 4
and 6 through 10 relative to the control bands (arbitrarily set at 100)
(lane 1, 100 ± 3; and lane 5, 100 ± 3) were 154 ± 5,
219 ± 10, and 285 ± 15 (lanes 2 through 4, respectively)
and 286 ± 16, 336 ± 18, 345 ± 15, 215 ± 9, and
343 ± 16 (lanes 6 through 10, respectively) (means ±
SD for three determinations). B, Effect of ER on the rate
of Sp1-[32P]Sp1 retarded band formation. The
time-dependent Sp1-DNA complex formation in absence
(left) or presence (right) of ER was also
investigated using procedures as described above. There was a
significant increase in Sp1-DNA complex formation after incubation for
2, 5, 10, or 15 min, and the overall on rate was increased 2.0- to
3.2-fold in the presence of ER at these time points.
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Although wild type hER does not bind to the [32P]Sp1
oligonucleotide in gel mobility shift assays, this does not exclude the
possibility of a weak ER-DNA association that stabilizes ER-Sp1
interactions. The results summarized in Fig. 6
compare the effects of wild type hER
and mutant 11C-ER, in which the ER-DNA-binding domain has been deleted,
on Sp1-[32P]Hsp 27-Sp1/ERE short retarded band formation.
Both wild type hER and 11C-ER (lanes 4 and 6, respectively) caused a
2-fold increase in formation of the retarded band, whereas the mutant
15C-ER and 19C-ER (containing C- and N- terminal deletions,
respectively) do not affect retarded band formation (lanes 8 and 10,
respectively). The 11C-ER interactions with Sp1 were observed in the
absence of E2, and addition of the hormone did not affect
the interaction (data not shown). These results indicate that the DNA-
binding domain of the ER is not required for enhancement of Sp1-DNA
complex formation. The binding data also complement transactivation
assays in ER-negative MDA-MBA-231 cells transiently transfected with
Hsp-CAT
or Sp1-TATA-CAT (Fig. 7
)
plasmids and hER or 11C-ER expression plasmids. CAT activity is not
induced by 10-8 M E2 using either
Hsp-CAT
Sp1/ERE or Sp1-TATA-CAT plasmids, whereas hormone
inducibility is restored with both plasmids after cotransfection of
wild type or 11C-ER. In contrast, hormone inducibility was not observed
using 15C-ER or 19C-ER expression plasmids.

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Figure 6. Enhanced Binding of Sp1 to Wild Type
[32P]Hsp 27-Sp1/ERE Short Oligonucleotide by in
Vitro Translated ER
In vitro translated proteins were obtained as described
in Materials and Methods. Equivalent amounts of
in vitro translated UPL (lanes 1 and 2), WT-ER (lanes 3
and 4), 11C-ER (lanes 5 and 6), 15C-ER (lanes 7 and 8), and 19C-ER
(lanes 9 and 10), as determined by [35S]methionine
labeling, were incubated with 2 x 10-8 M
E2 on ice for 15 min. Sp1 (30 ng) was added to the
preincubated proteins (lanes 2, 4, 6, 8, 10, 11, and 12) and then
subjected to gel electrophoretic mobility gel shift assay using
[32P]Hsp 27-Sp1/ERE short oligonucleotide. The retarded
Sp1-DNA bands (see arrow) were visualized by
autoradiography and quantitated by densitometry. The intensity values
of bands in lanes 4, 6, 10, 11, and 12 relative to the control band
(arbitrarily set at 100) (lane 2, 100 ± 1) were 234 ± 2,
226 ± 6, 115 ± 4, 126 ± 5, 10 ± 1, and 230
± 10 (lanes 4, 6, 10, 11, and 12, respectively) (means ±
SD for three determinations).
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Figure 7. Effect of Wild Type and ER Variants on Induction of
CAT Activity by E2 in MDA-MB-231 Cells Transiently
Transfected with Wild Type Hsp-CAT Sp1/ERE or Sp1-TATA-CAT Plasmids
MDA-MB-231 cells were cotransfected with Hsp-CAT Sp1/ERE or
Sp1-TATA-CAT with pCDNA3-NEO (as control) plasmids, WT-ER, 11C-ER,
15C-ER, or 19C-ER (total amount of DNA was kept constant). The
transient transfection and CAT assays were performed as described in
Materials and Methods. Cells were treated with
Me2SO or 10-8 M E2.
Results as compared with control(s) (arbitrarily set at 100) are
means ± SD for three determinations.
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The direct interactions between Sp1 and ER were investigated in
coimmunoprecipitation experiments. In vitro translated
[35S]ER and Sp1 were mixed and treated with the
bifunctional, reversible cross-linking reagent dithiobis(succinimidyl
propionate). The resulting complexes were then immunoprecipitated with
Sp1 or ER polyclonal antibodies, reduced to cleave the cross-linker,
and resolved by SDS-PAGE. The results in Fig. 8
demonstrate that ER can be reversibly
cross-linked to Sp1. The radiolabeled [35S]ER was
immunoprecipitated by both Sp1 and ER antibodies (Fig. 8
, lanes 2 and
5, respectively); [35S]ER alone was immunoprecipitated
with ER antibodies (lane 4). Incubation of Sp1 plus
35S-labeled unprogrammed lysate (UPL) followed by
immunoprecipitation with ER or Sp1 antibodies (lanes 3 and 6,
respectively) did not give a radiolabeled protein. The Sp1 antibody
(Ab) did not immunoprecipitate [35S]ER alone (data not
shown). Experiments performed without the utilization of cross-linking
reagents did not co-precipitate Sp1, indicating that the
protein-protein interaction between Sp1 and ER is transient under the
conditions used (data not shown). These data show that the ER and Sp1
proteins physically interact and form a protein-protein complex in the
absence of DNA. Moreover, the interaction of ER and Sp1 proteins was
observed in the presence or absence of E2 and therefore was
hormone-independent.

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Figure 8. Immunoprecipitation of Chemically Cross-Linked
35S-Labeled in Vitro Translated ER and Sp1
Proteins
35S-Labeled in vitro translated proteins
were obtained as described in Materials and Methods.
In vitro translated [35S]ER (3 µl) or
35S-labeled unprogrammed rabbit reticulocyte lysate (3
µl, UPL) were incubated with 2 x 10-8
M E2 on ice for 15 min and then treated with 5
mM dithiobis(succinimidyl propionate) in the presence
(lanes 2, 3, 5, and 6) or absence (lane 4) of 40 ng Sp1. The
cross-linked complexes were then immunoprecipitated with either Sp1 Ab
(lanes 2 and 3), ER Ab (lanes 4, 5, and 6), or preimmune serum (lane
7). The immunoprecipitated proteins were then eluted with 2x SDS
sample buffer to reverse the cross-links and resolved by SDS-PAGE. The
Sp1 Ab had no cross-reactivity with ER (data not shown).
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To further characterize the interaction between Sp1 and ER, glutathione
S-transferase (GST) pulldown experiments were performed. GST
alone or GST-Sp1 was incubated with UPL to show the absence of specific
interactions with 35S-labeled proteins in the lysate (Fig. 9
, lanes 3 and 5, respectively). As an
additional control, [35S]ER was incubated with GST alone,
and negligible binding was observed (lane 4). Sp1 and ER were shown to
interact through a GST-Sp1 fusion protein incubated with
[35S]ER (lane 6). To further examine interactions between
Sp1 and ER, truncated GST-Sp1 fusion proteins containing various
regions of the Sp1 were incubated with [35S]ER and
analyzed. Incubation of GST-Sp1 [amino acids (aa) 1293] with
[35S]ER showed undetectable binding to
[35S]ER; however, incubation of GST-Sp1 (aa 1621) and
GST-Sp1 (aa 622788) with [35S]ER showed that both
fusion proteins bound to [35S]ER (lanes 8 and 9,
respectively). These results indicate an interaction of
[35S]ER with two different GST-Sp1 fusion proteins;
however, the major site of interaction of ER with Sp1 protein is
associated with the C-terminal DNA-binding domain of Sp1.

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Figure 9. In Vitro Interaction between GST-Sp1
(Wild Type and Various Truncations) with [35S]ER
Equal molar amounts of GST, GST-Sp1, GST-Sp1 (aa 1293), GST-Sp1 (aa
1621), and GST-Sp1 (aa 622788) were incubated with either
35S-labeled unprogrammed lysate (UPL) or in
vitro translated [35S]ER as indicated. Samples
were then analyzed and visualized as described in Materials and
Methods. Lane 1: 33% of total amount of UPL used in GST
pulldown experiment. Lane 2: 2% of total amount of
[35S]ER used for experiment. Results from incubating GST
with either UPL or [35S]ER are shown in lanes 3 and 4 as
indicated. Results from incubating wild type GST-Sp1 or various
truncated GST-Sp1 fusion proteins with [35S]ER are shown
in lanes 5 through 9 as indicated.
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DISCUSSION
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The steroid hormone receptors are ligand-activated transcription
factors that induce gene expression upon interaction with
hormone-responsive elements, coactivators, and other proteins
associated with the general transcription apparatus. Interaction of the
liganded ER homodimer with both perfect and imperfect palindromic EREs
has been characterized for multiple estrogen-responsive genes (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43).
Recent studies have reported that Sp1(N)xERE half-site
motifs located within the 5'-promoter region of the CKB,
fos, cathepsin D, RAR
, and Hsp 27 genes are also
sequences that bind ER in association with the Sp1 protein (1, 24, 25, 26, 27, 28).
For example, the results illustrated in Fig. 1
confirm the estrogen
inducibility of wild type Hsp-CATs Sp1/ERE and Hsp-CAT
Sp1/ERE
plasmids (lanes 1, 2, 7, and 8), which contain the
Sp1(N)10ERE half-site (-108 to -84) identified in the Hsp
27 gene promoter (28). However, estrogen responsiveness was also
observed with the same plasmids containing mutations in the ERE
half-sites (lanes 3, 4, 9, and 10). These results suggested that
E2-induced transactivation may require only GC-rich
Sp1-binding sites, and the results illustrated in Figs. 2 through 5


demonstrate 1) E2-responsiveness of constructs or
oligonucleotides containing consensus Sp1 promoter sequences (Fig. 2
),
and 2) enhanced Sp1-[32P]Sp1 oligonucleotide retarded
band formation after coincubation with ER protein (Figs. 3 through 5

).
Interestingly, after incubation of Sp1 and ER proteins with
[32P]Sp1, a ternary Sp1/ER-[32P]Sp1 complex
is not observed in gel mobility shift assays; however, ER enhances the
on rate of Sp1-[32P]Sp1 formation (Fig. 5B
). This type of
protein (ER) enhancement of protein-DNA complex formation is comparable
to results of other studies showing that HTLV-1 Tax, SREPB, and cyclin
D1 enhance bZIP, Sp1, and ER binding to their respective enhancer
sequences without forming a ternary complex (44, 45, 46). The interactions
of ER with Sp1 are also similar to the functional synergy previously
reported for Sp1 and the erythroid cell transcription factor, GATA, in
which GATA synergistically enhances Sp1-induced transactivation in
Schneider cells using Sp1-dependent promoters (47, 48, 49). Moreover, in
the same cell line, RB protein and E2F1 enhanced Sp1-induced responses
utilizing several different constructs containing functional Sp1-
binding sites (50, 51).
The functional synergy between the ER and Sp1 nuclear transcription
factors was further investigated using mutant ERs containing deletions
in the DNA-binding (11C-ER), N-terminal (19C-ER), and C-terminal
(15C-ER) domains. The latter two ER mutants did not enhance Sp1 binding
in gel mobility shift assays using wild type [32P]Hsp
27-Sp1/ERE, which contains the Sp1(N)10ERE half-site motif
from the Hsp 27 gene promoter (Fig. 6
), or transactivation using
Hsp-CAT
Sp1/ERE or cSp1-TATA-CAT plasmids in MDA-MB-231 breast
cancer cells (Fig. 7
). In contrast, both wild type and 11C-ER enhance
both Sp1 binding and transactivation (Figs. 6
and 7
), indicating that
deletion of the DNA-binding domain of ER (11C-ER) does not abolish the
synergy of the ER with Sp1. These results are similar to those reported
for ER-AP1 interactions in which the AP1 complex serves as a tether,
when bound to its cognate DNA element, to target steroid receptors such
as the ER in the absence of a consensus ERE (52). This functional
interaction also occurs independently of ER binding to DNA and
parallels results observed for Sp1-ER interactions (Figs. 6
and 7
).
Both ER and Sp1 physically interact with other nuclear proteins
(45, 46, 47, 48, 49, 50, 51, 52), and the results illustrated in Fig. 8
show that an ER/Sp1
protein complex can be coimmunoprecipitated. The interaction of Sp1 and
ER proteins and enhancement of Sp1-DNA binding by ER (Figs. 3 through 5

) was observed in the presence or absence of E2, whereas
transactivation was hormone-dependent (Figs. 1
, 2
, and 7
). The effects
of E2 on interactions of ER with other nuclear proteins are
variable; for example, GATA-1 binds ER only in the presence of
E2 (47), whereas binding of cyclin D1 to the ER is
E2-independent (46). Cyclin D1 enhancement of ERE-dependent
reporter gene activity is also observed in the absence of hormone,
whereas ER-mediated inhibition of GATA-induced responses are
E2-dependent. The rationale for the hormone-independent or
dependent responses associated with ER interactions with other nuclear
proteins is unclear and requires further research. In this study,
[35S]ER also bound to GST-Sp1 fusion protein prebound to
GST-Sepharose beads. [35S]ER interacted with truncated
GST-Sp1 fusion proteins containing the C-terminal (aa 622 to 788) but
not the N-terminal region of the protein (Fig. 9
). Similar results have
been reported previously for interactions of Sp1 with E2F1 and GATA-1
in which binding is also mediated through the C-terminal DNA-binding
domain of Sp1 (47, 51). Our results also indicated that
[35S]ER interacted with regions of the B (partial) and C
domains (aa 294 to 621) of Sp1 (Fig. 9
), suggesting that interactions
of Sp1 with the ER may involve more than one domain of Sp1. Our results
showed that ER primarily interacted with the C-terminal region of Sp1,
and the importance of the weaker interactions with other domains in the
Sp1 protein requires further study.
The Sp1 protein plays a major role in regulating expression of diverse
cellular and viral genes, most of which are not affected by hormones.
Nevertheless, results of this study demonstrate that GC-rich binding
sites are potential targets for ER-mediated transactivation. This
suggests that hormone responsiveness via Sp1/ER interactions will be
highly promoter- and cell-specific, and current studies in this
laboratory are focused on identification of estrogen-responsive Sp1
enhancer sequences and their cell-specific hormone-induced
transactivation.
 |
MATERIALs AND METHODS
|
---|
Chemicals, Cells, Oligonucleotides, and Antibodies
MCF-7 and MDA-MB-231 cell lines were obtained from the American
Type Culture Collection (ATCC, Rockville, MD). Cells were maintained in
MEM with phenol red and supplemented with 10% FBS plus 10 ml
antibiotic-antimycotic solution (Sigma Co., St. Louis, MO) in an
air-carbon dioxide (95:5) atmosphere at 37 C. Cells were grown in
DMEM/F12 medium without phenol red and 2.5% stripped FBS 2 days before
dosing. The hER was kindly provided by Dr. Ming Tsai (Baylor College of
Medicine, Houston, TX). The ER deletion mutants 11C-ER, 15C-ER, and
19C-ER were kindly provided by Dr. Pierre Chambon. The wild type and
mutant ERE for gel mobility shift assays has previously been described
(26). The Hsp 27 and Sp1 oligonucleotides (see below) were synthesized
by the Gene Technologies Laboratory, Texas A&M University (College
Station, TX). Human ER Ab (H222) was purchased from Abbott Laboratories
(North Chicago, IL). Sp1 monoclonal antibody (Sp1 Ab) was purchased
from Santa Cruz Biotech (Santa Cruz, CA). Dimethyl sulfoxide
(Me2SO) was used as solvent for E2 and the
antiestrogens. All other chemicals and biochemicals were the highest
quality available from commercial sources.
The oligonucleotides structures and their descriptors are given below
and used throughout the manuscript to identify the specific
oligonucleotide. The Sp1 and ERE half-sites are underlined,
and mutated bases are indicated with an asterisk. Hsp
27-Sp1/ERE Short Oligo (Sense Strand):
5'-AGCTTGGAGGGGCGGCCCTCAAACGGGTCATTGCG-3'
Hsp 27-Sp1/ERE short oligo (sense strand):
5'-AGCTTGGAGGGGCGGCCCTCAAACGA*A*TCATTGCG-3'
Hsp 27-Sp1 oligo (sense strand):
5'-AGCTTGGAGGGGCGGCCCTCG-3' consensus Sp1
oligo (sense strand):
5'-AGCTTATTCGATCGGGGCGGGGCGAGCG-3' Hsp
27-Sp1/ERE long oligo (sense strand):
5'-AGCTTGGAGGGGCGGCCCTCAAACGGGTCATTGC-CATTA
ATAGAGACCTCAAACACCGCCTGCTAAAAATACCCGA-CTGG
AGGAGCATAAAAGCGCAGCCGAGCCCAGCGCCCCGC-ACTT
TTCTGAGGT-3' Hsp 27-Sp1/ERE long oligo
(sense strand):
5'-AGCTTGGAGGGGCGGCCCTCAAACGA*A*TCATTGC-CATTA
ATAGAGACCTCAAACACCGCCTGCTAAAAATACCCGA-CTGG
AGGAGCATAAAAGCGCAGCCGAGCCCAGCGCCCCG-CACTT
TTCTGAGGT-3' E1B-TATA oligo (sense strand):
5'-GATCCGTCGACGCTGTAGGGGTATATAATGGTTGC-GGATC-3'
Cloning
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-TATA oligonucleotide
containing complementary 5'-overhangs was then inserted into the
corresponding sites. The wild type Hsp 27-Sp1/ERE short and mutant Hsp
27-Sp1/ERE short and consensus Sp1 oligonucleotides were
cloned into the pBLTATA-CAT at the HindIII and
BamHI sites as previously described (28) to give the
Hsp-CATs Sp1/ERE, Hsp-CATs Sp1/ERE, and Sp1-TATA-CAT
plasmids, respectively. Wild type Hsp-CAT
Sp1/ERE and mutant
Hsp-Sp1/ERE-CAT
plasmids were constructed using the Hsp
27-Sp1/ERE long and Hsp 27-Sp1/ERE long oligonucleotides
(see above) which were cloned into the
HindIII/BamHI site of pBLCAT2 as previously
reported (28). The thymidine kinase (TK) promoter was then removed by
digesting wild type Hsp-CAT
Sp1/ERE and mutant Hsp-CAT
Sp1/ERE plasmids with BamHI and XbaI
and religating the complementary sites. Ligation products were
transformed into DH5
cells, and clones were verified by
sequencing.
Transient Transfection Assay
Cultured MCF-7 and MDA-MB-231 cells were transfected by the
calcium phosphate method with 10 µg reporter plasmid and 5 µg of
either the appropriate hER plasmid or empty construct (pCDNA3-Neo, In
Vitrogen, Inc., Carlsbad, CA) as a control. After 18 h, the media
was changed and the cells were treated with Me2SO (0.2%
total volume) or E2 (10-8 M) in
Me2SO for 44 h. Cells were then washed with PBS and
scraped from the plates. Cell lysates were prepared in 0.16 ml of 0.25
M Tris-HCl, pH 7.5, by three freeze-thaw-sonication cycles
(3 min each). Cell lysates were incubated at 56 C for 7 min to remove
endogenous deacetylase activity. CAT activity was determined using 0.2
mCi
d-threo-[dichloroacetyl-1-14C]chloramphenicol
and 4 mM acetyl-CoA as substrates. The protein
concentrations were determined using BSA as a standard. After TLC,
acetylated products were visualized and quantitated using a Betascope
603 Blot analyzer (Intelligenetics, Mountain View, CA). CAT activity
was calculated as the percentage of that observed in cells treated with
Me2SO alone (arbitrarily set at 100%), and results are
expressed as means ± SD. The experiments were carried
out at least three times for each treatment group.
Electrophoretic Mobility Shift Assays Using in Vitro
Translated Proteins
Plasmids containing the WT-ER, 11C-ER, 15C-ER, and 19C-ER were
used to in vitro transcribe and translate the corresponding
proteins in a rabbit reticulocyte lysate kit (Promega, Madison, WI).
Parallel reactions with [35S]methionine were also
performed to monitor translational efficiency and control for loading.
Gel electromobility shift assays were performed by assembling the
appropriate in vitro translated proteins in 1x binding
buffer (20 mM HEPES, 5% glycerol, 100 mM
potassium chloride, 5 mM magnesium chloride, 0.5
mM dithiothreitol, 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 the labeled oligonucleotides (30,000 cpm) were then added
to the reaction mixtures in the presence of 1 µg
poly(deoxyinosinic-deoxycytidylic)acid, and the mixtures were incubated
for 15 min at 25 C. Samples 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. Protein-DNA binding was visualized by autoradiography and
quantitated by densitometry using the Scanalytics Zero-D software
package (Scanalytics, Billerica, MA) and a Sharp JX-330 scanner
(Mahwah, NJ).
Immunoprecipitation and Protein Cross-Linking
35S-labeled ER was synthesized and incubated with
Sp1 as described above. The reaction mixture was diluted with 1x
binding buffer and cross-linked with 5 mM
dithiobis(succinimidyl propionate), a bifunctional, reversible
cross-linker, for 1 h at 25 C, then quenched with 0.22
M lysine as described by Lin et al. (50).
Radioimmunoprecipitation (RIPA) was carried out by adding 500 µl of
RIPA buffer (PBS-1% NP400.5% sodium deoxycholate-0.1%DNA-10 mg/ml
phenylmethylsulfonyl fluoride (PMSF)-aprotinin 30 µl/ml-sodium
orthovanadate 10 µl/ml) and 1 µg of antisera. After incubating for
1 h at 4 C, 20 µl of Agarose A (Santa Cruz Biotechnology, Santa
Cruz) was added and incubated (rocking) for 1 h at 4 C. The bound
complex was then washed four times with RIPA buffer containing 2
M urea. The precipitated proteins were then eluted with 2X
SDS sample buffer to reverse the crosslinks and resolved on a 6%
SDS-polyacrylamide gel, dried and visualized by autoradiography.
GST Pulldown Experiment
GST, GST-Sp1, or GST-Sp1 (truncated) fusion proteins were
purified essentially as described by the manufacturer in GST: Gene
Fusion System (Pharmacia Biotech). DH5
bacterial cells transformed
with either pGEX-4T-1 or with plasmids pGEX-2TK-MCS-Sp1,
pGEX-2TK-MCS-Sp1 (1293), pGEX-2TK-MCS-Sp1 (1 -621), or
pGEX-2TK-MCS-Sp1 (622788) kindly provided by Professor Erhard
Wintersberger (51) were grown overnight in Lauria broth + 50 µg/ml
ampicillin at 37 C. Cultures were then diluted 1:10 with Lauria broth +
50 µg/ml ampicillin and grown at 37 C until A600 reached
between 0.5 to 0.7 (about 23 h). Protein expression was induced with
0.05 mM isopropyl-D-thiogalactopyranoside, and
cultures were allowed to grow for an additional 1.5 h; 1.5 ml were
transferred to an eppitube and centrifuged, and the pellet was
resuspended in 300 µl of sonication buffer [150 mM KCl,
40 mM HEPES (pH 7.5), 0.5 mM EDTA, 5.0
mM MgCl2, 1.0 mM dithiothreitol,
0.05% Nonidet P-40] supplemented with 1.0 mM PMSF, and 10
µg/µl aprotinin. Cells were lysed by sonication, and the crude
bacterial extract was either frozen at -80 C or used immediately. Ten
microliters of a 50% slurry of glutathione-Sepharose 4B beads were
added to bacterial extracts containing either GST, wild type, or
truncated GST-Sp1 fusion proteins and incubated at room
temperature for 30 min with shaking; the beads were then washed twice
with sonication buffer and once with hER wash buffer (250
mM NaCl, 0.1% Nonidet P-40, 50 mM HEPES (pH
7.5), 5.0 mM EDTA]. After the final wash, 80 µl of hER
binding buffer (hER wash buffer supplemented with 0.5 mM
dithiothreitol, 1.0 mM PMSF, 10 µg/µl aprotinin) and 3
µl of transcription and translation rabbit reticulocyte lysate system
(Promega) and in vitro translated hER were added to the
beads. This reaction was incubated at 4 C for 2 h and the beads
were washed four times with hER wash buffer. Ten microliters of SDS
loading buffer were added to the beads and heated at 100 C for 3 min,
and samples were analyzed on a 10% SDS polyacrylamide gel. Proteins
were fixed to the gel with 30% methanol/10% acetic acid solution for
20 min. The gel was then treated with EN3HANCE (DuPont),
dried and exposed to film.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, Texas 77843-4466.
This work was supported by NIH Grant ES-04176, the Robert A. Welch
Foundation, and the Texas Agricultural Experiment Station.
Received for publication January 6, 1997.
Revision received June 24, 1997.
Accepted for publication July 17, 1997.
 |
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