Allosteric Modulation of Estrogen Receptor Conformation by Different Estrogen Response Elements
Jennifer R. Wood,
Varsha S. Likhite,
Margaret A. Loven and
Ann M. Nardulli
Department of Molecular and Integrative Physiology (J.R.W., M.A.L.,
A.M.N.) Department of Biochemistry (V.S.L.) University of
Illinois Urbana, Illinois 61801
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ABSTRACT
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Estrogen-regulated gene expression is dependent on
interaction of the estrogen receptor (ER) with the estrogen response
element (ERE). We assessed the ability of the ER to activate
transcription of reporter plasmids containing either the consensus
vitellogenin A2 ERE or the imperfect pS2, vitellogenin B1, or oxytocin
(OT) ERE. The A2 ERE was the most potent activator of transcription.
The OT ERE was significantly more effective in activating transcription
than either the pS2 or B1 ERE. In deoxyribonuclease I (DNase I)
footprinting experiments, MCF-7 proteins protected A2 and OT EREs more
effectively than the pS2 and B1 EREs. Limited protease digestion of the
A2, pS2, B1, or OT ERE-bound receptor with V8 protease or proteinase K
produced distinct cleavage products demonstrating that individual ERE
sequences induce specific changes in ER conformation. Receptor
interaction domains of glucocorticoid receptor interacting protein 1
and steroid receptor coactivator 1 bound effectively to the A2, pS2,
B1, and OT ERE-bound receptor and significantly stabilized the
receptor-DNA interaction. Similar levels of the full-length p160
protein amplified in breast cancer 1 were recruited from HeLa nuclear
extracts by the A2, pS2, B1, and OT ERE-bound receptors. In contrast,
significantly less transcriptional intermediary factor 2 was recruited
by the B1 ERE-bound receptor than by the A2 ERE-bound receptor. These
studies suggest that allosteric modulation of ER conformation by
individual ERE sequences influences the recruitment of specific
coactivator proteins and leads to differential expression of genes
containing divergent ERE sequences.
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INTRODUCTION
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The hormone estrogen is critical for normal development and
maintenance of the female reproductive system. In addition, estrogen
plays a role in prevention of cardiovascular disease and osteoporosis,
proliferation of breast cancer cells, and concentration of sperm in the
male reproductive tract (1, 2, 3, 4). Estrogens actions are mediated by the
estrogen receptor (ER). The ER belongs to a large superfamily of
nuclear receptors that function as ligand-inducible transcription
factors (5). The ER activates transcription by binding to estrogen
response elements (EREs), which are generally located in the
5'-flanking region of estrogen-responsive genes. The consensus ERE
(GGTCAnnnTGACC), which is present in the Xenopus laevis
vitellogenin A2 gene, is an inverted palindromic sequence separated by
three intervening nucleotides (6). Numerous studies have examined the
interaction of the ER with the A2 ERE in in vitro binding
experiments, x-ray crystallographic studies, and transient transfection
assays (6, 7, 8, 9, 10). If the ERE sequence deviates from the consensus
sequence by even a single base pair, the receptor exhibits reduced
affinity for the ERE and decreased ability to activate transcription
(6, 7, 9, 11). However, relatively few studies have examined the
ability of imperfect ERE sequences to mediate transcription activation.
The fact that the majority of known estrogen-responsive genes contain
imperfect EREs that differ from the consensus ERE sequence by one or
more base pairs provides compelling impetus to better understand how ER
interaction with these imperfect ERE sequences influences gene
expression.
Multiple factors influence the ability of an ERE to activate
transcription, including the recruitment of transcription factors to
the DNA-bound receptor and to other cis elements present in
target genes. Earlier studies suggested that ER interaction with the
basal transcription factors TBP (TATA binding protein), TFIIB
(transcription factor IIB), and TAFII30 may play
a role in activating estrogen-responsive genes (12, 13, 14). However, since
the interaction of these proteins with ER is not altered by ligand,
they cannot confer estrogen responsiveness to target genes. Thus,
direct interaction between the receptor and these general transcription
factors may be necessary, but is not sufficient, for ligand-dependent
regulation of target gene expression. Interaction of the receptor with
coactivator proteins appears to be a crucial step in mediating
estrogen-regulated gene expression. Estrogen-bound ER interacts
directly with numerous coactivator proteins, including the p160
proteins SRC-1/NCoA-1, TIF2/GRIP1/NCoA-2, and
pCIP/ACTR/AIB1/RAC3/TRAM-1 in in vitro transcription
experiments and enhances expression of ERE-containing promoters (Refs.
15, 16 and references therein). Recruitment of the transcription
adapter proteins CBP/p300 may also play a role in ligand-dependent
activation by ER (17, 18).
Complementary techniques have demonstrated that ER conformation is
different when the receptor is bound to estrogen or antiestrogen
(19, 20, 21, 22). Our laboratory has also demonstrated that ER conformation is
different when the receptor is bound to the A2 or pS2 ERE (23). Thus,
ER conformation is dependent on both ligand- and DNA-induced
conformational changes. It seems plausible that these ligand- and
DNA-induced changes in ER conformation could lead to differential
association of proteins with the receptor. DNA-induced
conformational changes have also been observed with several
trans acting factors including other nuclear receptors,
NF
B (nuclear factor-
B), Pit-1, TATA-binding protein, and the
yeast protein phermone/receptor transcription factor (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35).
Two receptors, ER
and ERß, have been described and may be involved
in differential expression of target genes (36, 37). We have examined
the interaction of one of these receptors, ER
, with the vitellogenin
A2 ERE and three imperfect EREs. The Xenopus laevis B1 ERE2
[AGTCAnnnTGACC (38)] and the human oxytocin ERE
[GGTGAnnnTGACC (39)] differ from the A2 ERE sequence in
the 5'-half site while the human pS2 ERE [GGTCAnnnTGGCC
(40)] differs from the A2 ERE sequence in the 3'-half site. We find
that ER conformation is different when the receptor is bound to these
four different ERE sequences and demonstrate that these alterations in
receptor conformation influence the association of transcriptional
intermediary factor 2 (TIF2) with the ERE-bound receptor.
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RESULTS
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Ability of the A2, pS1, B1, and Oxytocin (OT) EREs to Activate
Transcription
Transient cotransfection assays were carried out to assess the
abilities of the A2, pS2, B1, and OT EREs to serve as enhancer elements
for ER-mediated transcription activation. HeLa cells were cotransfected
with a chloramphenicol acetyltransferase (CAT) reporter plasmid, a
human ER
expression vector, and a ß-galactosidase reporter plasmid
and exposed to 10 nM E2 or ethanol.
The CAT reporter plasmids contained a single A2, pS2, B1, or OT ERE
upstream of a TATA sequence. The most potent activator of transcription
was the A2 ERE. CAT activity was 10.5-fold higher when cells
transfected with the A2 ERE reporter plasmid were exposed to
E2 than when cells were treated with ethanol
(Fig. 1
). When cells were transfected
with a pS2, B1, or OT ERE-containing reporter plasmid, CAT activity was
2.7-, 1.6-, and 9.5-fold higher, respectively, when cells were exposed
to E2 compared with ethanol controls. Although
the A2 ERE consistently activated transcription to higher levels than
the OT ERE, the increased basal CAT expression with the A2 ERE resulted
in similar fold induction with these two EREs. CAT activity was
unaffected by hormone treatment when the parent vector, which contained
a TATA sequence but no ERE, was used (Fig. 1
, -), demonstrating that
the EREs were responsible for the increased CAT activity in the
presence of E2. These data indicate that the
three imperfect EREs, which have sequences that differ from the A2
sequence, but similar affinities for the receptor (11, 41), have very
different abilities to activate transcription.

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Figure 1. E2-Dependent Activation of the A2, pS2,
B1, and OT ERE
A, HeLa cells were cotransfected with a human ER expression vector, a
ß-galactosidase expression plasmid, and a CAT reporter plasmid
containing a TATA sequence alone (-) or in combination with an A2,
pS2, B1, or OT ERE. Cells were treated with ethanol vehicle or 10
nM E2. Values are presented as the mean ±
SEM. Students t tests demonstrated that
all E2-treated samples were statistically different from
the corresponding ethanol-treated samples, except for the parent
plasmid. B, Sequences of the A2, pS2, B1, and OT ERE are shown. ERE
half -sites are capitalized. Nucleotides that differ
from the A2 ERE half-sites are underlined. The 3-bp
spacer and nucleotides at the 5'- and 3'-ends of each ERE are from the
endogenous gene sequences.
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Protection of the A2, pS2, B1, and OT EREs by MCF-7 Nuclear
Proteins
Although the promoters of the four reporter plasmids used in the
transient transfection assays were identical in nucleotide sequence
except for the ERE sequence, the reporter plasmids containing the A1,
pS2, B1, and OT EREs were activated to very different extents. To
understand how this might occur, we examined the interaction of the
ERE-containing promoters with MCF-7 nuclear proteins in DNase I
footprinting experiments. MCF-7 nuclear extracts contain ER as well as
other nuclear proteins involved in estrogen-mediated transactivation.
32P- labeled DNA fragments containing a TATA
sequence and either the A2, pS2, B1, or OT ERE were incubated with
increasing amounts of MCF-7 nuclear extract and exposed to limited
DNase I digestion. Since competition assays with purified ER have
demonstrated that the affinity of the receptor is 2-fold higher for the
A2 ERE than its affinity for any of the imperfect EREs (41), twice as
much nuclear extract was included in the binding reactions with the
imperfect EREs than with the A2 ERE shown in Fig. 2A
. The ERE sequences were protected by
proteins in the ER-containing nuclear extracts, but the extent of
the protection was dependent on the sequence of the ERE (Fig. 2A
).
Phosphorimager analysis demonstrated that both A2 consensus ERE
half-sites were effectively protected when increasing amounts of
nuclear proteins were included in the binding reactions (Fig. 2B
).
Surprisingly, the OT consensus and imperfect ERE half-sites were also
equally protected. In contrast, only the consensus ERE half-site was
substantially protected in the B1 ERE, and the two pS2 ERE half-sites
were minimally protected. The enhanced protection of the A2 and OT EREs
compared with the pS2 and B1 EREs was most apparent when 10 µg of
MCF-7 proteins were included in the binding reaction. In addition to
the protection of the EREs, regions of DNase I hypersensitivity were
observed flanking the EREs. This hypersensitivity was most evident with
the A2 and OT EREs. Only minimal hypersensitivity was observed in the
area flanking the pS2 ERE. Significant protection was also observed at
the TATA sequence with each of the four DNA fragments. Similar
protection and hypersensitivity patterns were observed with two
different MCF-7 nuclear extracts in four independent footprinting
experiments. In addition, similar footprinting patterns were obtained
when the other DNA strand was exposed to DNase I digestion in the
presence of MCF-7 nuclear extracts (data not shown).

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Figure 2. DNase I Footprinting with A2, pS2, B1, or OT
ERE-Containing DNA Fragments and MCF-7 Nuclear Extracts
A, 32P-labeled DNA fragments containing a TATA sequence and
an A2, pS2, B1, or OT ERE were combined with the indicated amounts of
MCF-7 nuclear extracts (NE), exposed to limited DNase I digestion, and
run on a sequencing gel. The positions of the TATA box, consensus ERE
half-sites (open rectangles), and imperfect ERE
half-sites (shaded rectangles) were determined by
piperidine cleavage of DMS-treated, 32P-labeled DNA
fragments (G). B, Phosphorimager analysis was used to quantitate the
fraction of 5'- (open squares) and 3'- (closed
squares) ERE half-sites protected by 0, 5, 10, or 50 µg MCF-7
nuclear extract. Data from four independent experiments were combined
and each value is expressed as the mean ± SEM.
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Interaction of the ER with the A2, pS2, B1, and OT EREs
To further define how the A2, pS2, B1, and OT EREs differentially
activate transcription, we initiated a series of studies to examine the
ER-ERE interaction in detail using highly purified receptor.
Baculovirus-expressed ER, which was fused at the amino terminus to a
flag epitope, was purified by immunoadsorption. The ability of the
purified ER to bind to each of the four EREs was assessed in gel
mobility shift assays. Inclusion of increasing concentrations of ER in
the binding reaction resulted in a dose-dependent increase in the
formation of the receptor-DNA complex (Fig. 3
). Thus, the baculovirus-expressed,
purified receptor bound effectively to each of the EREs tested.

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Figure 3. Binding of ER to the A2, pS2, B1, and OT EREs
Increasing amounts of purified ER (0, 15, or 30 fmol) were combined
with 32P-labeled DNA fragments containing the A2, pS2, B1,
or OT ERE and fractionated on a nondenaturing acrylamide gel.
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Protease Sensitivity of ERE-Bound Receptor
We previously demonstrated that the conformation of partially
purified, yeast-expressed human ER was different when the receptor was
bound to the A2 and pS2 EREs (23) and hypothesized that this change in
ER conformation might influence the ability of the receptor to activate
transcription. To determine whether a DNA-induced change in ER
conformation was a common characteristic of the ER-DNA interaction and
whether changes in receptor conformation might influence transcription
activation, protease sensitivity assays were carried out to examine the
conformation of the receptor when it was bound to the A2, pS2, B1, or
OT ERE. Protease sensitivity assays rely on the ability of a protease
to cleave a protein at accessible amino acids. Thus, if the DNA-bound
receptor is subjected to limited proteolysis and the resulting
receptor-DNA complexes migrate with different mobilities, one can infer
that different amino acids are exposed and that ER conformation varies
when the receptor is bound to different ERE sequences.
Purified ER was combined with 32P-labeled DNA
fragments containing the A2, pS2, B1, or OT ERE and digested with
increasing concentrations of Staphylococcus aureus V8
protease, which cleaves at aspartic and glutamic acids. Digestion of
the A2 ERE-bound receptor with V8 protease produced three major (V2,
V3, and V4) and two minor (V5 and V6) receptor-DNA complexes (Fig. 4
). This digestion pattern was distinctly
different from the digestion pattern formed with the OT ERE-bound
receptor (V1, V2, V5, and V6). While digestion of the pS2 ERE-bound
receptor with V8 protease produced four equally represented complexes
(V2, V3, V5, and V6), digestion of the B1 ERE-bound receptor produced
three major (V2, V3, and V5) and two minor (V4 and V6) complexes.

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Figure 4. V8 Protease Digestion Patterns of the ERE-Bound
Receptor
32P-labeled DNA fragments containing the A2, pS2, B1, or OT
ERE were combined with 100 fmol purified ER, digested with 0, 0.25,
0.5, 1.0, 2.5, 3.75, or 5.0 µg V8 protease, and fractionated on a
nondenaturing acrylamide gel. The V8 protease-digested receptor-DNA
complexes are indicated (V1V6).
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To determine whether a protease with different specificity could
produce different digestion patterns when the receptor was bound to
each of the four EREs, protease sensitivity assays were carried out
with proteinase K, which preferentially cleaves at aliphatic and
aromatic amino acids. The digestion patterns of the A2 and OT ERE-bound
ER differed significantly from each other and from the digestion
patterns of the pS2 and B1 ERE-bound receptor (Fig. 5
). Digestion of the A2 ERE-bound
receptor with proteinase K produced one major (P5) and two minor (P4
and P7) receptor-DNA complexes. In contrast, when the receptor was
bound to the OT ERE, six receptor-DNA complexes were observed (P1, P2,
P3, P4, P6, and P7). Digestion of the pS2 and B1 ERE-bound receptor
resulted in formation of protein-DNA complexes with similar mobilities
(P3, P4, P5, P6, and P7). However, protein-DNA complexes P4 and P5 were
more prominent with the B1 ERE than with the pS2 ERE. Since our
protease sensitivity assays used highly purified ER and DNA fragments
that were identical in size and nucleotide composition, except for the
ERE sequence, these findings demonstrate that the differences observed
in receptor-DNA complex formation were due to differences in
conformation of the receptor when bound to the A2, pS2, B1, and OT
EREs. In spite of the fact that five different proteases were used to
examine conformation of unoccupied, E2-occupied
receptor and antiestrogen-occupied receptor bound to the A2 ERE, no
differences in digestion patterns were observed (data not shown).

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Figure 5. Proteinase K Digestion Patterns of the ERE-Bound
Receptor
32P-labeled DNA fragments containing the A2, pS2, B1, or OT
ERE were combined with 100 fmol purified ER, digested with 0, 1.0, 2.5,
5.0, 10, 25, or 50 ng proteinase K, and fractionated on a nondenaturing
acrylamide gel. The proteinase K-digested receptor-DNA complexes are
indicated (P1P7).
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Association of ER with Receptor Interaction Domains (RIDs)
It has become increasingly clear that the ER does not function in
isolation but requires the participation of other proteins to
effectively regulate transcription of target genes. Since we had
demonstrated that the ERE sequence could alter the accessibility of
amino acids in the DNA-bound receptor, we next wanted to determine
whether DNA-induced changes in receptor conformation could alter the
ability of the receptor to associate with two coactivators that are
known to interact with ER, glucocorticoid receptor interacting protein
1 (GRIP1, Ref. 42) and steroid receptor coactivator 1 (SRC-1, Ref. 43).
Both of these coactivators have three central RIDs (Fig. 6A
,
), which contain LXXLL motifs and
are essential for interaction with nuclear receptors. SRC-1, unlike
GRIP1, contains an additional RID in the carboxy terminus (31, 44).

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Figure 6. Interaction of GRIP1 and SRC-1 GST Fusion Proteins
with A2, pS2, B1, or OT ERE-Bound ER
A, Schematic representation of GRIP1, SRC-1, and GST fusion proteins.
Corresponding amino acid numbers and RIDs ( ) are indicated. B,
Purified ER (57 fmol) was incubated with 32P-labeled DNA
fragments containing an A2, pS2, B1, or OT ERE in the presence of 100
nM E2. GST or GRIP1
(GRIP15-766,
GRIP1563-1121, and
GRIP1730-1121) and SRC-1
(SRC1595-780 and
GST-SRC-11237-1440) GST fusion proteins were
included in the binding reactions as indicated, and samples were run on
nondenaturing acrylamide gels.
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GRIP1 and SRC-1 glutathione-S-transferase (GST) fusion
proteins were tested for their abilities to interact with the DNA-bound
ER in gel mobility shift assays. Three GRIP1 GST fusion proteins, which
contained either three or one of the central RIDs and varying amounts
of amino- or carboxy-terminal flanking sequence, and two SRC-1 GST
fusion proteins, which contained either the three central RIDs or the
carboxy-terminal RID, were used. When the SRC-1 or GRIP1 GST fusion
proteins were combined with 32P-labeled
ERE-containing DNA fragments and purified ER, each of the
RID-containing GST fusion proteins interacted with the DNA-bound ER and
supershifted the receptor-DNA complexes (Fig. 6B
). The receptor-DNA
complex was unaffected by inclusion of GST alone in the binding
reaction. Interestingly, the amount of coactivator-receptor-DNA complex
formed was significantly greater than the amount of receptor-DNA
complex, suggesting that interaction of the RID-containing peptides
with the receptor stabilized the ER-ERE interaction.
Association of Coactivators with the ERE-Bound Receptor
Although the GRIP1 and SRC-1 RIDs failed to distinguish between
the A2, pS2, B1, and OT ERE-bound receptor, it seemed possible that
coactivator regions outside the RIDs might be required to detect subtle
changes in ER conformation. Therefore, we examined the abilities of the
ERE-bound receptors to recruit full-length p160 proteins from HeLa
nuclear extracts. As seen in Fig. 7A
, although AIB1 (amplified in breast cancer 1) and TIF2, the human
homolog of GRIP1, were readily detected in HeLa nuclear extracts, SRC-1
was barely detectable. As expected, no ER was present in the HeLa
nuclear extracts. The association of these p160 proteins with the
ERE-bound receptor was assessed in DNA pull-down assays. Biotinylated
oligos containing either the A2, pS2, B1, or OT ERE or a nonspecific
nucleotide sequence were adsorbed to streptavidin magnetic beads and
incubated with the baculovirus-expressed, purified ER and HeLa nuclear
extracts. After washing away nonspecifically bound proteins, the
DNA-bound ER and associated proteins were eluted, run on an SDS
acrylamide gel, transferred to nitrocellulose, and probed with
antibodies. As seen in Fig. 7B
, ER bound to each of the four
ERE-containing oligos, but very little ER was bound to the oligo
containing a nonspecific sequence (NS). When the same blot was
probed with an AIB1-specific monoclonal antibody, significant levels of
AIB1 were detected when the oligos contained an ERE, but not when the
oligo lacked an ERE sequence. When a TIF2-specific monoclonal antibody
was used, the levels of TIF2 recruited to the A2, pS2, B1, and OT
ERE-bound ER were again significantly more than observed in the absence
of the ERE. However, the levels of TIF2 associated with the four
different EREs appeared to vary. To determine whether different levels
of AIB1 or TIF2 were recruited to the four different EREs, data from
five independent pull-down experiments were combined and quantitated.
To account for any differences in the affinity of the receptor for
consensus and imperfect EREs (41) and to ensure that the level of
coactivator protein did not simply reflect the level of ERE-bound
receptor, all data were expressed as the relative ratio of coactivator
to ER. Although all four of the ERE-bound receptors recruited similar
amounts of AIB1, the B1 ERE-bound receptor recruited statistically
lower levels of TIF2 than the A2 ERE-bound receptor (Fig. 7C
). ER
binding to the nonspecific DNA was extremely low and resulted in nearly
undetectable levels of AIB1 and TIF2. Thus, recruitment of AIB1 and
TIF2 required an ERE and the ER. No significant binding of SRC-1, CBP,
p300, TFIIB, TBP, or nuclear receptor corepressor (NCoR) was
detected with any of the four EREs (data not shown).

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Figure 7. Interaction of the ERE-Bound Receptor with HeLa
Nuclear Proteins
A, HeLa nuclear extracts were run on an SDS acrylamide gel, transferred
to nitrocellulose, probed with a monoclonal antibody to AIB1, TIF2,
SRC-1, or ER, and visualized with a chemiluminescent detection system.
The position of mol wt standards run on the same gel are indicated. B,
Immobilized oligos containing a nonspecific (NS) sequence or the A2,
pS2, B1, or OT ERE were incubated with baculovirus-expressed, purified
ER and HeLa nuclear extracts. ER and associated proteins were eluted
and subjected to Western blot analysis using antibodies to ER, AIB1, or
TIF2. C, Results from five independent pull-down experiments were
combined, and the ability of the ERE-bound receptors to recruit AIB1
and TIF2 were assessed. The coactivator/ER ratios are presented as the
mean ± SEM. Different letters indicate
significant differences in the ability of the ERE-bound receptor to
recruit coactivators as determined by ANOVA (P <
0.05) followed by Bonferroni correction.
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DISCUSSION
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To determine whether ERE-induced changes in receptor conformation
could play a role in differential expression of estrogen-responsive
genes, we assessed the ability of the ER to activate transcription of
reporter plasmids containing four different EREs, examined the
interaction of the receptor with these consensus and imperfect EREs,
and tested the ability of the ERE-bound receptors to recruit
coactivator proteins.
Allosteric Modulation of ER Conformation
Using highly purified ER
, protease sensitivity assays were
carried out to demonstrate that the conformation of the receptor was
different when it was bound to four different ERE sequences. These
findings suggest that individual EREs function as allosteric modulators
of receptor conformation. Previous x-ray crystallographic studies have
documented that a localized change in the ER DNA binding domain must
occur when it binds to the A2 and B1 EREs (10, 45). Our protease
sensitivity assays suggest that changes must also occur in the DNA
binding domain of the receptor when it is bound to the pS2 and OT EREs
and that the changes in the DNA binding domain must be translated into
more global changes in the full-length receptor. The differential
association of antibodies with the DNA binding domain and the amino and
carboxy termini of the A2 and pS2 ERE-bound receptor lend further
support to this idea (23). Our studies provide strong evidence that the
ER displays substantial structural flexibility and that a change in one
region of the receptor may alter conformation in other regions of the
receptor. These findings emphasize the importance of examining the
full-length, DNA-bound receptor when defining mechanisms by which the
ER modulates transcription activation.
A number of studies have examined ligand-induced conformational changes
in the ER ligand binding domain and in the full-length receptor (19, 20, 46). Interestingly, we were unable to detect ligand-induced
differences in conformation of the A2 ERE-bound receptor in our
protease sensitivity assays using five different proteases (data not
shown). This could mean that the DNA-induced change in receptor
conformation obscures the ligand-induced change in receptor
conformation, that DNA occludes amino acids that are exposed in the
absence of DNA, or that the assay used does not have the sensitivity
required to detect ligand-induced changes in receptor conformation.
Receptor-DNA interactions
Distinct differences in the sensitivity of the A2, pS2, B1, and OT
EREs to DNase I digestion were detected in our in vitro
footprinting experiments. These differences most likely result from
changes in amino acid-nucleotide interactions that occur when the
nucleotide sequence deviates from the A2 ERE. The presence of an
adenine in the vitellogenin B1 ERE 5' half-site
(AGTCAnnnTGACC), rather than the guanine found in the A2
ERE 5'-half-site, requires the rearrangement of a local hydrogen bond
network (10, 45). Similar amino acid rearrangement may be required when
the adenine in the A2 ERE 3'-half-site (GGTCAnnnTGACC), which forms a
hydrogen bond with an glutamic acid in the ER DNA binding domain, is
replaced by a guanine in the pS2 ERE 3'-half-site
(GGTCAnnnTGGCC). Although the cytosine present in the A2 ERE
5'-half-site does not form a hydrogen bond with the ER DNA binding
domain in x-ray crystallographic studies, the complementary guanine
nucleotide does form a hydrogen bond with an arginine. This 1-bp change
in the OT ERE 5'-half-site (GGTGAnnnTGACC)
apparently represents a less detrimental change in nucleotide sequence
than those found in the imperfect pS2 and B1 ERE half-sites and does
not substantially decrease the protection of the OT ERE compared with
the A2 ERE. The effective protection of both A2 and OT ERE half -sites
may partially account for the enhanced ability of these two sequences
to function as more potent transcriptional enhancers.
DNase I hypersensitivity was observed flanking each of the ERE
sequences. We have previously shown that ER binding induces
conformational changes in DNA fragments containing A2 and imperfect
EREs (11, 47). These ER-induced changes in DNA structure could increase
the accessibility of sequences flanking the ERE to DNase I cleavage.
Taken together, our combined results indicate that the ER-ERE
interaction is a dynamic process involving changes in receptor
conformation and in DNA structure.
Interaction of ER with Coactivators
A number of coactivator proteins have been identified that
interact with nuclear hormone receptors in a hormone and AF-2-dependent
manner including the highly related p160 family members SRC-1/NCoA-1,
TIF2/GRIP1/NCoA-2, and pCIP/ACTR/AIB1/RAC3/TRAM-1 (15, 48). We have
demonstrated that SRC-1 and GRIP1 GST fusion proteins interacted with
purified ER when it was bound to A2, pS2, B1, and OT EREs. Thus,
DNA-induced changes in receptor conformation did not interfere with the
ability of the receptor to interact with GRIP1 and SRC-1 RIDs. In fact,
the SRC-1 and GRIP1 fusion proteins substantially increased the amount
of receptor-DNA complex formed. The stabilization of the ER-ERE
interaction may, in part, help explain the abilities of these
coactivators to enhance estrogen-mediated transcription activation.
While we have not addressed the abilities of individual SRC-1 and GRIP1
RIDs to stabilize the ER-DNA interaction, others have demonstrated that
the second of the three central SRC-1 and GRIP1 RIDs have a higher
affinity for ER and an increased capacity to activate transcription
than the other two central RIDs (44, 49). We found the fusion proteins
containing the three central GRIP1 RIDs or the third of the central
GRIP1 RIDs (GRIP1730-1121)
were both quite effective in stabilizing the ER-DNA interaction.
Many groups have reported that the interaction of ER with p160 proteins
is ligand dependent and plays an important role in transcription
activation (Refs. 15, 48 and references therein). However, the
effect of DNA binding on the receptor-coactivator interaction has
generally not been addressed. Although AIB1 was recruited equally to
all four of the ERE-bound receptors, significantly less TIF2 was
recruited to the B1 ERE-bound receptor compared with the A2 ERE-bound
receptor. Increased expression and recruitment of TIF2 has been
correlated with enhanced activation of estrogen-responsive reporter
plasmids (50). Thus, it is quite intriguing that the B1 ERE was the
least potent transcriptional enhancer in HeLa cells in vivo
and that the B1ERE- bound receptor was the least efficient in
recruiting TIF2 from HeLa nuclear extracts in vitro. These
findings suggest that ER conformation and the association of the
receptor with coactivator proteins are influenced by DNA binding.
Although the differential recruitment of TIF2 to the ERE-bound receptor
may help to enhance gene expression, it seems likely that the exposure
of distinct receptor epitopes would also lead to the recruitment of
other ERE-specific coregulatory proteins and thereby assist in
modulating transactivation. This is, to our knowledge, the first
demonstration that differences in ERE sequence alter the association of
ER with a coregulatory protein. However, DNA-induced effects on
recruitment of coregulatory proteins are not restricted to the ER.
Takeshita et al. (31) have suggested that DNA binding
influences the association of the thyroid hormone receptor with
SRC1.
Regulation of Estrogen-Responsive Genes
Our transfection studies used simple promoters containing a single
ERE and a TATA sequence. Certainly naturally occurring
estrogen-responsive promoters contain numerous cis elements
and require the participation of multiple trans acting
factors to effectively regulate transcription. However, our
transfection experiments have clearly demonstrated that the abilities
of A2 and imperfect EREs to regulate transcription varied
substantially. The affinity of the ER is 2-fold higher for the A2 ERE
than for the pS2, B1, or OT EREs (11, 41). Thus, the decreased affinity
of the receptor for the imperfect EREs may partially account for the
decreased ability of the imperfect EREs to activate transcription.
However, since the affinity of the receptor for the imperfect EREs is
similar (11, 41), differences in affinities of the receptor for the
imperfect EREs could not explain the differences in the abilities of
the three imperfect EREs to activate transcription. We propose that
ERE-induced changes in receptor conformation and the differential
recruitment of coregulatory proteins by the ERE-bound receptor may lead
to differential expression of genes possessing distinct ERE sequences.
Additional regulatory versatility could be provided by the ability of
the receptor to detect subtle differences in ERE sequence and bind
preferentially to specific ERE half-sites. These studies have
identified mechanisms that could mediate differential expression of
estrogen-responsive genes in a single cell and provided new insight to
define how imperfect EREs regulate transcription activation.
DNA-induced changes in receptor conformation have now been documented
with a number of nuclear receptor family members. Estrogen,
glucocorticoid, vitamin D, progesterone, retinoic acid, retinoid X, and
thyroid hormone (23, 27, 28, 29, 30, 31, 32, 33, 34) receptors undergo conformational changes
on binding to their cognate recognition sequences. Given the high
degree of structural and functional homology of nuclear receptor
superfamily members, it seems plausible that DNA-induced changes
in receptor conformation and sequence-specific recruitment of
nuclear proteins could play a role in regulating transcription of other
hormone-responsive genes.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
HeLa cells were maintained in DMEM/F12 containing 10% FCS and
transferred to media containing 10% charcoal dextran (CD)-treated (51)
FCS 2 days before harvest. MCF-7 cells were maintained in phenol red
free Eagles MEM containing 5% CD-treated (51) calf serum and
transferred to serum-free media (52) 3 days before harvest.
Plasmids
OT ERE oligos
5'-CTAGATTACCGGTGACCTTGACCCTACTCA-3' and
5'-GATCTGAGTAGGGTCAAGGTCACCGGTAAT-3' were
annealed and inserted into pCY7 (53) as previously described for the
B3ERE circular permutation vectors (11) to produce B3OT ERE. B3consERE,
B3pS2ERE, B3ERE2 (11), and B3OT ERE contained identical nucleotide
sequence, except for the ERE sequence. Annealed OT ERE oligos were also
inserted into TATA-CAT (54) as described previously (11) to construct
the CAT reporter plasmid OT ERE TATA-CAT. OT ERE+10 TATA-CAT was
prepared as described for the ERE+10 TATA-CAT vectors (11). The CAT
reporter plasmids contained promoters that were identical in nucleotide
sequence except for the ERE sequence. Thus, the sequence flanking the
EREs and position of the promoters were identical and would not
influence transactivation. All plasmids were sequenced and purified on
two cesium chloride gradients.
SRC-1 (pGEX NBD1and pGEX NBD2) and GRIP1 (pGEX-T1GRIP1
5-766, pGEX
GRIP1563-1121, pGEX
GRIP1730-1121) GST
expression vectors were generously provided by Akira Takashita
(Toranomon Hospital, Tokyo, Japan) and Michael Stallcup (University of
Southern California, Los Angeles, CA), respectively.
Preparation of Expressed Proteins and Nuclear Extracts
Viral stock for production of ER
in Sf9 cells was generously
provided by James Kadonaga (University of California, San Diego, CA)
and Lee Kraus (Cornell University, Ithaca, NY). Cells were infected
with the ER
-containing recombinant baculovirus for 72 h,
exposed to 10 nM E2 17ß-estradiol
(E2) 20 min before harvest and immunopurified
essentially as described (55). Flag-tagged ER was eluted from the M2
antibody resin with elution buffer [20 mM Tris, pH 7.5,
100 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.1%
NP-40, and 2 mM dithiothreitol (DTT)] containing 0.5 mg/ml
ovalbumin and 0.2 mg/ml flag peptide (University of Illinois
Biotechnology Center, Urbana, IL). To determine ER concentration,
purified ER was combined with 30 nM
[6,7-3H] estradiol (52 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) with or without a
150-fold excess of unlabeled E2 in PTGG buffer (4
mM Na2HPO4, pH
7.4, 0.08% mono-thioglycerol, 10% glycerol) with protease inhibitors
(50 µg/ml leupeptin, 5 µg/ml PMSF, 1 µg/ml pepstatin, and 5
µg/ml aprotinin) and incubated at room temperature for 30 min.
Hydroxylapatite resin (100 µl) was added and incubated for 25 min at
4 C. The resin was washed with PTGG buffer four times and resuspended
in 1 ml ethanol. The ethanol-solubilized 3H
E2 was quantitated and the level of bound ER was
determined by subtracting nonspecific counts per min from total counts
per min.
SRC-1 and GRIP1 GST fusion proteins were expressed in the BL21DE3 pLys
S strain of Escherichia coli. Cells were induced with 1
mM
isopropylthio-ß-D-galactoside for 3 h at
37 C, pelleted, frozen, and lysed in 3 volumes of TEGND (50
mM Tris, pH 7.9, 1 mM EDTA,
10% glycerol, 0.5 M NaCl, 5
mM DTT) with protease inhibitors. Sodium
deoxycholic acid was added to 0.05% and rotated at 4 C for 15 min. The
cell lysate was clarified by centrifugation at 180,000 x
g for 30 min. The supernatant was incubated with glutathione
sepharose beads (Amersham Pharmacia Biotech) for 30 min at
4 C. After washing beads with PBS containing 0.1% Triton X-100, 5
mM DTT, and protease inhibitors, the GST-fusion
proteins were eluted with 10 mM reduced
glutathione in 50 mM Tris, pH 8, and 0.2% Triton
X-100.
To prepare MCF-7 nuclear extracts, cells were harvested, exposed to10
nM E2 for 20 min at 37 C, and
homogenized in TEG buffer (50 mM Tris, pH 7.9, 7.5
mM EDTA, and 10% glycerol) containing protease inhibitors.
Nuclei were pelleted and resuspended in TEG buffer containing 0.5
M KCl with protease inhibitors and incubated for 20 min at
4 C with vortexing at 10-min intervals. Nuclear lysates were spun
180,000 x g for 30 min at 4 C. The supernatant
containing ER and other nuclear proteins was aliquoted and stored at
-80 C. Protein concentrations of the MCF-7 nuclear extracts were
determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Richmond, CA) with BSA as a standard. HeLa nuclear
extracts were prepared in similar fashion except that cells were not
exposed to E2.
HeLa Cell Transfections
HeLa cells were seeded in six-well plates at a density of
850,000 cells per well. After 16 h, the cells were changed to
serum free media and transfected by combining 5 µg ERE+10 CAT
reporter plasmid (11), 250 ng cytomegalovirus (CMV)ß-gal
(CLONTECH Laboratories, Inc., Palo Alto CA), and 5 ng of
the human ER expression vector pCMV5 hER (56) with 10 µg lipofectin
(Life Technologies, Inc., Gaithersburg, MD) and 32 µg
transferrin (Sigma, St. Louis MO) in HBSS. The
DNA/lipofectin/transferrin mixture was incubated for 10 min at ambient
temperature and added to the cells. After 67 h at 37 C, the
DNA/lipofectin/transferrin mixture was removed and the cells were
maintained on DMEM/F12 with 10% CDFCS ± 10 nM
E2 for 24 h. Cells were harvested in TNE (40
mM Tris, pH 7.5, 140 mM NaCl, and 1.5
mM EDTA), pelleted, and resuspended in 250 mM
Tris-HCl. The cells were lysed during three freeze/thaw cycles and the
supernatant was cleared by centrifugation.
ß-Galactosidase activity was determined (57) and used to normalize
each sample for transfection efficiency. To determine CAT activity, 130
µl cell extract were combined with 41.4 µg acetyl CoA and 0.1 µCi
[14C] chloramphenicol, adjusted to a final
volume of 150 µl with 20 mM Tris, pH 7.5, and incubated
1.5 h at 37 C. Acetylated chloramphenicol was separated from
nonacetylated chloramphenicol on Sil G TLC plates (Alltech, Deerfield,
IL) using 5.3% methanol in chloroform. Levels of nonacetylated and
acetylated chloramphenicol were quantitated on a Molecular Dynamics
Phosphorimager with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale CA). Students t tests were used to
determine whether statistical differences between ethanol and
E2-treated groups existed.
DNase I Footprinting
The CAT reporter vectors consERE-CAT, pS2ERE-CAT, ERE2-CAT (11),
and OT ERE-CAT, which contained the A2, pS2, B1, and OT EREs,
respectively, were digested with BamHI. The 1.8-kb
ERE-containing DNA fragments were gel purified, labeled as described
(23), and digested with NcoI. The 128-bp
32P-labeled ERE-containing DNA fragments were gel
purified and combined with the indicated amounts of MCF-7 nuclear
extract in binding reaction buffer (15 mM Tris,
pH 7.9, 0.2 mM EDTA, 10% glycerol, and 4
mM DTT) containing 45 mM
KCl, 1 µg poly(dI-dC), 1.25 mM
MgCl2, and 0.5 mM
CaCl2 in a final reaction volume of 50 µl.
Ovalbumin was included as needed to maintain constant protein
concentrations. The binding reactions were incubated for 15 min at room
temperature and then digested with RQ1 ribonuclease RNase-free DNase
(Promega Corp., Madison WI) for 0.59 min. The resulting
DNA fragments were separated on a sequencing gel and visualized by
autoradiography. The protection of 5'- and 3'-ERE half-sites was
quantitated using the method of Brenowitz et al. (58). The
5'- and 3'-ERE half-site boundaries were determined using the
dimethylsulfate (DMS)-treated, piperidine-cleaved
32P-labeled DNA fragments, and the levels of
radioactivity in each 5'- and 3'-ERE half -site were quantitated from
four independent experiments using a phosphorimager and ImageQuant
software (Molecular Dynamics, Inc.). To account for
differences in sample loading, the amounts of radioactivity present in
each 5'- and 3'-half site were normalized to a region of the gel that
was unaffected by the addition of nuclear proteins. The same region of
each lane was used for normalization purposes. The level of protection,
fraction protected, was calculated by comparing the radioactivity
remaining in each ERE half-site after addition of MCF-7 nuclear
proteins with the level of radioactivity present in each ERE half -site
in the absence of nuclear proteins.
Gel Mobility Shift and Protease Sensitivity Assays
For characterization of the purified ER, the circular
permutation vectors B3consERE, B3pS2ERE, B3ERE2 (11), and B3OTERE
containing the A2, pS2, B1, and OT EREs, respectively, were digested
with EcoRI and BamHI. DNA fragments were
32P labeled as described previously (23). The
55-bp ERE-containing DNA fragments were isolated and combined with
purified, E2-occupied ER in binding reaction
buffer, 20 mM KCl, and 50 ng of poly(dI-dC) in a
final volume of 20 µl and incubated 15 min at room temperature. For
the coactivator studies, 55 bp 32P-labeled
ERE-containing DNA fragments were incubated with 57 fmol of purified,
E2-occupied ER for 10 min. Ovalbumin (20 µg)
was added to all binding reactions and GRIP1 and SRC-1 GST fusion
proteins or GST were added as indicated. Partial proteolysis of
DNA-bound ER was carried out with 55 bp ERE-containing DNA fragments
and 100 fmol of purified, E2-occupied ER
as
described (23). After a 10-min incubation, the indicated amounts of
S. aureus V8 protease (Worthington Biochemical Corp., Freehold, NJ) or proteinase K (Promega Corp.) were added to the binding reactions. Free and complexed
DNAs were separated on low ionic strength nondenaturing acrylamide gels
(59).
DNA Pull-Down Assays
The 34-bp oligos used in pull-down assays were prepared by
annealing a 5'-biotinylated forward strand to the reverse strand.
Assays were carried out essentially as described (60). Four picomoles
of annealed oligos containing either the A2, pS2, B1, or OT ERE, or a
nonspecific sequence were immobilized on 100 µg of streptavidin
paramagnetic beads (Dynal, Lake Success, NY) in 10 µl of
buffer T (10 mM Tris, pH 7.5, 1 mM EDTA, 1
M NaCl, 0.003% NP40) for 1 h at room temperature with
constant agitation. After one wash with buffer T at 1 mg beads/ml
buffer and one wash with transcription buffer (10 mM HEPES,
pH 7.6, 100 mM potassium glutamate, 2.5 mM DTT,
10 mM magnesium acetate, 5 mM EGTA, 3.5%
glycerol) with 0.003% NP40, the immobilized DNA was incubated with
transcription buffer containing 2.5 mg/ml BSA, 5 mg/ml
polyvinylpyrrolidone, and 2.5 mM DTT for 30 min at room
temperature. The immobilized DNA was washed twice with transcription
buffer containing 0.5 mg/ml BSA and 0.05% NP40 and incubated with 750
fmol of purified ER in 50 µl of transcription buffer containing
0.001% NP40, 5 µg of BSA, and 10-6
M E2. 50 µl transcription buffer
containing 100 µg of HeLa nuclear extract, 250 ng poly(dIdC), 1
mM ATP, 0.001% NP40, 80 mM KCl, and
10-6 M E2 was
incubated for 10 min at 4 C and spun in a microfuge for 2 min at 4 C.
The supernatant was added to the ER-DNA mixture. After rotation for
4 h at 4 C, the nonadsorbed proteins were removed and the DNA was
washed three times with 300 µl of transcription buffer containing 0.5
mg/ml BSA, 0.05% NP40, and 10-7
E2. The ER and its associated proteins were
eluted in 10 µl of SDS sample buffer, separated on 10% SDS gel, and
electroblotted onto a nitrocellulose membrane. Western analysis was
carried out with monoclonal antibodies directed against TIF2 (BD
Transduction Laboratories, Inc. Lexington, KY), AIB1 (BD
Transduction Laboratories, Inc.), SRC1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or the FLAG epitope
(Sigma, St. Louis, MO). A chemiluminescent detection
system (Pierce Chemical Co., Rockford, IL) was used to
detect the proteins. Autoradiograms were scanned and quantitated using
ImageQuant 5.0. Coactivator/ER ratios from five independent experiments
were combined. To minimize inter experimental variation, each
coactivator/ER ratio was divided by the mean coactivator/ER ratio for
that experiment and multiplied by mean coactivator/ER ratio for all
experiments. ANOVA was carried out using InStat 1.0 software (Louisiana
State University, Baton Rouge, LA).
 |
ACKNOWLEDGMENTS
|
---|
We thank James Kadonaga, Lee Kraus, Michael Stallcup, and Akira
Takeshita for generously providing reagents used in these studies. We
thank Penelope Pitch and Yvonne Ziegler for technical assistance, Kurt
Kwast for assistance with statistical analysis, and Larry Petz for
helpful comments during the preparation of this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: anardull{at}life.uiuc.edu
This work was supported by NIH Grant DK-53884 (to A.M.N.) and an
American Heart Association Predoctoral Fellowship (to J.R.W.).
Received for publication January 12, 2001.
Revision received March 16, 2001.
Accepted for publication April 2, 2001.
 |
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