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J Biol Chem, Vol. 273, Issue 45, 29321-29330, November 6, 1998
From the Department of Biochemistry and Biophysics and the
University of Rochester Cancer Center, The University of Rochester
School of Medicine and Dentistry, Rochester, New York 14642
The estrogen receptor (ER) is a transcription
factor that binds to a specific DNA sequence found in the regulatory
regions of estrogen-responsive genes, called the estrogen response
element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an
inverted repeat. A number of genes have multiple copies of these
imperfect EREs. In order to understand why natural EREs have developed
in this manner, we have attempted to define the basic sequence
requirements for ER binding. To this end, we measured the binding of
homodimeric ER to a variety of nonconsensus EREs. We discovered that an
ERE containing even a single change from the consensus may be unable to
bind ER. However, an ERE with two changes from the consensus may be
capable of binding avidly to ER in the context of certain flanking
sequences. We found that changes in the sequences flanking a
nonconsensus ERE can greatly alter ER-ERE affinity, either positively
or negatively. Careful study of sequences flanking a series of EREs
made it possible to develop rules that predict whether ER binds to a
given natural ERE and also to predict the relative amounts of binding
when comparing two EREs.
The estrogen receptor
(ER)1 is a transcription
factor that regulates the expression of estrogen-responsive genes by
binding to a specific DNA sequence found in their regulatory regions. This sequence is called the estrogen response element (ERE).
Many reports analyzing estrogen-responsive genes revealed a common
sequence motif that was anticipated to be the ERE (1-6). Experiments
that placed this sequence upstream of a reporter gene confirmed that a
13-bp inverted repeat was an essential element for estrogen
responsiveness (3). The essential ERE was determined to have the
consensus sequence 5'-GGTCAnnnTGACC-3' (4, 7). The symmetry of the
sequence was found to facilitate the binding of ER as a homodimer (8,
9). However, only a handful of the most highly estrogen-responsive
genes contain perfect consensus EREs. Many genes have been found to
contain sequences that appear to be EREs, but most of these vary from
the consensus by one or more nucleotides. Studies of ER binding showed
that one or more changes from the consensus sequence resulted in lower
ER-ERE affinity (10-12) and that sequences immediately flanking the
ERE impact ER-ERE binding (13). However, none of these reports
delineated the requirements for ER binding to nonconsensus EREs. The
results presented here define characteristics of nonconsensus EREs that determine whether or not productive ER-ERE binding occurs.
Preparation of Calf Uterine ER--
ER was purified from calf
uteri as described previously (14-17), modified as follows. Ammonium
sulfate-precipitated uterine cytosol was dissolved in TDPEK 175, 20%
glycerol, 0.1% Nonidet P-40 buffer (40 mM Tris, pH 7.6, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 175 mM KCl, 20% glycerol,
0.1% Nonidet P-40) and either batch absorbed to heparin-agarose resin
(Affi-Gel Heparin, Bio-Rad) or dissolved in TDPEK 100, 20% glycerol,
and 0.1% Nonidet P-40 and layered onto Yellow 86 resin (Sigma).
Proteins were eluted from heparin-agarose with a linear 175-500
mM KCl gradient and from Yellow 86 with a 300 mM step gradient. The peak ER-containing fractions eluted
at 300 mM KCl from heparin-agarose and at 200 mM KCl from Yellow 86. ER-containing fractions were pooled
and diluted to 100 mM KCl for use in assays.
ER was either liganded with
17 Highly Purified Human ER ERE-containing Oligomers--
Both strands of all oligomers were
synthesized and polyacrylamide gel electrophoresis-purified by Genosys
Biotechnologies (The Woodlands, TX). The regions flanking all sequences
tested were identical (Fig. 1A). The oligomers were
annealed, and 0.5 µg was 32P-end-labeled by
Escherichia coli DNA polymerase I Klenow fragment incorporation of [ Gel Shift ERE Binding Assays--
32P-End-labeled
DNA (60,000 cpm) and 0, 10, 20, 30, 40, or 50 µl of ER were incubated
with poly(dI-dC) (0.45 µg, Midland Certified Reagents, Midland, TX)
and an ER-supershifting antibody (either c-311 from Santa Cruz
Biotechnologies, Inc., Santa Cruz, CA or H222, a gift from Abbott) in a
total reaction volume of 60 µl. Reactions were incubated at 0 °C
for 60 min and loaded onto a 5% nondenaturing polyacrylamide gel.
Electrophoresis was performed at 150 V for 1.5 h at room
temperature in 1× TBE (100 mM Tris base, 0.831 mM boric acid, 1 mM EDTA). The gel was dried at
80 °C under vacuum and exposed to film overnight.
ERE Competition Assays--
6 µl of 3400 nM
recombinant human ER was diluted with 1040 µl of TDPEK 100, 20%
glycerol, 0.1% Nonidet P-40 containing 8.5 µg of poly(dI-dC). 15 µl of the ER-poly(dI-dC) mixture was added to tubes containing 6 µg
of 32P-labeled p17 ERE that had been premixed with
increasing concentrations of the appropriate competitor. Total reaction
volume was 18 µl. The mixture was incubated on ice for 1.5 h. 10 µl was loaded onto a 5% native polyacrylamide gel and separated by
electrophoresis at 150 V for 1.5 h. The gels were dried at
80 °C under vacuum and exposed to a PhosphorImager screen (Molecular
Dynamics, Inc., Sunnyvale, CA) overnight. DNA counts in the appropriate
bands were quantitated and graphed.
Gel Shift-based Determinations of Kd--
Oligomers
were dissolved at concentrations above 1000 ng/µl, as determined by
fluorimetry (Hoefer DynaQuant 200, Hoefer Instruments), and
32P-end-labeled using E. coli DNA polymerase I
Klenow fragment (3'-5' exo-) (New England Biolabs, Beverly, MA)
incorporation of [ DNA Substrates Used for ER Binding--
We designed a series of
ERE oligomers varying in sequence in our effort to define the
requirements for stable binding to ER. Single-stranded oligomers
containing variants of the ERE core consensus sequence were
synthesized, gel-purified, and annealed, to create double-stranded DNA.
Each substrate consisted of a test segment, having a sequence
resembling the core consensus ERE, embedded in a larger oligomer. We
call the additional length of oligomer the "background sequence."
The background nucleotides were arranged to avoid any sequences related
to the core consensus ERE. Therefore, the background is not able to
stabilize ER-ERE interaction, except by providing a natural DNA
extension on either side of the ERE. The background sequence is shown
in Fig. 1A, and the test
sequences are shown in Fig. 1B. The rationale for choice of
the test sequences will be explained below. Binding was measured by gel
shift, under conditions previously found to allow efficient ER-ERE
binding (15).
ER Binding and Competition Assays--
We performed binding assays
using partially purified ER obtained from calf uteri, at concentrations
of ER under 10 nM. We did this for two reasons: first, to
have our in vitro assays approximate the levels of ER found
inside the cell (21) and, second, to have the ER produced in, modified
in, and obtained from a mammalian source in a mixture of proteins that
naturally occur with ER. The binding assays then served to test whether
the results obtained using the highly purified recombinant
baculovirus-expressed ER apply to ER-purified directly from calf.
Antibody was used to supershift the ER-containing complexes in the
binding assays, which resulted in a clear separation of the
ER-containing shifts from those that were the result of non-ER
proteins. Because of concerns that antibodies could affect the amounts
of shifted complex, two different antibodies were used, in separate
experiments. Both antibodies, H222 and c311 (see "Experimental
Procedures"), gave identical results. One set of results is shown.
The results of the experiments containing antibody were identical to
those performed without antibody, except that the supershift allowed
clear visualization of low levels of ER-ERE binding. For ERE
competition experiments and for determinations of ER-ERE affinity, pure
recombinant baculovirus-expressed human ER from Panvera was used
because it was critical to have pure ER at high concentration. The
correlation of results between ER-ERE binding assays, ER-ERE
affinities, and ERE competition assays was absolute, showing that gel
shift is a very robust and sensitive method for comparing ER binding to
consensus and nonconsensus EREs. In addition, the results of
experiments using human ER mirrored those seen with calf ER. Both
estradiol-liganded and -unliganded ER behaved in an identical manner in
gel shift assays using the consensus ERE (Ref. 15 and data not shown).
All assays shown were performed with unliganded ER. Nucleotides within
the test sequences are defined by the numbering system shown in Fig.
1D.
A Single Change in the Consensus ERE Abolishes ER-ERE
Binding--
Fig. 2, lanes
1-11, contains gel shift results comparing the binding of
ER to two EREs that differ by a one-base pair substitution. Oligomer
p13, shown in lanes 1-6, contains the consensus
5'-GGTCAnnnTGACC-3' inverted repeat. ER-ERE binding produced a
prominent supershifted band. The sequence 13d1 in lanes
7-11 (Fig. 2) contained a T to G base substitution in the
Because ER binds ERE sequences as a homodimer (8, 9, 22-26) and each
ER molecule contacts a different arm of the ERE inverted repeat (27),
any change made to an arm of the ERE should increase or decrease ER-ERE
affinity in a manner identical to a change in the same position of the
other arm of the inverted repeat. This was tested by comparing 13d1,
which contains a T to G change at the Appropriate Flanking Sequences Allow ER Binding to Variants of the
Core Consensus ERE--
We considered whether sequences flanking the
core 13-bp ERE could alter ER-ERE binding. The first experiment (Fig.
2, lanes 7-11) involved the labeled 13d1
oligomer (Fig. 1B), which has a single base change from the
consensus 13-bp ERE. No supershifted ER was visible, indicating that ER
was unable to bind this element. Lanes 12-16
(Fig. 2) contained the 15d1 ERE, which has the same change from the
consensus as 13d1. However, the nucleotides on either side of the 13-bp
ERE have been substituted with nucleotides identical to those that
immediately flank the core inverted repeat of the potent vitellogenin
A2 ERE. ER binding to this new ERE produced an intense supershifted
band. Lanes 17-21 show the binding of ER to
17d1, a 17-bp inverted repeat containing a single change from the
consensus, with two nucleotides on either side of the core ERE
converted to those of the vitellogenin A2 ERE. The appearance of a dark
supershifted band confirmed that ER was also able to bind well to this
ERE sequence. Although a single change from the consensus was enough to
abolish ER binding in the presence of 13d1 background sequences, the
sequences adjacent to the core in 15d1 and 17d1 rescued ER-ERE binding.
5'-Flanking A Residues Rescue ER Binding to Nonconsensus EREs and
Contribute to ER Affinity--
Fig. 3
shows gel shift assays that contain substrates designed to explore the
contributions of 5' A residues immediately adjacent to the core 13-base
pair ERE. As was seen previously, there was no binding of ER to 13d1
(Fig. 3, lanes 1-6). The addition of a single A
residue adjacent to the 13d1 sequence (substrate 13d1 + A, Fig. 3,
lanes 7-12) resulted in the recovery of ER
binding. Lanes 13-18 show ER binding to 15d1,
which has a 5' A residue present on both strands of the oligomer. The
sensitive nature of this assay allowed the determination that there was
clearly more ER binding to 15d1 than to 13d1 + A. This result
demonstrates that ER-ERE binding is influenced by the sequence at both
of these sites.
A Single 5'-Flanking G Residue Reconstitutes ER Binding to a
Nonconsensus ERE, Showing Purines Are Preferred Immediately Flanking
the 13-bp ERE--
ER binding to an ERE with a single base change
depends on the sequence immediately flanking the 13-base pair inverted
repeat. The base oligomer around the 13d1 ERE included T and C at the Substitutions That Are Two Nucleotides Distant from the 13-bp Core
Consensus Influence ER-ERE Binding--
We also needed to know whether
sequences beyond those immediately flanking the core ERE affect ER
binding affinity. The 17d1 oligomer was used to test whether increasing
the inverted repeat beyond the A at Flanking Sequences Can Compensate for One or Two, but Not Three
Changes from the Consensus ERE--
The gel shift in Fig.
5 demonstrates the decrease in binding of
ER as the ERE sequence varies from the consensus. In lanes 1-6, where the ERE has only a single change from the
consensus, a strong ER supershift was seen. In lanes
7-12, where the ERE has two changes from the consensus,
there was a relative decrease in ER-ERE binding. In lanes
13-18, where the sequence incorporates three changes from
the consensus, there was no ER binding. Similar results were seen for
17d1, 17d2, and 17d3 (data not shown). These experiments reveal that,
with the appropriate flanking sequences, up to two changes from the
core 13-bp consensus can support ER-ERE binding. However, there was no
case where we observed ER-ERE binding when the ERE contained more than
two changes from the core consensus.
A Single A in the Immediately Adjacent 5'-Flanking Sequence
Compensates for One, but Not Two, Changes in the Core
Consensus--
Can one flanking nucleotide compensate for two changes
in the core sequence? The experiment in Fig.
6 was designed to test the binding of ER
to a sequence that contained the same two changes from the consensus as
the construct 15d2 but had only one compensatory A residue, at the
Competition with Selected EREs Confirms Results of Direct Binding
Assays--
Fig. 7A
demonstrates how different unlabeled EREs compete with labeled p17 for
binding to ER. This experiment was repeated at least three times for
each oligomer, with similar results. The EREs with the highest affinity
for ER, p15 and p17, effectively competed away the shifted complex. At
a 40-fold excess of unlabeled over labeled ERE, less than 5% of the
initial complex remained. The essentially indistinguishable curves show
that the affinities of both of these EREs for ER are similar. The 15d1
ERE, which has the 15-bp inverted repeat with a single change from the
consensus, did not compete as efficiently as the intact 15- and 17-bp
inverted repeats. Interestingly, however, 15d1 competed more
efficiently than the perfect 13-bp inverted repeat, showing that this
imperfect ERE has higher affinity for ER than the perfect p13 sequence. The 15d2, with two changes from the p15 consensus, and the 17d1AT, which has only the outermost vitellogenin A2 flanking sequences intact
at ±8-positions, have much lower, but similar affinities for ER. 15d2
and 17d1AT were the constructs that had the lowest levels of detectable
binding in the gel shift assays performed at physiological levels of
ER, as above. Neither 15d3, which had three changes from the p15
consensus, nor 13d1, which had a single change from the p13 consensus,
bound detectable levels of ER in the binding assays. The competition
experiments revealed that they both have extremely low affinity for ER.
After a 160-fold excess of 15d3 was added, 90% of the binding activity
remained. We were unable to detect any competition by 13d1 for ER
binding, even at a 320-fold excess of ERE. It was surprising that the
effect of this single change was so profound.
ER-ERE Affinity with Selected Consensus and Nonconsensus
EREs--
Gel shift assays were used to calculate the binding affinity
of p13, p15, and 15d2 for ER. Increasing concentrations of ERE were
added to a constant amount of ER. The bound and free ERE bands were
quantitated using the PhosphorImager. Semilogarithmic plots of ERE
titrations resulted in three distinctly different S-shaped saturation
curves. The experiment was repeated three times for each construct, and
the average value ± S.E. was graphed (Fig. 7B).
Affinities were determined as outlined under "Experimental Procedures." The affinity of ER for the p17 inverted repeat (0.25 nM) has been previously determined by other methods (15).
Because both the binding and competition assays showed little
difference between p17 and p15 affinities for ER, we expected the
calculated affinity of p15 to be close to 0.25 nM. In fact,
the calculated affinity was 0.54 nM, very close to the
expected value. Because 15d2 bound ER with low affinity, it was not
possible to achieve saturation, even at maximal concentrations of 15d2.
However, at saturation the amount of bound DNA equals the amount of
active ER. Because the amount of ER used in each experiment was
identical, the maximum possible amount of bound 15d2 equals the amount
of active ER. Since we could use this anticipated saturation value, the
points on the curve at high concentrations of 15d2 were not necessary
for an accurate determination of Kd. The
Kd for 15d2 was calculated to be 20.0 nM. Given the previous results seen for the ER-ERE binding
and competition experiments, we expected the binding curve for p13 to
position between that of p15 and 15d2. The plot of the binding data
showed that this was in fact the case, and the Kd
for p13 was determined to be 6.9 nM.
We have identified sequence requirements for ER to recognize EREs.
It had been reported that the 13-bp inverted repeat 5'-GGTCAnnnTGACC-3' ERE is the minimum length sequence having two essential qualities, the
ability to bind in a stable complex with ER and to mediate the
induction of estrogen-responsive genes (3). This 13-bp palindromic
structure was termed the core consensus ERE. However, most
estrogen-responsive genes contain variations of the core ERE. Our
experiments were designed to reveal how natural genes evolved to
contain variant, yet functional, EREs, and to explore the contributions
of flanking sequences to ER-ERE affinity. Our hypothesis is that
sequences surrounding the variant core consensus EREs in natural genes
allow them to retain ERE function despite the core sequence variations.
We found that the core ERE placed in an inert sequence background must
be perfect to retain function. However, within the appropriate
background, the entire sequence would be operational despite changes in
the core.
It is of interest to know whether a particular sequence is an ERE. We
show here that sequences resembling EREs may not bind ER or function in
hormone response. Sequences that vary from the core consensus by as
little as a single nucleotide can lose the ability to bind ER. For
example, even in huge excess, 13d1 did not bind ER and failed to
compete for ER binding with the consensus ERE. Many ERE-like sequences
may be the target of other transcription factors that bind "GGTCA"
motifs. Because many genes contain ERE candidates and the exact
flanking sequences that specify functional nonconsensus EREs were
unknown, a number of studies were performed to measure the binding and
transcriptional activity of various EREs (2, 12, 28-33). It was found
that a gene in one species may contain a functional ERE, whereas the
ERE in the analogous gene of a second species is nonfunctional. A goal
of our work is to predict functional ER-ERE binding on the basis of ERE sequence.
An essential feature of an ERE is the ability to bind ER. We measured
the ability of ER to retard an ERE-containing DNA segment in gel
electrophoresis and also the capacity of various EREs to compete for
binding to ER. Affinities were calculated for consensus and
nonconsensus EREs. To determine whether the core consensus ERE
represents the minimal element for ER binding, we designed an oligomer
called the "background sequence," which lacked any series of
nucleotides present in the core consensus ERE and was unable to
stabilize ER-ERE interaction (Fig. 1A). Test EREs were embedded in this background. Results showed that variation of a single
nucleotide in the palindromic 13-bp core ERE prevented ER-ERE
interaction. Furthermore, the single change abolished its ability to
compete, even at a 300-fold excess, with a functional ERE for binding
ER. We concluded that ER binding to nonconsensus EREs depended on
nucleotides flanking the core.
Flanking nucleotides would most likely contribute to ERE function by
direct interaction with the ER. Candidate nucleotides were chosen from
the ERE of the vitellogenin A2 gene, a highly inducible
estrogen-regulated gene (2-4). Its ERE is an extended 17-bp
palindrome, containing an A residue at the We considered the possibility that the symmetry of the inverted repeat
sequence contributed to ER affinity. If so, flanking nucleotides that
extend the core inverted repeat would promote binding, and the identity
of the nucleotides would then be of lesser importance. Construct
15d1A-T exchanged the positions of the flanking A and T residues found
in 15d1, while maintaining the inverted repeat. This sequence was
identical to the 13d1 construct, except that there was an A residue
placed 3' of the core inverted repeat. No ER binding was observed. This
demonstrated that the identity of individual nucleotides is important
and that a simple expansion of the inverted repeat does not increase ER binding.
It is important to know the effective distance of flanking base
sequences from the core sequence. It was clear that the Stabilization of ER binding by flanking sequences at position A variety of ERE-containing oligomers were tested for their ability to
compete with the p17 consensus for ER binding. These experiments
provided an independent and corroborative assessment of relative
affinities. Based on both binding and competition assays, relative
binding affinities were ranked in the following order: p15 ~ p17 > 15d1 ~ 17d1 > p13 > 17d1AT ~ 15d2 ~ 17d2 > 15d3 ~ 13d1.
The dissociation constant (Kd) for p17 was reported
as 0.25 nM. Affinity measurements for p15 yielded a
Kd of 0.54 nM, comparable with p17
within experimental variance. Therefore, Kd
determinations using gel shift analyses could accurately estimate
affinities of different EREs for ER. The affinities of 15d1 and 17d1
for ER were higher than that of p13, which was calculated to be 6.9 nM. The Kd for 17d2 was 20 nM, and the affinities of 15d2 and 17d1AT should be
similar, based on binding and competition experiments.
Could we define the limit of base substitutions in the core sequence
that could be rescued by flanking sequences? The answer is determined
by the position of the substitutions. Our results indicate that two,
but not three, substitutions in the same arm of the inverted repeat
will function in the appropriate flanking sequence context. Can all
double substitutions be rescued? Reports by others provide evidence
that ER cannot bind to a subset of EREs with two changes from the
consensus, although they have flanking sequences expected to rescue
ER-ERE binding. The murine c-fos oncogene contains an ERE
with two changes from the consensus, one on each arm of the inverted
repeat, and does not bind ER (37). This sequence,
5'-AAGGTCTaggAGACCCC-3'
(38), is incapable of binding despite a stimulatory sequence at the
It appears that ER binding to EREs can be described by a general set of
rules. For ER to bind, at least 10 of the 12 nucleotides located
between two and seven nucleotides from the center of the inverted
repeat have to be of the correct sequence. The furthest flanking
positions eight nucleotides from the center also contribute to ER
binding affinity, but both correct bases are required to reconstitute
binding when the rest of the ERE has only nine of the required bases.
Any ERE with two changes from the core consensus is nonfunctional when
the changes are on different arms of the inverted repeat. EREs with
three or more changes from the core consensus do not bind ER. Also,
between From these rules, one can predict accurately whether a putative ERE
sequence will bind to ER and also determine the relative affinity of ER
to different EREs. For example, the vitellogenin B1 gene contains two
EREs in the regulatory region. We designated them vitellogenin 1B1
(5'-CCAGTCActgTGACCCA-3') and
vitellogenin 2B1
(5'-CAAGTTAtcaTGACCTC-3') (Fig.
1C). Vitellogenin 1B1 has a single change from the
consensus, and 2B1 has two changes from the consensus. The core
sequence is underlined, and changes from the consensus are in boldface
type. Applying our rules for ER-ERE binding, we would predict that
vitellogenin 1B1 will bind ER because it contains a 5' G residue at The ERE sequence in the hsp70-related gene has a single nucleotide
change from the consensus,
5'-CTGGTCActcCGACCAG-3' (42).
Krawczyk et al. (42) predicted that this ERE would confer estrogen responsiveness to a reporter gene. This prediction was based
on its similarity to the EREs found in the rabbit uteroglobin gene
(5'-CAGGTCAccaTGCCCTC-3') (43), the
human pS2 ERE (5'-CAGGTCActgTGGCCCT-3') (12), and
other sequences that had a single core substitution and mediated an
estrogen response. Based on flanking sequence requirements for ER-ERE
binding, we would predict that the rabbit uteroglobin ERE and the human
pS2 ERE would bind ER with approximately the same affinity as the 13-bp
core ERE, while the hsp70-related gene ERE would bind barely detectable
amounts of ER. In fact, the authors reported that the hsp70-related
gene ERE did not confer hormone responsiveness to reporter genes. They
concluded that the T nucleotide in the right half inverted repeat was
the essential element for estrogen responsiveness. While that
explanation is consistent with their results, we would predict that the
combination of a single core substitution with inappropriate flanking
sequences makes this apparent ERE inactive. One value of our findings
is that the same modified core sequence might be active in another
gene, having the correct flanking sequence. Expectation of an essential
role of the T nucleotide would then predict the wrong result.
Predictions based on principles determined here extend to other
examples in the literature. ERE sequences of some genes differ between
species, and it is important to know whether ER binds to these EREs.
The ERE sequences of the mouse and human lactoferrin gene are
different. The mouse lactoferrin gene contains an ERE with one change
from the consensus,
5'-CAGGTCAAGGTAACCCA-3' (32). We
predict that the 5' A residue compensates for that change, allowing
ER-ERE binding. Teng (32) reported that ER bound to this ERE and that
the ERE was functional in transfection experiments. The human
lactoferrin gene ERE sequence is
5'-CAGGTCAAGGCGATCTT-3'. Because
this ERE contains a 5' A residue on both strands, we expect this ERE to
bind ER although there are two changes from the consensus. Teng (32)
reported that this ERE also binds ER and functions in transfection
experiments. Although no direct comparison was made between the two
sequences, we expect that the mouse ERE binds more ER than the human
ERE, based on the number of changes from the consensus.
There are numerous reported ERE sequences to which the binding of ER is
predictable by our rules. These include rat and bovine oxytocin genes
(30); cathepsin D ERE (33); calbindin D-9K gene ERE (41); vitellogenin
Bl, vitellogenin A2, and chicken apo-very low density lipoprotein gene
EREs (2); rabbit uteroglobin ERE (43); hsp70-related gene ERE (42); and
Hageman factor XII gene ERE (44). The 5'-flanking region of the
tst-1/oct6 gene has a single change from the consensus
combined with an A residue at the Although we report here that purified, homodimeric ER does not bind
EREs with more than two changes from the consensus and others have seen
the same result (28, 30, 45, 48), genes are known where estrogen
response is facilitated through ERE elements that contain three or more
changes from the consensus. These are c-Fos and
c-Jun, transcription factors that bind to the AP-1 site and
rat creatine kinase B. It has been suggested that ER binding to EREs
that vary by three or more nucleotides is dependent on heterodimerization or other novel mechanisms. For example, mouse proto-oncogene c-jun contains
5'-AAGCAGAgcaTGACCTT-3' (13) that
binds ER. Mutation of this ERE so that the left arm of the inverted
repeat contains only a single base in common with the consensus
(5'-AATTAGAgcaTGACCTT-3') did not
diminish ER binding. Given these unexpected results, the authors
concluded that ER does not bind to these sequences as a homodimer, as
was seen with the consensus ERE. They suggested that ER interacts with
an unidentified protein factor to regulate estrogen induction of
c-jun expression in a cell-specific pattern. The sequence of the rat creatine kinase B ERE is
5'-AGGTCAgaaCACCCT-3', which also
binds ER (49). Estrogen regulation of the rat creatine kinase B gene is
complex, depending on Sp1 binding and a second regulatory region that
has no ERE. The mouse c-fos proto-oncogene was reported to
have an ERE, capable of ER binding, similar to the rat creatine kinase
B ERE. The sequence of the c-fos ERE is 5'-CAGGTCAccaCAGCCCA-3', containing
three changes from the consensus (50, 51). The authors report that gel
shift experiments show a difference in mobility for ER binding to the c-fos ERE compared with the consensus ERE (51). This is a
significant finding, because gel shifts for homodimeric ER·ERE
complexes show consistent migration behavior. The authors note that the
complex they observe may contain a heterodimer, where ER associates
with an unknown protein, similar to their findings for
c-jun. In fact, ER binding to degenerate ERE/Sp1 sequences
was reported to involve heterodimerization (52). Heterodimerization of
ER may be a prerequisite for ER binding to nonconsensus EREs with three
or more changes. Adding another level of complexity to ER regulation,
ER has been shown to interact with Fos-Jun complexes directly at the
protein-protein level, activating transcription through DNA sequences
that contain GGTCA-like elements, without binding to the DNA itself
(53)
We would expect the DNA sequence requirements for heterodimeric ER
binding or for ER binding as part of a multiprotein complex to be
different from those for homodimeric ER. This hypothesis places ER
action into two models. In the first, ER binds as a homodimer directly
to EREs that contain two or fewer changes from the consensus. Here, ER
acts alone to up-regulate a gene. In the second model, ER binds as a
heterodimer or as part of a multiprotein complex. These two models can
be distinguished by the ERE sequence. With ERE sequences containing
more than two changes from the consensus, ER regulation would depend on
the presence of other transcription factors, allowing for a more
complex response. Furthermore, the more mutations one arm of an ERE
contains, the more it resembles an ERE half-sequence. Half-ERE
sequences have been observed to bind ER and regulate the expression of
certain genes (54-58). Although we could not detect ER binding to a
half-ERE (1/2 ERE p17, Fig. 1) (data not shown), the
conditions under which ER binds to half-EREs seem to require high ER
concentrations and multiple half-sites in tandem (59). In other
reports, ER regulates gene expression through half ERE sequences
without binding directly to these sequences (58). Experiments exploring
the nature of ER-ERE binding to ERE-like inverted repeats that have
three or more changes from the consensus are currently being conducted.
In summary, our study emphasizes that the sequence requirements for a
"minimal" consensus ERE are more complex than previously thought.
The minimal sequence required for ER binding encompasses a wide variety
of possible combinations, which display stable ER binding. The
optimal binding sequence is
5'-C(A/G)GGTCAnnnTGACC(T/C)G-3'. Any change
from this optimal consensus diminishes ER-ERE affinity. The underlined
core consensus sequence represents the minimum length for binding
activity. However, at that length, no variation is allowed. Our
experimental results are consistent with the hypothesis that ER can
bind as a homodimer to any ERE with up to two changes in one arm of the
core consensus if appropriate flanking sequences are present. One
change in the core requires a corresponding single correct nucleotide
in the immediate flanking sequence at the We thank Layla Saidi for providing
information about antibody titration experiments. We are grateful to
Abbott for generously supplying anti-ER H222 antibody, and we thank
Julie Hehn at Abbott for expediting the process of obtaining H222.
*
This work was supported by National Institutes of Health
Grant HD 24459.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Dept. of Biochemistry, School of Medicine,
University of Louisville, Louisville, KY 40292.
¶
To whom correspondence should be addressed. Tel.:
716-275-0999; Fax: 716-271-2683.
The abbreviations used are:
ER, estrogen
receptor; ERE, estrogen response element; bp, base pair(s).
Sequence Requirements for Estrogen Receptor Binding to Estrogen
Response Elements*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-[2,4,6,7,16,17-3H]estradiol (141 Ci/mmol; NEN Life
Science Products) or unliganded. Typical ER concentrations ranged from
0.5 to 5 nM. All concentrations of ER refer to the amount
of homodimeric ER. Final concentrations of ER were determined by
hydroxylapatite assay (18).
--
Recombinant human ER from
baculovirus-infected insect cells was purchased from Panvera Corp.
(Madison, WI) at a concentration of 5000 or 3400 nM for use
in gel shift-based determinations of ER-ERE affinity and ERE
competition assays.
-32P]dATP and
[-32P]TTP (3000 Ci/mmol, NEN Life Science Products).
-32P]dGTP (3000 Ci/mmol, NEN Life
Science Products). Labeling was stopped by heating the mixture to
70 °C for 10 min, as per the manufacturer's instructions. The
specific activity of labeled DNA was determined as follows.
One-fortieth volume of the crude labeling mixture was spotted onto a
TLC plate (polyethyleneimine-cellulose, EM Science, Gibbstown, NJ), and
the plate was developed in an aqueous solution of 0.37 M
HCl with 0.1 M sodium pyrophosphate. The unincorporated
counts migrated with the solvent front, whereas the labeled DNA
remained at the origin. The plate was divided into thirds, and each
third was counted separately in scintillation vials containing EcoScint
A (National Diagnostics, Atlanta, GA). The incorporated radioactivity
at the origin and the quantity of DNA in the reaction were used to
calculate the specific activity of each ERE. Increasing concentrations
of each [32P]DNA sample (1-µl volume) were mixed with a
constant amount (30 µl) of diluted ER (6 µl of 5000 nM
Panvera ER, 1294 µl of TDPEK 100, 20% glycerol, 0.1% Nonidet
P-40 containing 3.0 µg of poly(dI-dC)). Reactions were incubated for
1.5 h at 0 °C, loaded onto a 5% native gel, and separated by
electrophoresis at 150 V for 1.5 h. The gels were dried at
80 °C under vacuum and exposed to a PhosphorImager screen overnight.
DNA counts in the appropriate bands were quantitated and graphed. A
semilog plot of the saturation curves was used to determine the ERE
concentration at the midpoint of the maximal amount of ER·ERE
complex. Actual Kd values were calculated using the
equation,
where [EREmidpoint] is the concentration of
labeled ERE that results in a gel shift of half-maximal intensity (15,
19, 20). The concentration of ER had to be determined independently, because a significant proportion of the recombinant Panvera ER was
incapable of binding ERE. The concentration of active ER was measured
by titrating known ERE concentrations against a constant but unknown
concentration of ER and subjecting the reactions to gel shift. In the
shifted complex, the amount of ERE bound equaled the amount of ER
bound. Since both the bound and free ERE were quantitated using the
PhosphorImager, and the total amount of ERE was known, the molar
quantity of bound and free ERE could be determined. At a saturating
concentration of ERE, the maximal amount of ERE bound equaled the
concentration of ER. This value was used to calculate
Kd.
![K<SUB>d</SUB>=[<UP>ERE<SUB>midpoint</SUB></UP>]−½[<UP>ER</UP>]](/content/vol273/issue45/fulltext/29321/img001.gif)
(Eq. 1)
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (45K):
[in a new window]
Fig. 1.
A-D, sequences of ERE constructs.
A, the base oligomer sequence that surrounds the ERE test
sequences. Braces enclose the region into which the test
sequence is inserted. B, the test sequences, from 5' to 3',
surrounded by braces, that were inserted into the base
oligomer. Only the upper strand of the test sequences is shown. The 15- and 17-bp consensus sequences were based on the sequence of the highly
estrogen-responsive Xenopus vitellogenin A2 (vit
A2) gene. A change in one base was denoted as d1, two changes as
d2, and three as d3. Underlining emphasizes changes from the
core consensus. Boldface type indicates bases
that have been altered from the vitellogenin A2 sequence in the
flanking region. Lowercase type highlights
nucleotides in the spacer region. Except for 13d1, EREs with a single
change from the consensus were numbered using the base designations in
D. C, the vitellogenin B1 ERE oligomers are shown in their
entirety. Changes from the core 13-bp consensus ERE are
underlined. The sequence flanking the ERE is that which is
found in the vitellogenin A2 gene (2). The complementary strand (not
shown) contained a 5' overhang sequence 5'-TAA-3'. D, the
extended consensus inverted repeat. The 13-bp core consensus is
underlined. The central 3-bp spacer is shown in
lowercase. The flanking sequences are not underlined. The
bases in the ERE are numbered according to their distance from the
nucleotide at the center of the inverted repeat, with negative numbers
on the left of the center, and positive numbers on the right.
Labeled arrows below the sequence
delineate the sizes of the inverted repeats referred to in the
text.
4-position (Fig. 1D). No supershifted band was evident,
indicating that ER was not able to bind to this oligomer. Therefore, we
concluded that ER-ERE binding can be disrupted by a single base change
from the consensus. We extended these findings by testing ER binding to
other EREs with single base substitutions at each position in one arm
of the inverted repeat (Fig. 1B). In no case was any
supershifted ER·ERE complex visible (data not shown).
View larger version (80K):
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Fig. 2.
ER binding to EREs with a single change from
the consensus. The ability of ER to bind to a consensus or
nonconsensus ERE in the context of the same flanking sequences was
tested. The sequence of each ERE is shown, and the 13-bp core ERE
sequence is boxed. The position of supershifted ER·ERE
complex and free DNA is indicated to the left. The position
of an ERE complex that does not contain ER is also indicated. Oligomer
p13 contained the 13-base pair consensus ERE, and 13d1 contained the
minimal consensus with a single base change (Fig. 1B), 15d1
had a 15-bp inverted repeat with the same base change, and 17d1 had a
17-bp inverted repeat also with the same base change.
32P-End-labeled ERE was incubated with 0, 10, 20, 30, 40, or 50 µl of Yellow 86 purified ER, 0.45 µg of poly(dI-dC), and
c-3ll antibody. The mixture was incubated at 0 °C for 1 h and
loaded onto a 5% nondenaturing polyacrylamide gel. Following
electrophoresis, the gel was dried, and the bands were detected by
autoradiography.
4-position of the ERE, to
13G1, where the +4-position of the 13-bp ERE was changed from an A to a
G. Both changes resulted in a loss of ER-ERE binding. Also, the
4-position in 13d1 was changed from a pyrimidine to a purine, whereas
the +4-position was changed from a purine to another purine. It was possible that ER-ERE affinity could be dependent on the general structure of the nucleotide, but this did not appear to be the case. We
concluded that any change made in the left arm of the inverted repeat
can be considered to have an effect identical to the mirror change made
in the right arm of the inverted repeat. Although all possible base
substitutions were not tested, these results are fully consistent with
the hypothesis that all changes in the core consensus are equivalent in
their disruption of binding.
View larger version (90K):
[in a new window]
Fig. 3.
A single flanking A residue rescues ER
binding to a nonconsensus ERE. ER binding to EREs that contained
no 5'-flanking A (13d1), one 5'-flanking A (13d1 + A), or two
5'-flanking A residues (15d1), were compared. The sequence of each ERE
is shown, and the 13-bp core ERE sequence is boxed. Oligomer
13d1 contained the minimal consensus with a single base change of T to
G at position 4; 13d1 + A had the same base change plus a 5' A
residue added at position 7. 15d1 had a 15-bp inverted repeat with
the same base change, with a 5' A residue on both strands of the ERE.
The position of supershifted ER·ERE complex and free DNA is indicated
to the left. The position of an ERE complex that does not
contain ER is also indicated. ER/32P-ERE mixtures were
incubated with poly(dI-dC) and antibody and resolved as in Fig.
2.
7-position of the upper and lower strand, respectively, but ER was
unable to bind. This indicated that pyrimidines at this position inhibited ER-ERE binding. The addition of an A at the 7-position of
either strand, however, supported ER binding (Fig. 3). In order to test
whether the sequence preference for optimal ER-ERE binding included
only A at the 7 position, ER-ERE binding to 13d1 + G was tested (Fig.
4). In fact, the addition of a G residue
at the 7-position reconstituted approximately the same level of ER
binding as an A residue. For both 13d1 + A and 13d1 + G, the level of ER bound was significant but was below the level seen for ER binding to
15d1 (Figs. 3 and 4). This result suggests that a 15-bp extended ERE
consensus sequence includes either G or A at the 7-position of each
strand.
View larger version (104K):
[in a new window]
Fig. 4.
A flanking G residue rescues ER binding to a
nonconsensus ERE. ER binding to EREs that contained a 5'-flanking
G (13d1 + G) or two 5'-flanking A residues (15d1), were compared. The
sequence of each ERE is shown, and the 13-bp core ERE sequence is
boxed. Oligomer 13d1 + A contained the 13-bp consensus with a single
base change T to G at position 4 plus a 5' G residue added at
position 7. 15d1 had a 15-bp inverted repeat with the same base
change, with a 5' A residue on both strands of the ERE (Fig.
1B). The position of supershifted ER·ERE complex and free
DNA is indicated to the left. The position of an ERE complex
that does not contain ER is also indicated. ER/32P-ERE
mixtures were incubated with poly(dI-dC) and antibody and resolved as
in Fig. 2.
7 in 15d1 contributes to ER
affinity. However, ER binding to 17d1 and 15d1 was not noticeably
different. The high affinity binding of ER to either sequence resulted
in a very stable ER·ERE complex. This made it difficult to determine
whether additional vitellogenin A2 flanking sequences present in 17d1 resulted in more ER-ERE binding than was seen for 15d1 (data not shown). To overcome this difficulty, ER binding to 17d1AT was compared
with ER binding to 15d1AT. Reduced overall ER binding allowed a better
assessment of possible low level binding enhancement to 17d1AT
versus 15d1AT. Gel shift assays were performed using oligomer 15d1AT, in which the 5' A residue in position 7 on both strands, immediately flanking the imperfect core ERE, was exchanged for
a T. This substitution conserved the length of the inverted repeat and
the presence of an A-T base pair flanking the imperfect sequence on
both sides but put the A-T base pair in an opposite orientation with
respect to 15d1 (Fig. 1). This resulted in complete loss of ER binding,
identical to the result seen for 13d1. Because ER was unable to bind
15d1AT, any ER binding to 17d1AT can be attributed to the extended
flanking sequences. Subsequently, gel shifts were performed in parallel
using 17d1AT, the same sequence as 15d1AT, except for the addition of a
5' C residue at position 8 on both strands (Fig. 1), similar to the
sequence of the vitellogenin A2 ERE. The presence of the C in the 8
position of both strands resulted in a supershifted ER·ERE complex
(data not shown). A single C on one strand of the ERE at 8 was not
sufficient to reconstitute ER binding in 15d1AT. Recovery of ER-ERE
binding required the correct sequence at position 8 on both strands
of the 13d1 ERE. This shows that the identity of bases two residues distant from the core consensus is important for ER-ERE binding to
nonconsensus EREs.
View larger version (91K):
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Fig. 5.
The effect of one, two, and three changes
from the consensus ERE on ER binding. Lanes
1-6 show ER binding to 15d1, an ERE with one change from
the consensus. ER binding to 15d2 is shown in lanes
7-12. ER binding to 15d3 is shown in lanes
13-18. The sequence of each ERE is shown, and the 13-bp
core ERE sequence is boxed. The position of supershifted
ER·ERE complex and free DNA is indicated to the left. The
position of an ERE complex that does not contain ER is also indicated.
32P-End-labeled ERE was incubated with 0, 10, 20, 30, 40, or 50 µl of Yellow 86-purified ER, 0.45 µg of poly(dI-dC), and H222
antibody. The mixture was incubated at 0 °C for 1 h and
resolved as in Fig. 2.
7-position of one strand. As seen in Fig. 5, lanes
7-12, 15d2 was able to bind ER, showing that the presence of A residues at 7 on both strands was sufficient to compensate for
two changes to the sequence core. In Fig. 6, however, there was clearly
no ER binding to 13d2 + A, showing that a single flanking sequence
cannot compensate for two changes to the core. This result is
consistent with the pattern in which each single change in the 13-bp
inverted repeat requires a compensatory change in the immediate
flanking sequence.
View larger version (95K):
[in a new window]
Fig. 6.
A single flanking A residue does not
compensate for two changes in the consensus ERE. Lanes
1-6 show ER binding to 13d2 + A, an ERE with two changes
from the consensus and a 5' A residue at 7. For comparison, ER
binding to 15d1 is shown in lanes 7-12. The
sequence of each ERE is shown, and the 13-bp core ERE sequence is
boxed. The position of supershifted ER·ERE complex and
free DNA is indicated to the left. The position of an ERE
complex that does not contain ER is also indicated.
ER/32P-ERE mixtures were incubated with poly(dI-dC) and
antibody and resolved as in Fig. 2.
View larger version (17K):
[in a new window]
Fig. 7.
A, consensus and nonconsensus ERE
binding competition. 6 µg of 32P-labeled p17 ERE was
mixed with increasing amounts of the indicated unlabeled competitor
ERE, and each was added to 15 µl of 19.2 nM ER containing
48 ng of poly(dI-dC). The mixtures were incubated on ice for 1.5 h. 10 µl was subsequently loaded onto a 5% native polyacrylamide gel
and separated by electrophoresis at 150 V for 1.5 h. The gels were
dried at 80 °C under vacuum, and exposed to a PhosphorImager screen
for 12 h. Radioactivity in shifted DNA was quantitated and graphed
as a percentage of initial binding, where initial binding was the
intensity of the shifted band in the absence of competitor.
B, saturation plot of ER-ERE binding. Increasing
concentrations of the indicated 32P-labeled ERE were added
to a constant amount of recombinant human ER. The formation of the
ER·ERE complex was followed by gel shift, and the amount of binding
was quantitated using the PhosphorImager. The amount of bound ERE was
graphed versus the log of the concentration of ERE. The
experiment was done in triplicate, with the error
bars indicating ± S.E., as shown. The concentration of
active ER was equal to the amount of ERE bound at saturation.
Kd values were determined as they were under
"Experimental Procedures," by subtracting 1/2[ER] from the
ERE concentration at half-maximal binding.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
7-position of both strands
and a C residue at the 8-position of both strands. We asked whether
the vitellogenin A2 ERE contains the optimal flanking sequence or
whether outer nucleotides of this ERE represent a subset of sequences
that can compensate for changes in the core sequence. For example, the
presence of an A residue at 7, as in vitellogenin A2, was able to
compensate for a core variation, rescuing ER binding. Is the
5'-flanking A of vitellogenin A2 ERE necessary for ER binding to EREs
that have a single change from the consensus, or can other bases
substitute? We knew that 13d1, like some other 13-bp constructs that
did not bind ER, had a 5'-flanking T residue on the upper strand and a
5'-flanking C residue on the lower strand. This suggested that
pyrimidines could not substitute for the A residue at position 7.
However, when ER 13d1 + G was tested, ER binding was restored at about
the level seen for 13d1 + A. Apparently, purines are preferred for the
5'-position immediately flanking the core. Therefore, while the
vitellogenin A2 sequence served as a useful guide for testing
nucleotides, other base substitutions also restored ERE binding ability.
7 sequence
immediately flanking the 13-bp core had a large effect on ER-ERE
affinity. To test the influence of nucleotides further out, the
nucleotides at 8 were tested in the context of an ERE-like sequence
that did not bind ER, allowing minor contributions to be detected.
Although a correct nucleotide at 7 on either strand could compensate
for a single change in the core, correct nucleotides at 8 on both
strands were required to fully recover ER binding to an ERE having the
same core defect. A single C at 8 was not sufficient to recover ER
binding to 15d1AT. Detectable ER-ERE binding required a C at 8 on
both strands, as in 17d1AT. We suspect that both residues at the
8-position exert an equal small positive effect on ER-ERE binding
affinity and that both residues are needed to overcome the significant
negative effect of the changed core consensus on binding to EREs at
physiological concentrations of ER. In ranking the relative
contributions of flanking sequences to ER-ERE affinity, we conclude
that bases at the 7-position exert more influence than bases at the
8-position.
8 of
the inverted repeat is supported by additional evidence. The DNase I
footprint of ER spans 22 nucleotides (15), and the crystal structure of
the ER-DNA binding domain complexed with DNA shows contacts with 17 base pairs of core and flanking sequence (27). The crystal structure
shows that four side chains on the surface of the ER-DNA recognition
helix form hydrogen bonds with the GGTC bases in the ERE sequence.
Further contacts are made with the backbone of the DNA, suggesting that
these are not sequence-specific. However, comparisons of crystal
structures of ER and glucocorticoid receptor DNA binding domain
complexed with DNA revealed that even subtle changes in the DNA-protein
interface can substantially alter key contact points. Single DNA base
changes are likely to result in the rearrangement of side chains and
multiple stabilizing bonds (27). As a result, specific contacts made by
ER could vary extensively depending on the DNA sequence presented. The ER would not be unique in its ability to rearrange stabilizing contacts
for recognition of nonconsensus sequences. Other nuclear hormone
receptors can bind remarkably disparate DNA sequences; e.g.
not only will COUP-TF bind to inverted or direct repeats of 5'-GGTCA-3'
with spacing up to 12 nucleotides (34), but it can also tolerate
changes from the consensus (35, 36).
7-position. The 373 ERE found in the 5'-flanking region of
c-jun is similar. It has two changes from the consensus
sequence and a G residue at the 7-position, i.e.
5'-CGGGTCCgctGGACCTT-3', and is also unable to bind ER (13). ER would not bind an ERE-like sequence in the 5' regulatory region of the rat prolactin gene, 5'-TTTGTCActaTGTCCTA-3'
(39, 40). The rat calbindin D-9K gene contains a functional ERE with a
single change from the consensus, which when mutated to
5'-AGATCActgTGATCT-3', with a change in both arms of the inverted repeat, did not bind ER
(41). Together, these reports indicate that, although two changes from
the consensus may be tolerated in a single arm of the inverted repeat
in the presence of appropriate flanking sequences, a change in each arm
of the inverted repeat is not.
7 and +7, progressively more changes from the optimal
sequences result in lower affinity binding. Conversely, more bases in
agreement with the optimal sequences will result in tighter binding.
7
on the lower strand that compensates for the single base substitution
in the core consensus sequence. Vitellogenin 1B1 also has a 5' C
residue at position 8 in the upper strand, which contributes slightly
to binding affinity. Vitellogenin 2B1 has a 5' flanking A residue at
the 7-position on both strands, which compensates for the two changes
in the core consensus, allowing ER-ERE binding. As with vitellogenin
1B1, the 5' C residue in the 8-position has a small positive effect
on binding. Because vitellogenin 2B1 has two changes in the core
consensus and vitellogenin 1B1 has one, vitellogenin 1B1 should bind
more ER than vitellogenin 2B1. Gel shifts with these EREs (not shown)
confirmed these predictions.
7-position; it bound ER and
generated an estrogen response in reporter genes. The authors also
synthesized two EREs which had three changes from the consensus,
neither of which bound ER (45). Other EREs to which the rules
successfully apply are the 334 Xenopus vitellogenin ERE
(7), Xenopus ER gene ERE (46), and the salmon gonadotropin
II -subunit gene (47).
7-position or two changes
in the far flanking sequence at the 8-position. Two changes in the
core require two correct nucleotides in the immediate 7 flanking
sequence. If an ERE contains a change in each arm of the inverted
repeat, ER cannot bind. Because these rules apply to EREs with
one or two changes from the consensus but do not apply for EREs with
three or more changes, we suggest that ER does not bind alone as a
homodimer to EREs with more than two changes from the core consensus.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: ABL-FCRDC, P.O. Box B, Bldg. 539, Rm. 116, Fort
Detrick, Frederick, MD 21702.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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C M Klinge, S C Jernigan, K A Mattingly, K E Risinger, and J Zhang Estrogen response element-dependent regulation of transcriptional activation of estrogen receptors {alpha} and {beta} by coactivators and corepressors J. Mol. Endocrinol., October 1, 2004; 33(2): 387 - 410. [Abstract] [Full Text] [PDF]
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 R. Pilka, H. Domanski, S. Hansson, P. Eriksson, and B. Casslen Endometrial TIMP-4 mRNA is high at midcycle and in hyperplasia, but down-regulated in malignant tumours. Coordinated expression with MMP-26 Mol. Hum. Reprod., September 1, 2004; 10(9): 641 - 650. [Abstract] [Full Text] [PDF]
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 J. Palacios, E. T. Marusic, N. C. Lopez, M. Gonzalez, and L. Michea Estradiol-induced expression of Na+-K+-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1793 - H1800. [Abstract] [Full Text] [PDF]
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 A. J. Krieg, S. A. Krieg, B. S. Ahn, and D. J. Shapiro Interplay between Estrogen Response Element Sequence and Ligands Controls in Vivo Binding of Estrogen Receptor to Regulated Genes J. Biol. Chem., February 6, 2004; 279(6): 5025 - 5034. [Abstract] [Full Text] [PDF]
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 J-C Lambert, N Coyle, and C Lendon The allelic modulation of apolipoprotein E expression by oestrogen: potential relevance for Alzheimer's disease J. Med. Genet., February 1, 2004; 41(2): 104 - 112. [Abstract] [Full Text] [PDF]
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V. B. Bajic, S. L. Tan, A. Chong, S. Tang, A. Strom, J.-A. Gustafsson, C.-Y. Lin, and E. T. Liu Dragon ERE Finder version 2: a tool for accurate detection and analysis of estrogen response elements in vertebrate genomes Nucleic Acids Res., July 1, 2003; 31(13): 3605 - 3607. [Abstract] [Full Text] [PDF]
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 L. H. Wang, X. Y. Yang, X. Zhang, K. Mihalic, W. Xiao, and W. L. Farrar The cis Decoy against the Estrogen Response Element Suppresses Breast Cancer Cells via Target Disrupting c-fos not Mitogen-activated Protein Kinase Activity Cancer Res., May 1, 2003; 63(9): 2046 - 2051. [Abstract] [Full Text] [PDF]
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S. A. Jelinsky, H. A. Harris, E. L. Brown, K. Flanagan, X. Zhang, C. Tunkey, K. Lai, M. V. Lane, D. K. Simcoe, and M. J. Evans Global Transcription Profiling of Estrogen Activity: Estrogen Receptor {alpha} Regulates Gene Expression in the Kidney Endocrinology, February 1, 2003; 144(2): 701 - 710. [Abstract] [Full Text] [PDF]
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S. Zhou, G. Turgeman, S. E. Harris, D. C. Leitman, B. S. Komm, P. V. N. Bodine, and D. Gazit Estrogens Activate Bone Morphogenetic Protein-2 Gene Transcription in Mouse Mesenchymal Stem Cells Mol. Endocrinol., January 1, 2003; 17(1): 56 - 66. [Abstract] [Full Text] [PDF]
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M. J. Evans, H. A. Harris, C. P. Miller, S. K. Karathanasis, and S. J. Adelman Estrogen Receptors {alpha} and {beta} Have Similar Activities in Multiple Endothelial Cell Pathways Endocrinology, October 1, 2002; 143(10): 3785 - 3795. [Abstract] [Full Text] [PDF]
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 W. El-Sankary, V. Bombail, G. G. Gibson, and N. Plant Glucocorticoid-Mediated Induction of CYP3A4 is Decreased by Disruption of a Protein: DNA Interaction Distinct from the Pregnane X Receptor Response Element Drug Metab. Dispos., September 1, 2002; 30(9): 1029 - 1034. [Abstract] [Full Text] [PDF]
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P. Yi, S. Bhagat, R. Hilf, R. A. Bambara, and M. Muyan Differences in the Abilities of Estrogen Receptors to Integrate Activation Functions Are Critical for Subtype-Specific Transcriptional Responses Mol. Endocrinol., August 1, 2002; 16(8): 1810 - 1827. [Abstract] [Full Text] [PDF]
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D. Lopez, M. D. Sanchez, W. Shea-Eaton, and M. P. McLean Estrogen Activates the High-Density Lipoprotein Receptor Gene via Binding to Estrogen Response Elements and Interaction with Sterol Regulatory Element Binding Protein-1A Endocrinology, June 1, 2002; 143(6): 2155 - 2168. [Abstract] [Full Text] [PDF]
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D. Kressler, S. N. Schreiber, D. Knutti, and A. Kralli The PGC-1-related Protein PERC Is a Selective Coactivator of Estrogen Receptor alpha J. Biol. Chem., April 12, 2002; 277(16): 13918 - 13925. [Abstract] [Full Text] [PDF]
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P. Yi, M. D. Driscoll, J. Huang, S. Bhagat, R. Hilf, R. A. Bambara, and M. Muyan The Effects of Estrogen-Responsive Element- and Ligand-Induced Structural Changes on the Recruitment of Cofactors and Transcriptional Responses by ER{alpha} and ER{beta} Mol. Endocrinol., April 1, 2002; 16(4): 674 - 693. [Abstract] [Full Text] [PDF]
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K.-H. Lin, W.-J. Wang, Y.-H. Wu, and S.-Y. Cheng Activation of Antimetastatic Nm23-H1 Gene Expression by Estrogen and Its {alpha}-Receptor Endocrinology, February 1, 2002; 143(2): 467 - 475. [Abstract] [Full Text] [PDF]
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 C. J. Gruber, W. Tschugguel, C. Schneeberger, and J. C. Huber Production and Actions of Estrogens N. Engl. J. Med., January 31, 2002; 346(5): 340 - 352. [Full Text] [PDF]
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Y. Zhang and M. L. Dufau EAR2 and EAR3/COUP-TFI Regulate Transcription of the Rat LH Receptor Mol. Endocrinol., November 1, 2001; 15(11): 1891 - 1905. [Abstract] [Full Text] [PDF]
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S. A. Krieg, A. J. Krieg, and D. J. Shapiro A Unique Downstream Estrogen Responsive Unit Mediates Estrogen Induction of Proteinase Inhibitor-9, a Cellular Inhibitor of IL-1{beta}- Converting Enzyme (Caspase 1) Mol. Endocrinol., November 1, 2001; 15(11): 1971 - 1982. [Abstract] [Full Text] [PDF]
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C. M. Klinge Estrogen receptor interaction with estrogen response elements Nucleic Acids Res., July 15, 2001; 29(14): 2905 - 2919. [Abstract] [Full Text] [PDF]
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 P. de Santa Barbara, C. Méjean, B. Moniot, M.-H. Malclès, P. Berta, and B. Boizet-Bonhoure Steroidogenic Factor-1 Contributes to the Cyclic-Adenosine Monophosphate Down-Regulation of Human SRY Gene Expression Biol Reprod, March 1, 2001; 64(3): 775 - 783. [Abstract] [Full Text]
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P. S. Jones, E. Parrott, and I. N. H. White Activation of Transcription by Estrogen Receptor alpha and beta Is Cell Type- and Promoter-dependent J. Biol. Chem., November 5, 1999; 274(45): 32008 - 32014. [Abstract] [Full Text] [PDF]
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G. Urban, T. Golden, I. V. Aragon, J. G. Scammell, N. M. Dean, and R. E. Honkanen Identification of an Estrogen-inducible Phosphatase (PP5) That Converts MCF-7 Human Breast Carcinoma Cells into an Estrogen-independent Phenotype when Expressed Constitutively J. Biol. Chem., July 13, 2001; 276(29): 27638 - 27646. [Abstract] [Full Text] [PDF]
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