An Explanation for Observed Estrogen Receptor Binding to Single-Stranded Estrogen-Responsive Element DNA
Mark D. Driscoll,
Ganesan Sathya,
Layla F. Saidi,
Michael S. DeMott,
Russell Hilf and
Robert A. Bambara
Department of Biochemistry and Biophysics The University of
Rochester School of Medicine and Dentistry Rochester, New York
14642
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ABSTRACT
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Estrogen-inducible genes contain an enhancer
called the estrogen response element (ERE), a double-stranded inverted
repeat. The estrogen receptor (ER) is generally thought to bind to the
double-stranded ERE. However, some reports provide evidence that an ER
homodimer can bind a single strand of the ERE and suggest that
single-stranded ERE binding is the preferred binding mode for ER. Since
these two models describe quite different mechanisms of receptor
action, we have attempted to reconcile the observations. Analyzing DNA
structure by nuclease sensitivity, we found that two identical
molecules of a single strand of DNA containing the ERE sequence can
partially anneal in an antiparallel manner. Bimolecular annealing
produces double-stranded inverted repeats, with adjacent unannealed
tails. The amount of annealing correlates exactly with the ability of
ER to bind bimolecular EREs. Either strand of an ERE could anneal to
itself in a way that would bind ER. We conclude that ER binds only the
annealed double-stranded ERE both in vitro and in
vivo.
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INTRODUCTION
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The estrogen receptor (ER) is a soluble protein that
activates expression of responsive genes by binding a specific DNA
sequence, an inverted repeat called the estrogen response element
(ERE). Binding stability is thought to be largely sequence dependent,
although a number of variations on the binding consensus sequence of
5'-C(A/G)GGTCAnnnTGACC(T/C)G-3' accommodate stable binding. A
considerable amount of evidence suggests that the ER binds the
double-stranded, double-helical form of this sequence as a protein
homodimer (1, 2, 3, 4). When estradiol is liganded to the ER, the complex of
hormone, receptor, and DNA are capable of activating the transcription
complex of the responsive gene. They appear to act by a looping
mechanism, whereby the DNA-bound ER binds directly to the transcription
complex (5, 6, 7, 8, 9). This mechanism is consistent with the ability of the
ER-ERE to activate promoters from considerable distances upstream and
downstream of the transcription initiation site.
Since binding of the estrogen response element (ERE) is an important
step in ER action, a number of investigators have considered whether
ER-DNA interaction alters the DNA structure, and whether such
alteration has a biological role. ER-ERE binding was found to cause
moderate topological effects on the double-stranded DNA (10). Also, a
bend is induced in the DNA at the site of interaction (11). Such
effects are consistent with binding to double strands and the proposed
looping mechanism of activation.
Contrasting with these observations, a group of reports have provided
evidence that the ER interacts preferentially with only one strand of
the ERE (12, 13, 14, 15, 16). The authors concluded that after ER binding, the ERE
containing DNA unwound into single strands, and that this step may be a
critical feature of the mechanism of ER action. They suggested that ER
participates in the unwinding of the ERE. Furthermore, they proposed
that ER binding depends on the ability of the ERE to assume an unusual
three-dimensional conformation, inferred from the anomalous migration
of oligomers containing the ERE sequence in native gel electrophoresis.
These results suggest that the recognition of the ERE is structure
specific, as well as sequence specific. In this event, the mechanism of
ER induction of gene expression could involve a structural alteration
in the DNA propagated to the promoter through the helix. Another
possibility is that the basic mechanism of ER enhancement of gene
expression involves looping, but that single-stranded DNA in the
looping complex must interact with components of the transcription
complex. These suggestions have a significant bearing on the mechanism
by which ERE-containing genes are regulated by the ER.
Before further consideration of the possible contributions of a
single-stranded DNA-binding step in the mechanism of ER action, we have
attempted to determine whether ER truly binds a single strand of the
ERE.
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RESULTS
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Our experimental approach is based on the hypothesis that even
single strands of ERE-containing oligomers can readily assume partially
double-stranded structures. That is, identical oligomers with the
sequence of one strand of an ERE can interact in the inverted repeat
region to form a structure that has segments of double helix and
regions that are unannealed. We reasoned that if ER-ERE interaction
correlates with the formation of double strands in the experimental
environment, then ER can be assumed to bind double-stranded ERE.
However, if the amount of ER-ERE interaction is independent of, or
inversely related to, the amount of a DNA sample that is double
stranded, then ER could prefer binding to the single-stranded ERE.
Identical oligomers containing an ERE sequence were exposed to
conditions designed to either favor or disfavor the formation of double
strands. Specifically, the single-stranded DNA was prepared for ER
binding by heating, followed by either quick chilling or slow chilling.
The quick-chill procedure was anticipated to discourage intermolecular
formation of double-stranded structure, whereas the slow chilling would
encourage formation of double strands.
DNA Substrates Were Probed by Snake Venom Phosphodiesterase to Test
for Double-Stranded Regions
The constructs tested using this assay are shown in Fig. 1
. The first was a single-stranded
36-nucleotide oligomer, designed to have no inverted repeats or large
regions of possible foldback. Subsequently examined oligomers contained
an ERE sequence. The second was the forward ERE strand, designated F1.
The third was the backward ERE strand, designated B1. The fourth,
designated ds1, was an equimolar mixture of F1 and B1, that could
anneal to form a 38-bp double-stranded region with two 3-bp
5'-overhangs. Another pair of oligomers, designated F2 and B2, was also
tested. These were identical to F1 and B1, except that the central
nucleotide in the ERE spacer was transposed between the strands.

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Figure 1. DNA Constructs
The sequences of the DNA constructs are as shown; the 12 essential
nucleotides of the 15-bp consensus ERE are underlined.
Residues are numbered starting at the 5'-end, so the numbers correspond
to the sizes of the 5'-end-labeled DNA generated by snake venom
phosphodiesterase digestion. The annealed, double-stranded DNA (ds1 and
ds2) refers to annealed (F1-B1) and (F2-B2).
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In Figs. 2
and 3
, digestion with snake venom phosphodiesterase was used
to determine the extent of double-stranded character in the DNA
constructs that were subsequently tested for their ability to bind ER.
Because the phosphodiesterase digests exonucleolytically in a 3'- to
5'-direction, all DNA was 5'-end labeled. The nuclease rapidly digests
single-stranded DNA but pauses when it encounters a double-stranded
region. By observing where the nuclease pauses during digestion of
substrate DNA over time, one can infer where the DNA contained
double-stranded regions. It appeared that digestion products smaller
than six nucleotides in length could not be resolved using a 12%
denaturing polyacrylamide gel (Figs. 2
and 3
). Since these small
products could be resolved in an 18% gel (data not shown), we could
verify that the nuclease could eventually digest the tested oligomers
all the way to the 5'-end.

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Figure 2. Phosphodiesterase Treatment of Quick-Chilled DNAs
The DNA sequences (Fig. 1 ) consist of single-stranded 36-nucleotide
oligomer (36), the F1 strand of the ERE-containing oligomer, the B1
strand of the ERE-containing oligomer, and F1 mixed with an equimolar
ratio of B1 to create fully duplex ERE (ds1). All DNA was 5'-end
labeled and subsequently quick chilled by heating to 90 C for 3 min,
followed by quenching in an ice bath. The DNA was then subjected to
digestion by snake venom phosphodiesterase, loaded onto a 12%
denaturing gel, separated by electrophoresis, and exposed to film as in
Materials and Methods. The 15 nucleotide digestion
products tended to comigrate as the fastest spot in the 12% gel.
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Figure 3. Phosphodiesterase Treatment of Slow-Chilled DNAs
The DNA sequences (Fig. 1 ) consist of single-stranded 36-nucleotide
oligomer (36), the F1 strand of the ERE-containing oligomer, the B1
strand of the ERE-containing oligomer, and F1 mixed with an equal
amount of B1 to create the double-stranded ERE oligomer (ds1). All DNA
was 5'-end labeled and subsequently slow chilled by heating to 90 C for
3 min, and then allowed to come to room temperature over 6 h. The
DNA was then subjected to digestion by snake venom phosphodiesterase
for the indicated times, loaded onto a 12% denaturing gel, separated
by electrophoresis, and exposed to film as in Materials and
Methods. The sizes of the digestion products are indicated in
the figure. The 15 nucleotide digestion products tended to comigrate
as the fastest spot in the 12% gel.
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Nuclease Digestion Indicates That Quick-Chilled F1 or B1 ERE
Strands Exhibit Little Double-Stranded Character
The oligomers used in the experiment in Fig. 2
were heated to 90 C for 3 min to
dissociate the strands and plunged into ice. This quick-chill treatment
was designed to prevent bimolecular annealing, favoring either
remaining as single strands or unimolecular self-annealing,
i.e. foldbacks. After this treatment, the DNA was exposed to
snake venom phosphodiesterase for up to 30 min, as shown in Fig. 2
.
Lanes 14 contained the intact substrate DNA, which was incubated
without enzyme at 37 C for 30 min. Digestion of the ERE-containing F1
strand showed pausing after 10 min of digestion. At this point,
products ranged in size between 38 and 27 nucleotides, with none
smaller than 27. Evidently, an annealed region was slowing digestion.
Although conditions were not favorable to the formation of
bimolecular structures, either foldback helices in monomer strands or a
small amount of bimolecular double strands may have formed. The exact
structures leading to this pause in digestion cannot be determined by
the techniques that we employ. At later times, the products were
reduced in size, with a significant amount of the terminal 15
nucleotide products accumulating by 30 min. In contrast, the
single-stranded 36 mer was degraded steadily and uniformly over the
30-min digestion period, showing very little double strand-dependent
pausing. The B1 strand exhibited little double-stranded character and
was digested more rapidly. A small portion of the ds1 ERE was digested
over the course of the reaction, but most remained intact. This showed
that, in spite of reaction conditions unfavorable to double-stranded
binding of identical single strands, when equimolar amounts of exactly
complementary strands were present, they did anneal efficiently. This
observation raises the possibility that some F1 could be annealing in a
bimolecular, partly double-stranded form, even though the region of
complementarity is smaller than in ds1.
Nuclease Digestion Indicates That F1 and B1 EREs Have More
Double-Stranded Character When Slow Chilled Rather Than Quick
Chilled
The same DNA constructs were then tested for the presence of
double strands by nuclease digestion after slow cooling from 90 C (Fig. 3
). Lanes 14 contain substrates that
were not exposed to nuclease. In the presence of nuclease, the
single-stranded 36 mer (36) was digested in a uniform pattern,
demonstrating that it lacked significant double-stranded character. The
fully duplex oligomer (ds1) was anticipated to completely reanneal
during the slow cooling. It was digested to only a minimal extent over
the 30-min time course, verifying that annealing protects the oligomers
from phosphodiesterase attack. Digestion of the B1 was markedly slower
than in Fig. 2
. At 10 min a significant amount of slow-chilled ERE
digestion products remained resistant to degradation to sizes shorter
than 30 nucleotides. By 30 min, large B1 products were still visible on
the gel, whereas the quick-chilled B1 was completely digested before 20
min. The F1 oligomer was quickly digested to a 37 or 38 long product,
but then resisted further digestion. The phosphodiesterase was
effectively stalled between nucleotides 38 and 27, resulting in dark
bands on the exposure throughout this region. The term "stalling"
is used to denote a pause lasting up to the time that the reaction is
sampled. A small number of the DNA molecules were eventually digested
past position 27. At that point the rate of further digestion
increased, suggesting that the nuclease had passed the region of
annealing. Although the nuclease paused between nucleotides 38 and 27
when the DNA was quick chilled (see Fig. 2
), slow chilling of the DNA
resulted in a much more pronounced, stable, double-stranded region. It
should be noted that the regions in which digestion was impeded do not
precisely overlap the predicted annealed portion of the DNA. This may
be a result of steric interference between the structure of the enzyme
and the double-stranded ERE (see Discussion and Fig. 7
).

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Figure 7. Model of Snake Venom Phosphodiesterase Digestion of
Annealed Single-Stranded Oligomer
A, The sequence of the single-stranded forward oligomer is shown at the
top, with the ERE inverted repeat
underlined. Both the top and bottom
strands are of the same sequence, with unpaired tails extending
both 5' and 3' from the annealed region. The regions where the
phosphodiesterase pauses are indicated on the sequence. B, A diagram of
the phosphodiesterase and the annealed oligomers is shown. The 5'-end
label is denoted by a star. The phosphodiesterase is an
oval, and the phosphodiesterase active site is at the
tip of the triangle. The phosphodiesterase begins
digestion at the 3'-end of the DNA and continues until it encounters a
double-stranded region. Steric hindrance is presumed to prevent the
active site from efficient cleavage of the DNA closer than five
nucleotides from the annealed region. C, Digestion continues slowly
through the double-stranded region, and then begins to accelerate as
the double-stranded region becomes shorter. D, After digestion to the
position of three complementary nucleotides, the nuclease has resumed
its most rapid rate of cleavage.
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Nuclease Digestion Indicates That Quick-Chilled F2 Has
Double-Stranded Regions While B2 Is Single Stranded
Experiments from other laboratories, described in
Discussion, suggested that a change in the central
nucleotide of the ERE could alter ER binding affinity to
single-stranded DNA (13, 15, 16). Consequently, we performed additional
experiments to explore the ER-binding properties to such
single-stranded constructs. Different oligomers, Forward 2 (F2) and
Backward 2 (B2), were synthesized for this purpose (Fig. 1
). F2 and B2
were identical to F1 and B1, except that base 23 in the F1 oligomer was
changed from an A to a T in F2, and base 25 of B1 was changed from a T
to an A in B2, transposing the central ERE nucleotide between the two
strands. These oligomers were treated by either slow or quick chilling
as described above.
The first digestion measurements were made after quick chilling (Fig. 4
), similar to what was done in Fig. 2
.
Over the course of digestion, the duplex ERE (ds2) remained essentially
intact. By 30 min, B2 was almost completely digested, indicating that
it had no double-stranded regions capable of significantly impeding the
progress of the phosphodiesterase. Digestion of F2 was clearly
paused between nucleotides 27 and 36, with intermediate products
lingering in high concentration throughout the time course. This
indicates the presence of considerable double-stranded structure in
that region. Again, this may have been caused by foldback formation or
some bimolecular annealing. If short foldbacks were the major cause of
the pause, we predicted that an ERE in this conformation would be
inefficient at binding ER in subsequent gel shift experiments.

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Figure 4. Phosphodiesterase Treatment of Quick-Chilled F2 and
B2
The DNA sequences (Fig. 1 ) consist of single-stranded 36-nucleotide
oligomer (36), a 15-nucleotide marker oligomer, the F2 strand of the
ERE-containing oligomer, the B2 strand of the ERE-containing oligomer,
and F2 mixed with an equimolar ratio of B2 to create fully duplex ERE
(ds2). All DNA was 5'-end labeled and subsequently quick chilled by
heating to 90 C for 3 min, followed by quenching in an ice bath. The
DNA was then subjected to digestion by snake venom phosphodiesterase,
loaded onto an 18% denaturing gel, electrophoresed, and exposed to
film as in Materials and Methods. The 15 nucleotide
digestion products were resolved as single spots in the 18% gel.
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Nuclease Digestion of Slow-Chilled F2 and B2 Strands Reveals
Double-Stranded Regions in Both Oligomers
Digestion of slow-chilled F2 and B2 (Fig. 5
) occurred with substantial stalling of
the phosphodiesterase, resulting in the persistence of products 28 to
36 nucleotides in size. These large products were prominent even after
30 min of digestion. The digestion pattern was clearly different when
the DNA was slow chilled compared with quick chilled. After 30 min of
digestion, no large quick-chilled B2 products remained, but most of the
slow-chilled B2 remained between 28 to 36 nucleotides long. This
implies that, while quick chilling of B2 did not produce intramolecular
double strands capable of stalling digestion, slow chilling resulted in
the formation of intermolecular double strands.

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Figure 5. Phosphodiesterase Treatment of Slow-Chilled F2 and
B2
The DNA sequences (Fig. 1 ) consist of single-stranded 36-nucleotide
oligomer (36), a 15-nucleotide marker oligomer, the F2 strand of the
ERE-containing oligomer, the B2 strand of the ERE-containing oligomer,
and F2 mixed with an equal amount of B2 to create the double-stranded
ERE oligomer (ds2). All DNA was 5'-end labeled and subsequently slow
chilled by heating to 90 C for 3 min, and was then allowed to come to
room temperature over 6 h. The DNA was then subjected to digestion
by snake venom phosphodiesterase for the indicated times, loaded onto a
12% denaturing gel, electrophoresed, and exposed to film as in
Materials and Methods. The sizes of the digestion
products are indicated in the figure. The 15 nucleotide digestion
products tended to comigrate as the fastest spot in the 12% gel.
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The digestion of F2 paused between 28 and 36 nucleotides, a process
seemingly independent of quick or slow chilling. However, where
quick-chilled F2 had a substantial amount of 15 nucleotide product
appearing after only 10 min, slow-chilled F2 had almost no 15
nucleotide product, even after 30 min (lane 19, Fig. 5
). Evidently the
slow-chilled F2 is a longer, more stable double-stranded product than
the quick-chilled F2, that cannot be easily digested into smaller
products. This is consistent with F2 adopting a shorter unimolecular
foldback conformation after quick chilling, and a larger, more stable
bimolecular double-stranded region after slow chilling.
ER Does Not Bind to Quick-Chilled Single-Stranded ERE, but Binds
Efficiently to Slow-Chilled Single-Stranded ERE
Gel shift assays were used to monitor ER binding to quick- or
slow-chilled DNA substrates. A comparison of how chilling rates
affected the formation of ER-ERE complexes was anticipated to reveal
whether single-stranded or double-stranded oligomers were
preferentially bound by ER. ER binding to quick-chilled DNA was
measured in lanes 112 of Fig. 6A
.
ER-specific antibody H222 was added to supershift ER-bound DNA. No
shift in the DNA was evident for quick-chilled single-stranded F1, B1,
F2, or B2 (lanes 18). Quick-chilled double-stranded oligomer ds1
bound ER (lanes 9 and 10). There was a substantial amount of unannealed
single-stranded DNA in ds2 oligomer after quick chill (lanes 11 and
12). This resulted in very weak binding of ER to ds2, which was
observed only in longer exposures of the gel. However, slow cooling
resulted in ER binding to all four single-stranded ERE sequences (Fig. 6B
, lanes 18). Slow-chilled F1 and F2 oligomers bound substantially
higher amounts of the receptor compared with slow-chilled B1 and B2.
Using H222 antibody, we confirmed that these complexes contained ER
(lanes 2, 4, 6, and 8). The amount of ER binding to slow-chilled
double-stranded oligomers ds1 and ds2 was also significantly enhanced
compared with quick chilled (Fig. 6
, A and B, lanes 912).

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Figure 6. ER Binding to Quick- or Slow-Chilled DNAs
The 5'-end-labeled single-stranded oligomers or 3'-end-labeled annealed
double-stranded oligomers were heated to 90 C and quick chilled on ice
(A) or slow chilled in an aluminum heat block to room temperature (B).
Ten femtomoles of either slow chilled or quick chilled
[32P] end-labeled DNA was added to an ER premix [600
fmol Panvera ER, 1.0 µg poly dI-C (Midland Certified
Reagents), and 40 µl TDPEK 100++ (see Materials and
Methods)] with (lanes 2, 4, 6, 8, 10, 12) or without (lanes 1,
3, 5, 7, 9, 11) an ER supershifting antibody H222, in a total reaction
volume of 50 µl. Reactions were incubated at 37 C for 15 min, and
loaded onto a 5% nondenaturing polyacrylamide gel, separated by
electrophoresis, and exposed to film overnight. The migrations of free
single-stranded ERE (ssERE), double-stranded ERE (dsERE), shifted
ER-ERE (ERERE), and supershifted ER-ERE (AbERERE) are
indicated.
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These results clearly show that efficient ER binding to initially
single-stranded EREs depends on whether or not the single-stranded
oligomers were quick or slow chilled. The results reflect whether or
not two strands have an opportunity to anneal, rather than remaining as
single strands with foldback structures. Figure 6
also demonstrates
that although quick chilling was unfavorable to the formation of
bimolecular, antiparallel annealed EREs, when double-stranded oligomers
are provided, quick-chill treatment allows some proportion of these
complementary strands to anneal to form double-stranded DNA. The
distribution of double and single strands was different when ds1 and
ds2 were subjected to quick chill. The amount of ER binding to these
two oligomers correlated with the amount of double-stranded ERE formed
during the quick-chill process. It was clear that ER binds only to
double-stranded ERE. Slow-chill treatment allowed bimolecular annealing
of single-stranded ERE-containing oligomers to form regions of double
strandedness, allowing ER to bind such bimolecular ERE. Slow chill also
allowed the formation of double-stranded ERE from complementary single
strands, increasing the amount of ER binding. From these results, it is
clear that slow-chill conditions that favor double-stranded bimolecular
ERE formation result in increased ER binding, and quick chill
conditions that favor unimolecular foldback structure formation result
in decreased ER binding.
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DISCUSSION
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Previous reports have described high-affinity binding of the ER to
single-stranded DNA, specifically to one of the two strands of an
ERE.
Here we present compelling evidence that ER does not bind
single-stranded DNA. We measured ER binding to either one or the other
strand of an ERE and to the double-stranded ERE sequence. First, each
oligomer was heated to effect complete melting, and then either slow or
quick chilled. We anticipated that when ERE-containing strands were
quick cooled they would predominantly form short intramolecular
foldback structures. When slow cooled, these strands would be more
likely to form dimers in which the symmetrical regions of the ERE
annealed to form longer segments of double helix. We then compared how
ER-ERE binding was affected by the process of melting and reannealing.
Rate of digestion with snake venom phosphodiesterase was used as
diagnostic of the degree of double strandedness, since this exonuclease
is greatly impeded on a double-stranded substrate. Results showed that
quickly chilled ERE-containing single strands had little
double-stranded character. In gel shift experiments these did not bind
ER. Slow-chilled ERE-containing strands had more double-stranded
character and bound correspondingly more ER. We conclude that the
amount of ER binding correlates to the amount of double stranded
character of an ERE sample.
Binding of ER to double-stranded DNA is supported by a number of
studies. X-ray crystal structures of the DNA-binding domain of ER
complexed with DNA showed that the DNA was in an annealed,
double-stranded form (3). A previous study by our group measured the
degree of unwinding of ERE-containing plasmid DNA caused by binding of
ER. Topoisomerase I relaxation assays detected only minor alterations
in double helical structure (10). Those results were not consistent
with a local melting of the ERE and could only be interpreted as ER
binding to double-stranded DNA. This prompted us to consider that a
single strand of an ERE-containing segment might, nevertheless, form a
double helical structure, causing misinterpretation of supposedly
single-stranded DNA binding data. The core consensus ERE is a
symmetrical sequence with an inverted repeat structure. Because of this
sequence feature, a single strand should be able to form a foldback.
Two identical strands would be expected to interact to form a
double-helical region with single-stranded tails (see Fig. 7
). We anticipated that ER binding to
single strands could be explained by protein interaction with one of
these structures. The foldback that results from intramolecular
annealing is only half as long as the natural ERE. However, the
bimolecular annealed region should strongly resemble a natural ERE core
sequence, making it a good candidate for forming a stable complex with
the ER. Our results showing almost no ER interaction with quick-chilled
single strands of ERE-containing DNA, expected to form the short
foldbacks, were consistent with this expectation. Slow cooling was
expected to favor the bimolecular interaction. Results showing greater
double-stranded character and ER binding to the same DNA after slow
cooling further support this interpretation.
Other investigators reported ER binding to one strand of the ERE, but
not the complementary strand (15, 16). The ERE sequences F1 and B1 that
we tested in this report are identical to those of Mukherjee and
colleagues (15) with extended flanking sequences added to the 5'- and
3'-ends. They reported that ER binding depended on the presence of a T
in the center of the spacer region of the inverted repeat, and that
changing the central nucleotide to an A abolished binding. However,
Lannigan and colleagues (12, 14) reported preferential ER binding to
the strand that contained an A in the central position and observed no
binding to the strand with a T as the central nucleotide. It is
possible that, for any given ERE, one strand is capable of binding ER,
and the second is inert, by virtue of the central nucleotide in the
ERE. To test this possibility, we synthesized two pairs of
complementary oligomers (F1 and B1, F2 and B2) that were identical,
except that the central nucleotide was exchanged between strands. We
made F1 with a central T, and B1 with a central A. Subsequent
experiments showed that ER was able to bind to all single-stranded
oligomers after slow-chill annealing. It is clear that an A-T exchange
in the center of the ERE is not necessarily the determinant of whether
or not ER binds.
Our results showed that ER is capable of binding either the forward or
backward strand of an ERE. Lannigan and colleagues (15), exploring the
requirements for ER binding, found that ER binding to various
single-stranded EREs depended, at least in part, on sequences flanking
the ERE. We suggest that sequence context surrounding the ERE, combined
with sequences within the ERE, acted in concert to determine whether or
not efficient bimolecular annealing occurred without deliberate quick
or slow chilling, producing double-stranded ERE sites for ER to
bind.
We observed double-stranded regions in the quick-chilled ERE contained
in oligomer F1 using the snake venom phosphodiesterase assay (Fig. 2
).
While it was clear that the process of quick chilling the DNA inhibited
intermolecular interactions, it did not entirely prevent associations
between certain strongly complementary sequences within the same
oligomer. This allowed us to test whether ER has at least some affinity
for unimolecular foldback structures. If ER binding to foldbacks
occurs, then dark ER-ERE bands should have appeared in Fig. 6A
, lanes
18. However, we detected no ER binding to quick-chilled
single-stranded EREs. We also found that ER bound best to these
ERE-containing oligomers only after slow chilling, consistent with our
hypothesis that the formation of double-stranded bimolecular EREs is
critical for ER binding.
A powerful diagnostic feature of the digestion with venom
phosphodiesterase is that it reveals the position of annealing.
However, the double-stranded regions detected by the nuclease digestion
did not correspond exactly with the annealed bases predicted by the
sequence of the oligomer. As the phosphodiesterase tracked in a 3'- to
5'-direction, it paused six nucleotides 3' of the anticipated
double-stranded region. A likely explanation for this observation is
depicted in Fig. 7
. As the nuclease approached the double-stranded
region, the front edge of the enzyme contacted the junction of both
strands, slowing forward progress. The active site of the
phosphodiesterase is contained within the protein structure, so the
point where digestion was slowed was 3' of the actual point of
annealing. Digestion through the double-stranded region depended on
transient unannealing of the two strands, providing an opportunity for
the phosphodiesterase to move ahead. As the double-stranded region
became shorter, the likelihood of strand separation increased, allowing
an increased rate of digestion. After three complementary nucleotides
remained, digestion continued unimpeded through the remainder of the
oligomer. The figure represents the digestion of only one strand,
although both strands would be digested by the enzyme from the 3'-end.
This model is similar to what is seen in deoxyribonuclease I (DNase I)
footprinting experiments, where steric hindrance caused by enzyme
structure enlarges the apparent footprint of a protein on the DNA
(17).
The three-dimensional structure of the DNA strand may have a strong
influence on whether the ERE can pair with a second identical strand
and bind ER. It has also been suggested that ER binding to
single-stranded EREs is dependent on putative intramolecular non-B-form
tertiary structure adopted by the DNA. The unusual structure of the
single-stranded DNAs was inferred from the anomalous migration patterns
of the DNAs in gel shifts (12). It is interesting to note, however,
that ER binding to single-stranded EREs depended on retention of the
specific ERE-inverted repeat sequence (15). If the structure of the DNA
was the determining factor for ER binding, then changes in the sequence
that retain the proper structure should also retain ER-ERE binding.
However, all deviations from the consensus ERE resulted in diminished
ER binding, an indication that the sequence, not the structure, is
critical for ER-ERE binding. This is consistent with the interpretation
that interaction of the inverted repeat regions to form a bimolecular
helix is critical to interaction with the ER. The structure adopted by
two annealed strands of identical sequence is novel: it consists of a
central double- stranded region bracketed by four single-stranded tails
(Fig. 7
). Anomalous migration patterns could easily result from such a
structure. Unfortunately, it is difficult to determine whether a DNA
sequence is truly single or double stranded using gel shift. The
results of the snake venom phosphodiesterase digestions allowed us to
locate double- and single-stranded regions with reasonable accuracy
within our oligomers.
We have shown that ER binding to an ERE-containing oligomer correlates
with its ability to anneal to an identical copy of itself in the region
of the inverted repeat. The annealing produces a canonical
double-stranded ERE with a single-base mismatch in the center of the
3-bp spacer region. We conclude that the ER binds to the double strand
version of the ERE.
 |
MATERIALS AND METHODS
|
---|
Highly Purified Human ER
Recombinant human ER
from baculovirus-infected
insect cells was purchased from Panvera Corporation (Madison, WI) at a
concentration of 5000 nM or 3400 nM for use in
gel shift assays. ER was not liganded.
DNA Oligomers
Both strands of all oligomers were synthesized and PAGE purifed
by Genosys Biotechnologies (The Woodlands, TX). The regions flanking
all sequences tested were carefully chosen to contain no more than
three adjacent nucleotides of the core consensus ERE (see Fig. 1
).
Single-stranded oligomers were 5'-end labeled by incorporation of
[32P] from
[32P]ATP using T4
polynucleotide kinase according to the manufacturers instructions
(New England Biolabs, Inc., Beverly, MA). All oligomers
were dissolved in TE (10 mM Tris, pH 8.0, 0.1
mM EDTA). The labeled, double-stranded constructs were
formed by annealing labeled, fully complementary strands in equimolar
amounts.
Treatment of Single-Stranded Oligomers
Labeled, single-stranded oligomers were heated to 90 C for 3 min
in an aluminum block, after which the block was allowed to cool to room
temperature over 6 h. These conditions, which we call "slow
chilled," were designed to promote bimolecular associations between
two identical DNA molecules.
In parallel, oligomers were heated to 90 C for 3 min and immediately
cooled on ice. This quick-chill method was employed to discourage
bimolecular annealing and favor intramolecular annealing.
Gel Shift Assays
Single-stranded oligomers were 5'-end labeled by incorporation
of [32P] from
[32P]ATP using T4
polynucleotide kinase. Equimolar amounts of complementary oligomers
were annealed and 3'-end labeled using
[32P] dGTP and
the Klenow fragment of Esherichia coli DNA polymerase I,
according to manufacturers instructions (New England Biolabs, Inc.). The specific activity of the labeled DNA was determined
as follows. One-fortieth volume of the labeling reaction was spotted on
a TLC plate (polyethyleneimine-cellulose, EM Science, Gibbstown, NJ),
and the plate was developed in an aqueous solution of 1 M
HCl and 0.1 M sodium pyrophosphate. The unincorporated
counts migrated with the solvent front, and the incorporated label
remained at the origin. The plate was cut into thirds and counted in
EcoScint A (National Diagnostics, Atlanta, GA). The incorporated counts
at the origin and the quantity of DNA used for the labeling reaction
were used to calculate the specific activity of the labeled oliomer.
Ten femtomoles of either slow chilled or quick chilled
[32P] end-labeled DNA were added to an ER premix [600
fmol of purified ER (Panvera Corp), 1.0 µg poly dI-C (Midland
Certified Reagents, Midland, TX), and 40 µl TDPEK 100++ (40
mM Tris-HCl, pH 7.5, 1.0 mM dithiothreitol, 0.5
mM phenylmethylsulfonyl fluoride, 1 mM
EDTA, 100 mM KCl + 20% glycerol + 0.1% NP-40 +100 µg/ml
BSA] with or without an ER supershifting antibody (H222, a gift from
Abbott Laboratories, Abbott Park, IL)) for a total
reaction volume of 50 µl. Reactions were incubated at 37 C for 15 min
and loaded onto a 5% nondenaturing polyacrylamide gel. Electrophoresis
was performed at 150 V for 1.5 h at room temperature in 1x 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 at -80 C with an intensifying screen (Fisher
Biotech Lightning Plus, Springfield, NJ).
Snake Venom Phosphodiesterase Assays
Either slow-chilled or quick-chilled [32P]
end-labeled DNA (60,000 cpm,
10 ng, 384 fmol) was digested with 15
ng (3 µl of a 1:400 dilution of 2 mg/ml) of snake venom
phosphodiesterase (from Crotalus durissus, Boehringer Mannheim, Indianapolis, IN) for up to 30 min. The reactions were
stopped at various time points by the addition of gel loading buffer
(98% deionized formamide, 10 mM EDTA, pH 8.0, 0.025%
xylene cyanol, 0.025% bromophenol blue). The reactions were heated to
90 C for 3 min, loaded onto a 12% or 18% denaturing polyacrylamide
gel, and separated by electrophoresis at 60 watts for 1 h. The gel
was dried at 80 C under vacuum, and exposed to film overnight at -80 C
with an intensifying screen.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Robert A. Bambara, Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 575 Elmwood Avenue, Rochester, New York 14642. E-mail:
robert_bambara{at}urmc.rochester.edu
This work was supported by NIH Grant HD-24459.
Received for publication June 23, 1998.
Revision received March 19, 1999.
Accepted for publication March 19, 1999.
 |
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