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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go. 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).

 
In Figs. 2Go and 3Go, 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. 2Go and 3Go). 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. 1Go) 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 1–5 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. 1Go) 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 1–5 nucleotide digestion products tended to comigrate as the fastest spot in the 12% gel.

 
Nuclease Digestion Indicates That Quick-Chilled F1 or B1 ERE Strands Exhibit Little Double-Stranded Character
The oligomers used in the experiment in Fig. 2Go 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. 2Go. Lanes 1–4 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 1–5 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. 3Go). Lanes 1–4 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. 2Go. 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. 2Go), 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. 7Go).



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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.

 
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. 1Go). 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. 4Go), similar to what was done in Fig. 2Go. 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. 1Go) 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 1–5 nucleotide digestion products were resolved as single spots in the 18% gel.

 
Nuclease Digestion of Slow-Chilled F2 and B2 Strands Reveals Double-Stranded Regions in Both Oligomers
Digestion of slow-chilled F2 and B2 (Fig. 5Go) 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. 1Go) 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 1–5 nucleotide digestion products tended to comigrate as the fastest spot in the 12% gel.

 
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 1–5 nucleotide product appearing after only 10 min, slow-chilled F2 had almost no 1–5 nucleotide product, even after 30 min (lane 19, Fig. 5Go). 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 1–12 of Fig. 6AGo. 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 1–8). 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. 6BGo, lanes 1–8). 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. 6Go, A and B, lanes 9–12).



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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 (ER•ERE), and supershifted ER-ERE (Ab•ER•ERE) are indicated.

 
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 6Go 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 7Go). 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. 2Go). 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. 6AGo, lanes 1–8. 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. 7Go. 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. 7Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Highly Purified Human ER{alpha}
Recombinant human ER{alpha} 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. 1Go). Single-stranded oligomers were 5'-end labeled by incorporation of [32P] from {gamma}[32P]ATP using T4 polynucleotide kinase according to the manufacturer’s 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 {gamma}[32P]ATP using T4 polynucleotide kinase. Equimolar amounts of complementary oligomers were annealed and 3'-end labeled using {alpha}[32P] dGTP and the Klenow fragment of Esherichia coli DNA polymerase I, according to manufacturer’s 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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