©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intrinsically Bent DNA in a Eukaryotic Transcription Factor Recognition Sequence Potentiates Transcription Activation (*)

(Received for publication, July 6, 1994; and in revised form, September 12, 1994)

Jongsook Kim Sherry Klooster David J. Shapiro (§)

From the Department of Biochemistry, University of Illinois, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Many eukaryotic transcription factors induce DNA bending on binding to their recognition sequences. DNA bending could play a structural role by altering contacts between the protein and DNA. Alternatively, DNA bending could play a more direct role in transcription activation. To distinguish between these possibilities, we inserted two to eight copies of the intrinsic bending sequence, AAAAAACGTG, into a minimal promoter containing only a TATA box. The intrinsic DNA bending sequence was a potent activator of transcription in both in vivo transfection experiments and in a cell-free transcription system. A protein binds to the intrinsic bending sequence with high specificity in gel mobility shift assays and was required for its transcription in cell-free extracts. The intercalator, distamycin, which eliminates the ability of the sequence to bend, specifically reduced its transcription by about 60%. Mutations in the sequence which abolished DNA bending reduced transcription by approximately 70% in vivo. Competition gel mobility shift assays showed that the transcription factor bound equally well to mutants in which DNA bending was abolished and to the intrinsic bending sequence. These data indicate that DNA bending can play a direct role in the activation of eukaryotic transcription.


INTRODUCTION

Most models for the action of eukaryotic transcription factors have emphasized their ability to bind to specific DNA sequences and to interact via protein-protein contacts with other transcription factors and with components of the basal transcription apparatus(1, 2, 3) . Protein-protein contacts between transcription factors bound at nonadjacent sites require distortion of the structure of the DNA helix by DNA looping (4, 5) or by DNA bending(6) . DNA bending may be due to either the intrinsic properties of special base sequences that are repeated in phase with the DNA helical repeat, or it may be induced by binding of a DNA-binding protein to its recognition sequence(6) . Intrinsically bent DNA is often located near functionally important regions of genes(7, 8, 9, 10, 11, 12) .

Studies in which DNA bending appears to play a structural or architectural role in bringing nonadjacent sites on the DNA into closer proximity provide the clearest demonstration of a role for DNA bending. This was first shown in experiments in which the bacteriophage integration host factor-induced DNA bend was replaced by either an intrinsic DNA bend or by a DNA bend induced by another protein (13, 14, 15, 16) . A structural or architectural role for protein-induced DNA bending has also been proposed from studies of viral and cellular transcription regulatory proteins such as HMGI/Y(17) , the lymphocyte enhancer factor (LEF-1)(18) , YY1(19) , and the mammalian sex determining factor SRY(18) . Instead of acting as direct and independent activators of transcription, these proteins appear to function by providing a proper architectural framework in which other transcriptional regulatory proteins can function.

A more direct role for DNA bending in prokaryotic transcription regulation is suggested by numerous studies(20, 21, 22, 23, 24, 25) , including those in which the binding site of the CAP protein is replaced by an intrinsic DNA bending sequence, or by the binding site of another protein which induces a similar degree of DNA bending(20, 21, 22, 23, 24) . The evidence implicating DNA bending in eukaryotic transcription activation is less direct. A DNA bending protein facilitates interaction of progesterone receptor with its recognition sequence(26) . The transcriptionally active jun-fos heterodimer and the transcriptionally inactive jun-jun homodimer bend DNA in opposite directions(27) . Differences in the direction of DNA bending have also been reported for homo- and heterodimeric complexes of c-Myc and Max (28, 29) and could potentially affect interaction with the TATA-binding protein complex, which bends DNA sharply on binding to the TATA box(30, 31) . Many upstream activator proteins (reviewed in (32) ), several high mobility group box transcription factors(17, 18, 33) , and several members of the steroid/nuclear receptor gene superfamily(34, 35, 36, 37, 38, 39, 40) induce DNA bending on binding to their recognition sequences. For these proteins, which are independent transcription activators, protein-induced DNA bending could play a direct role in transcription activation, as is seen in some prokaryotic systems(10, 20, 21, 22, 23, 41) . Alternatively, the protein-induced DNA bend could increase contacts between the protein and the DNA, helping to stabilize binding of the protein to its recognition sequence(42) .

To test these possibilities and to evaluate the role of intrinsically bent DNA, which is present in the promoter regions of the estrogen-regulated vitellogenin genes(8, 37) , we generated a series of bending plasmids containing phased multimers of the synthetic A(6)CGTG intrinsic DNA bending sequence(14) , linked to a minimal promoter, containing only a TATA box. Binding of a transcription factor to this sequence makes it a potent eukaryotic transcription activator both in vivo and in vitro. Since transcription factor binding did not alter the DNA conformation, we were able to make a direct assessment of the role of intrinsically bent DNA in factor binding and transcription. Mutants in this sequence which do not bend DNA show a 3-4-fold reduction in transcription. Similarly, eliminating the DNA bend through the use of the intercalating agent, distamycin, reduced transcription in our HeLa cell extracts by 2-3-fold. In competition gel mobility shift assays the transcription factor bound equally well to mutants in which DNA bending was abolished and to the original intrinsic bending sequence. This indicated that the ability of the intrinsic DNA bend to potentiate transcription was not based on enhancement of DNA binding. DNA bending therefore appears able to play a direct structural role in eukaryotic transcription, presumably by bending the DNA and facilitating contacts between the bound transcription factor and components of the basal transcription apparatus.


EXPERIMENTAL PROCEDURES

Plasmid Constructions

The series of A(6) and T(6) intrinsic bending plasmids were constructed by inserting a single copy of a 31-base pair oligonucleotide (TCGAGCACAACGTGAAAAAACGCGAAAAAAC) containing two A(6) tracts (or two T(6) tracts, depending on the direction of insertion) and a new DraIII site into the SalI site of the TATA box containing plasmid ATC0(43) . The most 3` A(6) and T(6) tracts begin 31 and 40 nucleotides upstream of the TATA box, respectively. A 10-base pair oligonucleotide (GTGAAAAAAC) containing only one A(6) tract (or T(6) tract) was ligated to itself so that it could form multimers and was inserted into the DraIII site(14) .

The A(9), T(9), and A series of plasmids and the other control plasmids, 4(A(2)CA(3)CGTG), 4(A(2)C(2)A(2)CGTG), and 7(A(2)C(2)A(2)CGTG) were generated by inserting one or more copies of the following blunt ended, 20-base pair double-stranded oligonucleotides AAAAAAAAAGAAAAAAAAAG, AAAAAAAAAAAAAAAAAAAA, AACAAACGTGAACAAACGTG, and AACCAACGTGAACCAACGTG into the SalI site of ATC0. The control plasmid, 4(A(6)GCGC), was constructed by isolating a DNA fragment containing the 4(A(6)GCGC) sequence from the plasmid HN (44) fragment and inserting it into the ATC0 plasmid.

For cell-free transcription studies DNA fragments containing zero to eight copies of the A(6)CGTG intrinsic bending sequence were inserted into the TATA-C(2)AT plasmid (45) after digestion with SphI and BglII.

For DNA bending analysis using the circular permutation assay, the DNA fragments bearing 4(A(6)CGTG), 4(A(6)GCGC), 4(A(2)CA(3)CGTG), and 4(A(2)C(2)A(2)CGTG) were inserted into the multiple cloning site(SmaI/BglII) of the PCY7 DNA bending vector(46) .

Cell Culture, Transfections, and CAT^1 Assays

Chinese hamster ovary (CHO) cells were grown at 37 °C in Dulbecco's modified Eagle's medium/F-12 media (Sigma) supplemented with 5% fetal bovine serum. Transfection was performed by calcium phosphate-DNA coprecipitation with glycerol shock(38, 43) . The cells were transfected with a total of 8 µg of DNA, including 2 µg of bending plasmid, 0.5 µg of TK luciferase plasmid as an internal standard, and 5.5 µg of carrier DNA (pTZ18U plasmid). 48 h after a 3-min shock in Hanks' balanced salt solution containing 20% glycerol, the cells were harvested in cold phosphate-buffered saline and broken by three rounds of freeze-thawing in 50-300 µl of TNE (40 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA). Cell debris was sedimented by centrifugation for 10 min at 4 °C in a microcentrifuge, and the supernatant was assayed for luciferase activity and for CAT activity by our quantitative mixed phase assay(47) . CAT activity is reported as counts/min/µg of protein in cell extracts. Protein concentrations were determined with Coomassie Blue reagent (Bio-Rad).

In Vitro Transcription

HeLa cell transcription extracts were prepared as we have described(48) . Standard in vitro transcription reactions (total volume, 30 µl) contained 20 mM Hepes, pH 7.9, 2 mM dithiothreitol, 6% glycerol, 3 mM MgCl(2), 50 mM KCl, 5 mM creatine phosphate, 20 units of RNasin (Promega), 0.1 mM ATP, 0.1 mM CTP, 3 µM UTP, 500 µM 3-O-methyl-GTP (Pharmacia Biotech Inc.), and 25 µCi of [P]UTP, 1 µg of pTZ18U plasmid(49) , and 0.5 µg of sonicated herring sperm DNA as carrier DNAs, 12.5-50 ng of reporter template, 6.25-25 ng of an adenovirus major late promoter (pML300) internal control template. Reactions were initiated by adding 3 µl (60 µg of protein) of HeLa cell nuclear extract and incubated for 50 min at 30 °C. At the end of the incubation, 120 units of RNase T(1) was added, and the reaction was incubated for 10 more min. For in vitro competition transcription experiments, all of the components were added except for the NTP mix and preincubated on ice for 10 min. The reaction was started by addition of the NTP mix. The reaction was terminated by addition of 180 µl of stop buffer (7 M urea, 1% SDS, 10 mM EDTA, 20 mM Tris, pH 7.9, 250 µg/ml yeast tRNA), extracted twice with phenol/chloroform/isoamyl alcohol, precipitated, and separated on 6% polyacrylamide-7 M urea gels. Quantitation of radioactive bands was carried out with a PhosphorImager (Molecular Dynamics). Transcription activity of the reporter template (R) was normalized using the internal control template (IC).

Gel Mobility Shift Assays

DNA fragments containing the eight A(6) sequence were obtained by digesting the plasmid DNA with HindIII and BglII, filled in with Klenow in the presence of [alpha-P]dCTP, fractionated on an acrylamide gel, and eluted overnight in TE. Competitor DNAs were prepared by digesting plasmids containing the intrinsic bending sequences, 2A(6), 4A(6), and 8A(6), with HindIII and BamHI, and electroeluting the fragments.

To prepare CHO cell nuclear extracts, 2 packed cell volumes of 5 times TEG buffer (1 times TEG: 50 mM Tris-HCl(3), pH 7.4, 7.5 mM EDTA, 10% glycerol) containing a mixture of protease inhibitors (50 µg/ml phenylmethylsulfonyl chloride, 0.5 µg/ml aprotinin, 5 µg/ml pepstatin, and 50 µg/ml leupeptin) were added to the cell pellet, and the cells were broken with a Dounce homogenizer. After centrifugation at 10,000 rpm for 10 s, the supernatant was discarded. The nuclear pellet was resuspended in two-thirds of the original packed cell volume of 5 times TEG containing 0.5 M KCl. After incubation for 20 min at 4 °C, the nuclear extract was sedimented at 46,000 rpm for 20 min, and the supernatant was retained.

Gel mobility shift assays were carried out as described(37, 38) , with minor modifications. Briefly, 10,000 cpm of the P-labeled DNA fragment was incubated for 15 min at room temperature with the following: CHO cell nuclear extract, 1 µg of sonicated salmon testis DNA, 2 µg of sonicated herring sperm DNA, 10% glycerol, 80 mM KCl, 15 mM Tris, pH 7.9, 0.2 mM EDTA, and 0.4 mM dithiothreitol in a final volume of 20 µl. In the competition experiment, the indicated amounts of unlabeled DNA fragments were preincubated with the reaction mixture for 10 min on ice before the labeled DNA probe was added. Radioactive bands were quantitated with a PhosphorImager.

DNA Bending Analysis: Circular Permutation Analysis

The PCY7-derived bending plasmids were digested with EcoRI, EcoRV, and HindIII, which generated DNA fragments of identical size which differ only in the location of the intrinsic DNA bending sequences and the mutated sequences. The samples were then fractionated by acrylamide gel electrophoresis and stained with ethidium bromide.

To test whether binding of the protein factor induced DNA bending, DNA fragments containing the mutant sequence 4(A(2)CA(3)CGTG), which does not induce DNA bending, located near the end or the middle of the DNA fragment(37) , were incubated with CHO cell nuclear extract and analyzed for DNA bending as we have described(37) .


RESULTS

An Intrinsic DNA Bending Sequence Activates Transcription in Vivo and in a Cell-free System

Phased multimers of A(6)CGTG and CACGT(6) tracts have been shown to be highly effective inducers of DNA bending(14, 50) . We constructed plasmids containing two to eight A(6)CGTG or CACGT(6) sequences, repeated in phase at 10-nucleotide intervals upstream of the Xenopus vitellogenin TATA box. Since A(6)N(4) or T(6)N(4) DNA sequences appear to induce an 18 ° bend in the DNA(14, 50, 51, 52) , the bending angles induced by the intrinsic bending sequences ranged from 36° to 144°. Plasmids containing the A(6) sequences on the same DNA strand as the TATA box were designated the A(6) series.

The plasmids were transfected into CHO cells. Plasmids containing multiple A(6) or T(6) tracts were potent transcription activators and exhibited a progressive increase in transcription with increasing numbers of intrinsic DNA bending sequences (Fig. 1). The plasmids containing two to eight A(6) tracts consistently showed a higher level of transcription activation than the plasmids containing two to eight T(6) tracts. The most powerful transactivator tested, 8A(6), exhibited an increase of approximately 100-fold in transcription relative to the basal TATA-CAT (ATC0) plasmid. The 8A(6) plasmid exhibited a level of transcription activation greater than the thymidine kinase promoter(53) , but less than the adenovirus major late promoter (Fig. 1).


Figure 1: An intrinsic DNA bending sequence A(6)CGTG is a powerful activator of eukaryotic transcription in CHO cells. The bending plasmids and two other standard plasmids (TKCAT and MLCAT) were cotransfected with the TK luciferase plasmid (used as an internal standard) into CHO cells. After 48 h the cells were harvested and assayed for CAT activity as described under ``Experimental Procedures.'' The data represent the mean ± S.E. for four independent transfections.



Since intracellular nucleosome formation and supercoiling could potentially alter bending of the circular DNA used in the transfections, we evaluated the ability of the intrinsic bending sequence to activate transcription in a cell-free system. The intrinsic bending sequences were cloned into a plasmid containing the C(2)AT sequence (45) and assayed in a HeLa cell extract(48) . With either linear or circular DNA templates, there was a gradual increase in transcription with increasing numbers of intrinsic bending sequences. Interestingly, the linear templates (Fig. 2, right panel) were almost four times more effective in activating transcription than the supercoiled circular templates (Fig. 2, left panel). These data demonstrate that in both intact cells and in a cell-free extract the intrinsically bent DNA is a potent activator of eukaryotic transcription.


Figure 2: An intrinsic DNA bending sequence is a potent activator in a cell-free transcription system. DNA fragments containing 0-8 A(6) tracts were cloned into a TATA-C(2)AT plasmid(45) . Transcription reactions contained NTP mix, carrier DNAs, 6.25 ng of major late template, and 25 ng of a covalently closed circular template (left panel) or linear fragments of PstI-digested DNA (right panel), including the 400-500-nucleotide fragment containing the bending sequence, TATA box, and C(2)AT cassette. In vitro transcription experiments were performed as described under ``Experimental Procedures.'' In each sample, transcription of the reporter template (R) (which is transcribed to yield a 370-nucleotide C(2)AT RNA) was normalized by use of the major late promoter (which produces a 300-nucleotide C(2)AT RNA transcript) as an internal control template (IC). The data in the lower panel represent normalized synthesis of the 370-nucleotide RNA transcript from the C(2)AT cassette.



A Nuclear Protein Binds Specifically to the A(6)CGTG Intrinsic DNA Bending Sequence

In preliminary experiments we demonstrated that a protein in nuclear extracts from CHO, HeLa, and MCF7 cells, but absent in COS cell extracts, bound specifically to A(6)CGTG multimers (data not shown). The three gel-shifted bands we observed (Fig. 3, top panel) were most likely due to different levels of occupancy of the multiple A(6) binding sites. To determine if the increasing ability to activate transcription we observed with increasing numbers of A(6) tracts ( Fig. 1and Fig. 2) correlated with increased binding by the nuclear protein, we carried out competition gel mobility shift assays under conditions in which the samples contained the same total number of A(6) tracts. The most highly bent DNA fragment, the 8A(6) fragment was the most effective competitor. The DNA fragment containing 4A(6) tracts was also an effective competitor, reducing binding by 50% at a 12.5-fold molar excess (Fig. 3, lower panel). In contrast, even at a 100-fold molar excess the DNA fragment containing 2A(6) tracts did not reach 50% inhibition of binding. These data are consistent with the effect of increasing numbers of copies of the A(6) sequence on transcription.


Figure 3: CHO cell nuclear proteins bind specifically and cooperatively to the A(6)CGTG intrinsic bending sequence. Reaction mixtures were preincubated for 10 min on ice in the presence of 15 µg of CHO cell nuclear extract and the indicated amounts of unlabeled 2A(6), 4A(6), and 8A(6) competitor DNAs prior to addition of the P-labeled 8A(6) DNA fragment. The upper panel is an autoradiogram showing the protein-DNA complexes. The intensity of the bands was quantitated with a PhosphorImager and is plotted in the lower panel as the average intensity of the three bands. Binding in the absence of competitor A(6) tracts was set equal to 100%.



Transcription Activation Requires a Soluble Trans-acting Factor Which Binds to Multimers of the A(6)CGTG Sequence

To test whether the protein binding to the A(6)CGTG intrinsic bending sequence played a role in transcription activation we carried out a competition experiment in the cell-free transcription system. Increasing amounts of the unlabeled 8(A(6)CGTG) DNA fragment progressively inhibited transcription of the reporter DNA containing the 8A(6) sequence (Fig. 4), but not of the adenovirus major late promoter (Fig. 4, upper panel, IC). These data indicate that the nuclear protein which binds specifically to the A(6)CGTG sequence in gel mobility shift assays is essential for transcription activation.


Figure 4: A soluble nuclear factor which binds to the A(6)CGTG sequence is required for its transcription in a cell-free system. Twenty-five ng of a circular plasmid containing the 8(A(6)CGTG) intrinsic bending sequence was used as a reporter template (R) and 6.25 ng of major late plasmid was used as an internal control template (IC). Increasing amounts of an unlabeled DNA fragment containing the 8(A(6)CGTG) sequence were added into an in vitro transcription reaction mixture as a competitor. All of the reaction components (except the NTP mix), including template and competitor DNAs and HeLa extract, were combined and preincubated on ice for 10 min. The reaction was initiated by the addition of the NTP mix. Background transcription by a control template in the absence and presence of competitor is shown in the first two lanes (OA(6)). Transcription from the bending template was normalized to that of the major late template and is presented relative to the radioactivity from the reaction without unlableled competitor (third lane), which is set equal to 100%.



DNA Bending Enhances Transcriptional Activation by the Sequence-specific Binding Protein

These studies (Fig. 1Fig. 2Fig. 3Fig. 4) do not discriminate between the possibility that the protein recognizes and binds to a bent DNA structure, the possibility that the protein exhibits sequence-specific interaction with the bending sequence, and the possibility that a transcription activator protein binds to the multiple AT-rich sequences. Our observation using circular permutation analysis (37) that binding of the factor to its recognition sequence did not induce DNA bending (data not shown) greatly simplified our analysis of potential mechanisms. Since binding of the factor did not induce DNA bending, we were able to evaluate the effects of intrinsic DNA bending on factor binding and transcription in an experimental system in which binding of the protein does not alter DNA bending. To evaluate these hypotheses we prepared several variants of the A(6)CGTG intrinsic bending sequence. Multimers of (A(9)N(1))(n), and long homopolymeric A:T tracts with the sequence (A)(n), which cause only a very slight deformation of the DNA structure(49) , were cloned into the minimal promoter at the same site used for the A(6)CGTG sequence. To determine the contributions to transcription activation of the specific nucleotide sequence and of the distortion of the DNA structure produced by the intrinsic DNA bend, we also constructed plasmids which contain DNA bends, or binding sites for the nuclear protein, or both. We employed a widely used gel electrophoresis assay (11, 14) to measure DNA bending. The reduced electrophoretic mobility of DNA fragments containing the intrinsic binding sequences 4(A(6)CGTG) and 4(A(6)GCGC) near the middle indicates that they induce significant DNA bending (Fig. 5A). In contrast, the sequences 4(AACAAACGTG) and 4(AACCAACGTG) exhibited minimal DNA bending (Fig. 5A).


Figure 5: DNA sequence is critical for binding to the A(6)CGTG intrinsic DNA bending sequence. In A the ability of several mutant DNA sequences to bend the DNA was analyzed by circular permutation analysis. The DNA sequences to be tested were cloned into the circular permutation vector pCY7. When these plasmids are cut with the restriction enzymes EcoRI (lanes a, d, g, and j), EcoRV (lanes b, e, h, and k), and HindIII (lanes c, f, i, and l), three fragments of identical length are generated, which differ only in the location of the intrinsic bending sequence. The digested DNA fragments were fractionated by electrophoresis and stained with ethidium bromide. In B gel mobility shift assays with P-labeled DNA fragments containing the A(6)CGTG, A(6)GCGC, or 4(A(2)CA(3)CGTG) sequences in the presence of CHO cell nuclear extracts were used to analyze the ability of these sequences to bind the factor.



We then examined the ability of the nuclear protein to bind to these DNA sequences in gel mobility shift assays. Protein-DNA complexes were only detected with the DNA fragments in 4(A(6)CGTG), 4(AACAAACGTG) (Fig. 5B), and 4(AACCAACGTG) (data not shown), which all contained the potential protein binding motif ACGTGA. Since the protein binds to two fragments, 4(AACAAACGTG) and 4(AACCAACGTG), which are ineffective in inducing DNA bending and failed to bind to A(6)GCGC, which effectively induced DNA bending (Fig. 5A), binding of the proteins to the A(6)CGTG intrinsic bending sequence is sequence-specific rather than bend-specific.

Abolition of DNA Bending Reduces Transcription

In transient transfections all of the sequences which contained homopolymeric A or T tracts were ineffective in activating transcription (Fig. 6, A, A, 6A(9), and 8T(9)). The 4(A(6)GCGC) sequence which is effective in inducing DNA bending, but does not bind the nuclear transcription factor, was unable to activate transcription (Fig. 6), indicating that DNA bending alone is insufficient for transcription activation. The sequences which contained the factor binding site, but had little or no ability to induce DNA bending, all showed a 3-4-fold reduction in their ability to activate transcription relative to the A(6)CGTG sequence (Fig. 6, compare 4(A(6)CGTG) and 6(A(6)CGTG) to 4(A(2)CA(3)CGTG), 4(A(2)C(2)A(2)CGTG), and 7(A(2)C(2)A(2)CGTG). These data suggest that the presence of a bent structure adjacent to the transcription factor recognition sequence is important to its activity.


Figure 6: DNA bending contributes to transcription activation by the binding protein. Different A(6) tract bending plasmids, 4(A(6)CGTG), 6(A(6)CGTG), and 4(A(6)GCGC), mutated plasmids (4(A(2)CA(3)CGTG), 4(A(2)C(2)A(2)CGTG), 7(A(2)C(2)A(2)CGTG), A, A, 6A and 8T(9)), and the control ATC0 plasmid were transfected into CHO cells and assayed as described under ``Experimental Procedures.'' The activities represent the mean ± S.E. of four independent transfections.



The experimental data from the DNA bending analysis, the gel mobility shift assays, and the transfections are summarized in Fig. 7.


Figure 7: Summary of DNA bending and transcription activation by the various DNA sequences. The solid squares indicate the protein activators binding to the ACGTGA sequence, DNA bends are shown as a curved shape, and the transcription initiation complex is represented as a multicomponent complex. Transcription is presented relative to the basal level of ATC0, which was set equal to 1.



Because the two mutations which abolish DNA bending and reduce transcription by approximately 70% are in the middle of the oligo(A) tract, these mutations inevitably resemble each other. To avoid the possibility that the changes in the nucleotide sequence of the mutations, rather than their failure to bend the DNA, were responsible for the loss of transcription, we used a system in which the ability of the intrinsic DNA bending sequence to bend DNA was altered biochemically and assayed in the cell-free transcription system.

The oligopeptide antibiotic, distamycin, disrupts the structure of the DNA bend by preferentially binding to the oligo(A) sequence(6, 55) . It has been reported that at low doses, distamycin does not abolish protein-DNA interactions and selectively prevents DNA bending(6) . Low levels (6.6-20 ng) of distamycin, which abolish DNA bending, selectively inhibited transcription from the intrinsic bending template (Fig. 8). Since the data plotted represent the ratio of transcription of the intrinsic bending sequence to transcription of the adenovirus major late promoter internal control (Fig. 8, lower panel), it represents specific inhibition of transcription from the intrinsic bending sequence. These data lend additional support to the view that the ability of the intrinsic DNA bending sequence A(6)CGTG to induce DNA bending plays an important role in its ability to activate transcription.


Figure 8: Distamycin, which prevents DNA bending, specifically interferes with cell-free transcription of the A(6)CGTG sequence. Reaction mixtures containing both reporter and internal control templates, NTP mix, and increasing amounts of distamycin were preincubated on ice for 10 min and then the reaction was initiated by the addition of HeLa nuclear extract. In the lower panel, relative transcription (R/IC) is plotted as a function of increasing amounts of distamycin.



Abolition of DNA Bending Does Not Alter the Affinity of the Transcription Factor for the Recognition Sequence

Our data did not distinguish between the view that DNA bending acts by increasing the affinity of the nuclear transcription factor for its recognition sequence and the idea that DNA bending acts more directly to facilitate protein-protein contacts important in the assembly or function of the transcription complex. We therefore used competition gel mobility shift assays to determine if a mutation which virtually abolished DNA bending, and reduced transcription activation by 3-4-fold, altered the affinity of the nuclear protein for the 4(A(6)CGTG) intrinsic bending sequence. Increasing amounts of the unlabeled DNA fragments, 4(A(6)CGTG) and 4(A(2)CA(3)CGTG), were added as competitors. The competition curves for the 4(A(6)CGTG) DNA fragment, which both induces DNA bending and effectively activates transcription, and the 4(A(2)CA(3)CGTG) DNA fragment, which does not induce DNA bending and is 3-4-fold less effective in activating transcription, were virtually identical (Fig. 9, lower panel). These data clearly demonstrate that reduced transcription activation on abolition of DNA bending is not due to a reduced affinity of the transcription factor for the unbent DNA fragment and strongly suggest that DNA bending contributes in a more direct way to transcription activation.


Figure 9: DNA bending does not influence the affinity of the protein for its recognition sequence. DNA fragments containing 4(A(6)CGTG) sequences were labeled and used in competition gel mobility shift assays. Increasing molar excess of the unlabeled competitor DNA fragments, 4(A(6)CGTG) and 4(A(2)CA(3)CGTG), were added. After incubating for 15 min at room temperature, protein-DNA complexes were separated from the free probe by gel electrophoresis. Radioactivity of the three gel bands whose mobility was shifted was quantitated by PhosphorImager and is plotted relative to increasing amounts of unlabeled competitor DNA. The data were normalized to the radioactivity of the reaction without competitor, which was set equal to 100%.




DISCUSSION

Transcription Activation by the Intrinsic DNA Bending Sequence Is Mediated by Binding of a Protein Transcription Factor

Insertion of multimers of the sequence A(6)CGTG upstream of a TATA box is sufficient to create a powerful promoter in both intact cells and in cell-free extracts ( Fig. 1and Fig. 2). Transcription activation by the A(6)CGTG sequence might be due to the presence of multiple AT-rich sequences near the TATA box. Binding of alpha-subunit of bacterial RNA polymerase to an AT-rich region is important in prokaryotic transcription(56) . AT-rich sequences are found in several bacterial and yeast promoters(10, 57) . However, plasmids containing multimers of the A(9)N(1) and T(9)N(1) sequences and the A and A sequences did not activate transcription (Fig. 6), indicating that transcription activation by the A(6)N(4) and T(6)N(4) tracts did not result from either structural deformation of the DNA (57) or from activator proteins binding to the AT-rich sequences.

Although intrinsically bent DNA can directly activate transcription by purified Escherichia coli RNA polymerase(23) , the sequence A(6)GCGC, which retained the ability to bend DNA ( (44) and Fig. 5A), failed to bind the factor, and did not activate transcription (Fig. 5B and Fig. 6). These data demonstrate that intrinsically bent DNA is not an independent activator of transcription in a simple eukaryotic promoter.

Our observation that the intrinsic bending sequence can titrate out an essential factor required for transcription by the A(6)CGTG sequence lends strong support to the view that a protein transcription factor binds specifically to the intrinsic bending sequence and is responsible for its ability to activate transcription. The dramatically greater ability of multimers of the A(6)CGTG sequence to act as competitors in gel mobility shift assays is consistent with both transfection and cell-free transcription data showing that multimers of the sequence are far more effective in stimulating transcription ( Fig. 1and Fig. 2). Also, our observations that moving the intrinsic bending sequence an additional 55 or 110 nucleotides upstream had little effect on its ability to activate transcription and that changing the phase of the bend by inserting an additional 5 or 6 nucleotides reduced transcription by approximately 50% (data not shown), are consistent with the idea that a protein transcription factor binds to the intrinsic bending sequence. This conclusion is also supported by our data that all of our plasmids containing four to eight copies of the sequence ACGTGA show significant ability to activate transcription, and none of the plasmids which lack this sequence are able to activate transcription. Taken together, these data provide strong evidence that the ability of the intrinsic DNA bending sequence to activate transcription is based on binding of a sequence-specific transcription factor.

In scanning the DNA sequence data base for protein binding sites related to the binding motif in the intrinsic DNA bending sequence, we found only the sequence TCACGT which matches the ACGTGA sequence inverted. This sequence is located very close to the transcription initiation site (-23 to -27) in a chorion gene, is important in transcription activation(58) , and is conserved in several Drosophila chorion genes(58) .

The Intrinsic DNA Bend Is Important for Transcription Activation

One piece of data consistent with a role for DNA bending in transactivation is our observation that transcription of a linear DNA fragment containing the intrinsic DNA bending sequence is approximately four times more efficient than transcription of a supercoiled fragment (Fig. 2). Preferential transcription of a linear template is not a general feature of this cell-free system, as it is not observed with the benchmark Adenovirus major late promoter(47) . It is possible that the covalently closed circular template resists DNA bending. To more directly test the role of DNA bending in transcription activation, we constructed plasmids which contain interruptions in the A(6) tract and therefore have negligible ability to bend DNA, but preserve the factor binding site. In transient transfections, these mutants exhibited a 3-4-fold reduction in activity relative to the A(6)CGTG intrinsic bending sequence. Additional support for this conclusion stems from our observation that the intercalator, distamycin, which prevents the DNA from bending(6, 55) , specifically reduces transcription from the A(6)CGTG sequence 2-3-fold. Taken together, our data strongly support the view that the intrinsic DNA bend induced by the A(6)CGTG sequence plays an important role in transcription activation.

The interaction of a transcription factor with the basal transcription apparatus is often cell type and promoter specific. Our experiments were carried out with two different cell types, CHO cells and HeLa cells, and two different transcription initiation regions. The transfection experiments employ the Xenopus vitellogenin B1 promoter TATA box and its flanking DNA, whereas the C(2)AT cassette (45) used in the cell-free transcription studies uses the transcription start site from the adenovirus major late promoter. Since both the transfection and cell-free transcription studies yield similar effects of DNA bending, the data are likely to be generally applicable.

DNA Bending Affects Transcription Activation, Not Binding of the Protein to the Intrinsic Bending Sequence

DNA bending can alter protein-protein interactions by influencing the structure of the promoter region(17, 19, 59) . However, DNA bending near protein binding sites may increase or decrease protein binding(26, 54, 60) . To discriminate between effects of DNA bending due to altered affinity of the factor for its binding site rather than direct effects on transcription, we compared the ability of four copies of the A(6)CGTG intrinsic bending sequence and of the mutant sequence A(2)CA(3)CGTG to compete for binding in the gel mobility shift assay. The competition curves of the two DNA fragments were virtually identical (Fig. 9), demonstrating that the 3-4-fold reduction in transcription we observe with the A(2)CA(3)CGTG sequence is not due to a reduction in the affinity of the transcription factor for its recognition sequence. Since the A(2)CA(3)CGTG fragment exhibits identical binding to the transcription factor, greatly reduced ability to bend DNA, and a 3-4-fold reduction in its ability to activate transcription, the intrinsic DNA bend appears to play a significant role in the ability of the bound protein to activate transcription.

Whatever the mechanism by which DNA bending stimulates transcription, we have developed a system in which the effect of intrinsic DNA bending on transcription can be studied in the absence of protein-induced DNA bending. We demonstrated that the intrinsic bend in the DNA sequence potentiates transcription by severalfold. Finally, we find that in this system the intrinsic DNA bending does not stimulate binding of the transcription factor to its recognition sequence. This suggests that by altering the structure of the promoter region, DNA bending may bring the bound transcription factor closer to the basal transcription complex and thereby facilitate direct proteinprotein contacts with components of the basal transcription apparatus.


FOOTNOTES

*
This research was supported by Grant HD-16720 from NICHD. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801. Phone: 217-333-1788: Fax: 217-244-5858; djshapir{at}UIUC.edu.

(^1)
The abbreviations used are: CAT, chloramphenicol acetyltransferase; CHO, Chinese hamster ovary; TK, thymidine kinase.


ACKNOWLEDGEMENTS

We are grateful to A. Nardulli, M. Churchill, B. Kemper, R. Dodson, H. Kanamori, and K. Glenn for many helpful suggestions and for reading the manuscript, to Dr. M. Churchill for the gift of HN plasmid, and to Dr. R. Roeder for the gift of the C(2)AT plasmid.


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