(Received for publication, July 6, 1994; and in revised form, September 12, 1994)
From the
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.
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
ACGTG 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.
The A,
T
, and A
series of plasmids and the other
control plasmids, 4(A
CA
CGTG),
4(A
C
A
CGTG), and
7(A
C
A
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
GCGC), was constructed by isolating a DNA fragment
containing the 4(A
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 ACGTG intrinsic bending sequence were
inserted into the TATA-C
AT plasmid (45) after
digestion with SphI and BglII.
For DNA bending
analysis using the circular permutation assay, the DNA fragments
bearing 4(ACGTG), 4(A
GCGC),
4(A
CA
CGTG), and
4(A
C
A
CGTG) were inserted into the
multiple cloning site(SmaI/BglII) of the PCY7 DNA
bending vector(46) .
To prepare CHO cell nuclear extracts, 2 packed cell volumes of 5
TEG buffer (1
TEG: 50 mM Tris-HCl
,
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
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.
To test whether binding of the
protein factor induced DNA bending, DNA fragments containing the mutant
sequence 4(ACA
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) .
The plasmids were transfected into CHO cells. Plasmids
containing multiple A or T
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
tracts consistently showed a higher level of transcription
activation than the plasmids containing two to eight T
tracts. The most powerful transactivator tested, 8A
,
exhibited an increase of approximately 100-fold in transcription
relative to the basal TATA-CAT (ATC0) plasmid. The 8A
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
ACGTG 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 CAT 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 tracts were cloned into a
TATA-C
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
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
AT RNA) was normalized by use of the major
late promoter (which produces a 300-nucleotide C
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
AT
cassette.
Figure 3:
CHO cell nuclear proteins bind
specifically and cooperatively to the ACGTG 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
, 4A
, and
8A
competitor DNAs prior to addition of the
P-labeled 8A
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
tracts
was set equal to 100%.
Figure 4:
A soluble nuclear factor which binds to
the ACGTG sequence is required for its transcription in a
cell-free system. Twenty-five ng of a circular plasmid containing the
8(A
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
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
). 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%.
Figure 5:
DNA
sequence is critical for binding to the ACGTG 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
CGTG, A
GCGC, or
4(A
CA
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(ACGTG), 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
GCGC, which effectively
induced DNA bending (Fig. 5A), binding of the proteins
to the A
CGTG intrinsic bending sequence is
sequence-specific rather than bend-specific.
Figure 6:
DNA bending contributes to transcription
activation by the binding protein. Different A tract
bending plasmids, 4(A
CGTG), 6(A
CGTG), and
4(A
GCGC), mutated plasmids (4(A
CA
CGTG), 4(A
C
A
CGTG), 7(A
C
A
CGTG), A
, A
, 6A
and
8T
), 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 ACGTG 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
ACGTG 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.
Figure 9:
DNA bending does not influence the
affinity of the protein for its recognition sequence. DNA fragments
containing 4(ACGTG) sequences were labeled and used in
competition gel mobility shift assays. Increasing molar excess of the
unlabeled competitor DNA fragments, 4(A
CGTG) and
4(A
CA
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%.
Although intrinsically bent DNA can directly activate
transcription by purified Escherichia coli RNA
polymerase(23) , the sequence AGCGC, 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 ACGTG 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
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 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 CAT 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.
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.