Inhibition of Ets-1 DNA Binding and Ternary Complex Formation
between Ets-1, NF-
B, and DNA by a Designed DNA-binding Ligand*
Liliane A.
Dickinson
,
John W.
Trauger§,
Eldon E.
Baird§,
Peter
B.
Dervan§,
Barbara J.
Graves¶, and
Joel M.
Gottesfeld
From the
Department of Molecular Biology, The Scripps
Research Institute, La Jolla, California 92037, the
§ Division of Chemistry and Chemical Engineering and Beckman
Institute, California Institute of Technology,
Pasadena, California 91125, and the ¶ Huntsman Cancer Institute,
Department of Oncological Sciences, University of Utah School of
Medicine, Salt Lake City, Utah 84132
 |
ABSTRACT |
Sequence-specific pyrrole-imidazole polyamides
can be designed to interfere with transcription factor binding and to
regulate gene expression, both in vitro and in living
cells. Polyamides bound adjacent to the recognition sites for TBP,
Ets-1, and LEF-1 in the human immunodeficiency virus, type 1 (HIV-1),
long terminal repeat inhibited transcription in cell-free assays and
viral replication in human peripheral blood lymphocytes. The DNA
binding activity of the transcription factor Ets-1 is specifically
inhibited by a polyamide bound in the minor groove. Ets-1 is a member
of the winged-helix-turn-helix family of transcription factors and
binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by Ets-1, by occupying the adjacent minor groove. Full-length Ets-1 binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of NF-
B, and the Ets-inhibitory polyamide also blocks formation of ternary Ets-1·NF-
B·DNA
complexes on the HIV-1 enhancer. A polyamide bound adjacent to the
recognition site for NF-
B also inhibits NF-
B binding and ternary
complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are
powerful inhibitors of DNA-binding proteins that predominantly use
major groove contacts and of cooperative protein-DNA ternary complexes.
 |
INTRODUCTION |
Pyrrole-imidazole polyamides are a novel class of small molecules
that bind predetermined DNA sequences in the minor groove. Sequence-specific DNA recognition depends on side-by-side pairing of
pyrrole (Py)1 and imidazole
(Im) amino acids; a Py opposite an Im targets a C·G base pair, and an
Im opposite a Py targets a G·C base pair (1, 2). Py/Py,
Py/
-alanine (
), and
/
pairs binds both A·T and T·A base
pairs (2, 3). Recent studies have shown that A/T degeneracy can be
overcome by replacing one pyrrole ring of the Py/Py combination with
3-hydroxypyrrole (Hp), with the result that the Hp/Py prefers T·A
over A·T base pairs (4). In addition to their high specificity,
polyamides bind DNA with affinities comparable to or even higher than
those of natural DNA-binding transcription factors. Polyamides can be
designed to interfere with specific DNA-binding proteins and as a
result inhibit their function. We have shown that DNA binding of
transcription factor TFIIIA was inhibited by a polyamide that bound
within the recognition site of zinc finger four of this nine-finger
protein. TFIIIA is an important regulator of transcription of 5 S RNA
genes, and as a result, transcription of 5 S RNA genes was suppressed in vitro as well as in cultured Xenopus cells
with this specific polyamide (5). Mismatch polyamides, differing from
the match polyamide in either the sequence of Py and Im rings or a
single atom substitution, were without effect on either TFIIIA binding or transcription.
In an attempt to extend the application range of these small molecules
to messenger RNA/protein-coding genes, and to a medically relevant
system, specific polyamides were designed to interfere with
transcription of the human immunodeficiency virus type 1 (HIV-1) (6).
Transcription of HIV-1 is regulated by the 5' long terminal repeat
(LTR), which contains a series of cis-acting sequences responsible for
basal and inducible viral gene expression. These sequences are well
characterized and include recognition sites for upstream stimulatory
factor, the E-twenty six-specific (ets) family of proteins,
lymphoid enhancer-binding factor-1 (LEF-1), the nuclear factors
NF-
B, Sp1, and the TATA box-binding protein (TBP) (reviewed in Ref.
7). TBP is indispensable for initiation of transcription, and LEF-1,
considered to be an architectural protein, plays a central role in
coordinating activities of multiple transcription factors. LEF-1 was
shown to bend DNA, which facilitates protein-protein interactions
between transcription factors bound at distant sites in enhancers
(8-12). A polyamide was designed to bind a 7-base pair sequence
located on each side of the TATA box and immediately upstream of the
Ets-1 recognition site in the HIV-1 promoter (designated polyamide
1, see Fig. 1). This polyamide prevented TBP and Ets-1 from
binding to their recognition sites and blocked basal transcription from
the HIV-1 promoter, but not from an unrelated promoter, in
vitro. A second polyamide (designated polyamide 3, Fig.
1), which was targeted to a sequence immediately upstream from the
LEF-1-binding site and immediately downstream from the Ets-1-binding
site, prevented LEF-1 from binding and blocked activated transcription
in vitro. Polyamide 3, however, did not inhibit
Ets-1 DNA binding. Mismatch polyamides 2 and 4 had no effect on the DNA binding activity of TBP, LEF-1, or Ets-1. A
combination of the two polyamides together effectively blocked viral
transcription and replication in cultured human peripheral blood
lymphocytes (6). The transcriptional activity of a variety of major
T-cell-specific cellular genes (cytokine and growth factor genes) was
also examined, and the expression of none of these genes was affected
by the polyamides. Hence, it was concluded that inhibition of viral
replication was the result of direct interference of the polyamides
with transcription factor binding on the HIV-1 enhancer/promoter.
In the present study, we have focused on the differential inhibition of
Ets-1 DNA binding by two distinct Py-Im polyamides and on the
cooperative interaction between Ets-1 and the p50 subunit of NF-
B,
which is necessary for HIV-1 enhancer function (13). The ets
proteins are attractive candidates for polyamide targeting because of
their unique mode of binding to DNA; the recognition helix of the
winged helix-turn-helix domain is embedded in the major groove at the
center of the DNA-binding site, and loop regions flanking the
recognition helix are anchored to the phosphate backbone on both sides
of the DNA recognition site (14-16). So far, we have demonstrated
polyamide inhibition of DNA binding domains that exclusively contact
the minor groove of DNA, such as zinc finger four of TFIIIA (5), the
high mobility group domain of LEF-1, and the "saddle" of TBP (6).
Recently, it was shown that a polyamide containing a basic tripeptide
tail could inhibit the DNA binding activity of the basic helix-leucine
zipper protein GCN4 that contacts DNA exclusively in the major groove
(17). Since many transcriptional regulators contact DNA in the major groove with additional contacts in the minor groove and with the phosphate backbone, it is of interest to know the mechanism by which
polyamides can inhibit a member of this class of proteins.
Ets-1, like most other ets proteins, functions in
association with other proteins. Complex formation between
ets proteins and other factors can release the
autoinhibitory effects on DNA binding of full-length Ets proteins
(reviewed in Refs. 18 and 19). It was shown that Ets-1 physically and
functionally interacts with AP-1 in normal and activated T-cells (20).
Ets-1 does not bind the minimal T-cell receptor
-gene by itself, but
it binds DNA cooperatively with a factor called CBF
2 (11). Recently, Ets-1 was identified as a factor that physically associates with the
POU homeodomain protein GHF-1/Pit-1 to fully reconstitute prolactin
promoter activity (21). The p50 subunit of NF-
B was shown to
associate with Elf-1, and this association plays a role in regulating
cell type-specific and inducible expression of the interleukin 2 receptor
-chain gene (22). Significantly, physical interactions
between ets and NF-
B/NFAT proteins were shown to play an
important role in their cooperative activation of the HIV-1 enhancer in
T-cells (13). We show here that full-length Ets-1 and the p50 subunit
of NF-
B bind DNA cooperatively and form a ternary complex on the
HIV-1 enhancer. This cooperative binding is also effectively inhibited
by polyamide 1, which may be an important contributing
factor to the highly effective shut down of the HIV-1 promoter observed
in vivo (6). Taken together, these findings demonstrate that
the application range of the small Py-Im polyamides may be broader than
anticipated. Polyamides can inhibit DNA-binding domains that make both
major and minor groove DNA contacts, provided that the polyamide
interferes with the minor groove binding portion, and polyamides can
inhibit protein-protein-DNA ternary complexes, which may be an
important consideration in designing future therapeutic drugs.
 |
EXPERIMENTAL PROCEDURES |
Polyamides--
Polyamides were synthesized by solid phase
methods as described previously (23).
Ets-1 Protein Purification--
Recombinant full-length Ets-1
and the
N331 deletion polypeptide were expressed in bacteria and
purified as described previously (24, 25). The high affinity deletion
construct,
N331, contains the Ets-1 DNA-binding domain without the
auto-inhibitory regions and binds DNA with a 23-fold higher affinity
than native, full-length Ets-1 (25). Purified proteins were stored in
20 mM sodium citrate, pH 5.3, 1 mM EDTA, 500 mM KCl at 4 °C.
Gel Mobility Shift Assays--
Three sets of complementary
oligonucleotides were synthesized (Genosys Biotechnologies, Inc.) for
use as double-stranded probes in gel mobility shift experiments. A
43-bp oligonucleotide corresponded to positions
160 to
117 of the
HIV-1 enhancer and contained the binding sites for Ets-1, LEF-1, and
polyamides 1 and 3 in their natural sequence
context. A 38-mer corresponded to positions
160 to
144 of the HIV-1
enhancer, followed immediately by an NF-
B-binding site (Fig.
4A). This artificial probe contained the binding sites for
polyamide 1 and 3 and Ets-1 in their natural
sequence context, followed by the first NF-
B recognition site
located at position
104 to
92 of the HIV-1 enhancer. A 73-mer
oligonucleotide corresponded to positions
160 to
92 of the HIV-1
enhancer and contained the binding sites for polyamides 1 and 3, Ets-1, LEF-1, and NF-
B in their natural sequence
context plus four additional bases at the 3' end. Equimolar amounts of
two complementary oligonucleotides were combined and end-labeled with
[
-32P]ATP and T4 polynucleotide kinase. After
labeling, unincorporated nucleotides were removed using a Qiaquick
nucleotide removal kit (Qiagen). The labeled oligonucleotides were
annealed and used in gel mobility shift experiments at a final
concentration of 1 fmol/20 µl (50 pM). A 143-bp
polymerase chain reaction (PCR) product, corresponding to nucleotide
positions
175 to
33 of the HIV-1 LTR, was also used in gel mobility
shift experiments. Binding reactions were carried out in a 20-µl
reaction volume with 25 mM Tris/HCl, pH 7.9, 6 mM MgCl2, 65 mM KCl, 10 mM dithiothreitol, 0.5 mM EDTA, 10% (v/v)
glycerol, and 100 µg/ml bovine serum albumin. The reactions also
contained 0.5 µg of poly(dI)·poly(dC) as a nonspecific competitor.
Ets-1,
N331, and NF-
B p50 (Promega Corp.) were diluted in binding
buffer immediately before adding to the samples, which were then
incubated on ice or at ambient temperature for 20 min. The bound and
free DNA were resolved on a 6% non-denaturing polyacrylamide gel
containing 44 mM Tris borate, pH 8.3, and 1 mM
EDTA. The gels were dried and exposed to Kodak Biomax film. The results
were quantitated using an LKB Laser densitometer or with a Molecular
Dynamics PhosphorImager equipped with ImageQuant software.
DNase I Footprint Experiments--
The probe used in DNase I
footprint experiments was a 250-bp EcoRV/BglII
restriction fragment from the plasmid pHIV LTR-CAT (obtained from Dr.
K. A. Jones) (26), which was singly end-labeled at the 3' end of
the BglII site with the Klenow fragment of DNA polymerase.
Thus, the top strand was labeled. DNase I digestions were carried out
in a 50-µl reaction containing 50 pM labeled DNA and 1 µg of poly(dI)·poly(dC) in the same binding buffer that was used
for gel mobility shift experiments. The DNA was incubated with
polyamides for 20 min at ambient temperature, followed by addition of
N331, and incubation for 20 min on ice. DNase I digestion was
allowed to proceed in the presence of 2.5 mM
CaCl2 and 5 mM MgCl2 for 30 s
at ambient temperature with 66 × 10
3 or 33 × 10
3 units of DNase I (Roche Molecular Biochemicals) in
the samples with or without protein, respectively. Reactions were
stopped by the addition of 0.2% SDS and 10 mM EDTA,
extracted with phenol/chloroform, and precipitated with ethanol prior
to electrophoresis on a 6% denaturing polyacrylamide gel containing
8.3 M urea, 88 mM Tris borate, pH 8.3, and 2 mM EDTA. The dried gels were exposed to Kodak Biomax film
with DuPont Cronex Lightning Plus intensifying screens at
80 °C.
 |
RESULTS |
Polyamide-binding sites within the HIV Enhancer--
A set of four
Py-Im polyamides 1-4 (Fig.
1) was synthesized by solid phase
methods, and their structures have been published (6). Binding
affinities for each polyamide were previously determined by
quantitative DNase I footprinting experiments (6). Polyamide
1 (ImPy-
-ImPy-
-ImPy-
-ImPy-
-Dp; where
is
-alanine,
is
-aminobutyric acid, and Dp is
dimethylaminopropylamide) binds the sequence 5'-AGCTGCA-3' with an
equilibrium dissociation constant (Kd) of 0.05 nM, whereas the mismatch polyamide 2 (ImIm-
-ImIm-
-PyPy-
-PyPy-
-Dp) exhibits 100-fold lower affinity for binding that site. Polyamide 3 (ImPyPyPy-
-ImPyPyPy-
-Dp) binds the sequence 5'-AGTACT-3' with a
Kd of 0.06 nM, and the mismatch
polyamide 4 (ImPyPyPy-
-PyPyPyPy-
-Dp) binds the same
sequence with a 33-fold lower affinity.

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Fig. 1.
Polyamide and transcription factor-binding
sites in the HIV-1 promoter/enhancer. Top, DNA-binding
sites for transcription factors and polyamides 1 and
3 (indicated by numbers above vertical
arrows) in the HIV-1 promoter/enhancer. Nucleotide positions are
shown relative to the major start site for transcription (+1).
Middle, the double-stranded DNA sequence encompassing the
Ets-1 and LEF-1 recognition sites and flanking sequences is shown.
Polyamides are represented schematically between the two DNA strands at
their respective binding sites. The closed and open
circles represent Im and Py rings, respectively; the
diamonds represent -alanine ( ), and the curved
lines represent -aminobutyric acid ( ). The Ets-1- and
LEF-1-binding sites are boxed. DNA binding by Ets-1 involves
two types of sequence-specific DNA recognition; in the center of the
site, amino acids within the recognition helix directly contact the
5'-GGA(A/T)-3' core in the major groove, whereas backbone phosphates
are contacted on the two flanks (closed squares) (15, 16,
30, 31, 37). The selected consensus for an optimal Ets-1-binding site
is indicated below the duplex sequence (35). The bases of the
Ets-1-binding site are numbered, starting with 1 for the first G of the
GGA core (16). Bottom, binding models for mismatch
polyamides 2 and 4, where the mismatches are
boxed.
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Polyamide 1-binding sites are found in the HIV-1 promoter
and enhancer immediately flanking the TATA element and partially overlapping the upstream side of the Ets-1-binding site, at nucleotide positions
153 to
147 (relative to the start site for transcription at +1) (Fig. 1). The consensus recognition site for Ets-1 is located at
nucleotide positions
149 to
141 (Fig. 1). DNase I footprinting revealed additional binding sites for polyamide 1 located within adjacent vector DNA sequences (see Fig.
2C). Polyamide 3 binds to a
site at nucleotide positions
141 to
136 within the HIV-1 enhancer,
adjacent to the recognition sequence for LEF-1 (
135 to
126), and
partially overlapping the downstream side of the Ets-1 recognition site
(Fig. 1). Additional recognition sites for polyamide 3 are
found in the HIV-1 promoter/enhancer and coding sequence (see Ref. 6
and Fig. 2C). Therefore, both polyamides have the potential
to block DNA binding by Ets-1 in addition to LEF-1 and TBP.

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Fig. 2.
Inhibition of Ets-1 DNA binding by polyamide
1. A, polyamide 1 titration on the
N331·DNA complex. Autoradiogram of a representative gel mobility
shift assay showing the inhibitory effect of polyamide 1 on
N331 binding is shown. Positions of the free probe (F)
and bound probe (B) are indicated. The concentration of
N331 was constant (12 nM in lanes 2-10), and
the concentration of the 43-bp probe (position 160 to 117) was 50 pM. The polyamide concentrations were 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM in lanes 3-10,
respectively. The polyamide was preincubated with the DNA probe for 20 min at room temperature before addition of the protein, followed by
20-30 min incubation on ice. B, graphical
representation of the decrease of the fraction of bound probe as a
function of the polyamide concentration. Closed squares
represent the data points obtained for polyamide 1;
open squares represent data for polyamide 3. C, DNase I footprint titration experiment with polyamides 1 and 3 in the
absence ( ) or presence (+) of 9.6 nM N331 protein.
Polyamides were incubated with the radiolabeled probe prior to addition
of N331. The polyamide concentrations were 0 nM
(lanes 2, 6, 10, and 14), 4 nM
(lanes 3, 7, 11, and 15), 20 nM (lanes 4, 8, 12, and 16), and 100 nM (lanes 5, 9, 13, and 17).
Lanes 1 and 18 show a G + A sequencing ladder.
The regions protected by polyamides and by N331 are indicated
alongside the sequencing ladder. Note the two DNase I-hypersensitive
sites are characteristic of the N331 footprint.
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Differential Inhibition of Ets-1 DNA Binding by Polyamides
1 and 3--
We previously reported that the DNA
binding activity of the isolated high affinity DNA binding domain of
Ets-1 was specifically inhibited by polyamide 1 but not by
polyamide 3 (6). We wished to explore the basis for this
differential inhibition in detail. A set of two complementary
oligonucleotides corresponding to position
160 to
117 was
synthesized and end-labeled followed by annealing of both strands. This
43-bp probe contained the binding sites for polyamide 1, Ets-1, polyamide 3, and LEF-1. Gel mobility shift
experiments were done with a deletion peptide
N331, which contains
the DNA-binding domain but lacks the autoinhibitory regions present in
the full-length protein, and as a result binds DNA with substantially
higher affinity than full-length Ets-1 (24, 25). The reported
Kd for
N331 binding to a synthetic Ets site,
termed SC1, is 8.5 pM, a 23-fold higher affinity than that
observed for the full-length protein binding the same DNA probe (25).
When
N331 (at a concentration of 12 nM) was added to the
labeled, 43-bp HIV-1 oligonucleotide (at a concentration of 50 pM), approximately 80% of the probe was converted into a protein-DNA complex (Fig. 2A, lane 2). In separate protein
titration experiments with both the SC1 and HIV-1 probes, we found that
N331 had an ~10-fold lower affinity for the HIV-1 probe compared with the SC1 probe (data not shown). This difference in binding affinity is likely due to the different sequences of the Ets-1 sites
within these two probes rather than to the length of the two DNA probes
(23 bp for the SC1 probe versus 43 bp for the HIV-1 probe
and three nucleotide differences within the respective 9-bp Ets-1 sites
(25)). When polyamides were preincubated with the DNA probe before
adding
N331, polyamide 1 prevented Ets-1·DNA complex
formation (Fig. 2A, lanes 3-10). In contrast, polyamide 3 had no effect on Ets-1 DNA binding, even at a
concentration as high as 200 nM (Fig. 2B and
Ref. 6). When both polyamides were combined, the degree of inhibition
was very similar to that observed with polyamide 1 alone
(data not shown). Quantitation of these gel mobility shift experiments
revealed that polyamide 1 inhibited Ets-1
N331·DNA
complex formation by 50% at a concentration of approximately 6 nM, and nearly complete inhibition was achieved between 50 and 200 nM polyamide (Fig. 2B). Polyamide 3, and the two mismatch polyamides 2 and
4, had virtually no effect on
N331 DNA binding in the
same concentration range (Fig. 2B and Ref. 6).
Polyamide 1 Prevents Ets-1 DNA Binding, Whereas
Polyamide 3 Coexists with Ets-1 on Overlapping DNA-binding
Sites--
We employed DNase I footprinting to visualize the DNA
sequence contacts made by polyamides 1 and 3 and
by
N331. A labeled DNA fragment derived from the HIV-1 enhancer was
incubated with each polyamide either alone or followed by addition of
N331 and a further 30-min incubation. As expected, both polyamides bound their target sites within the HIV-1 enhancer with high affinity (Fig. 2C, lanes 3-5 and 11-13). Additional
sites for polyamide 1 are present in upstream vector DNA
sequence (lane 5), and additional sites for polyamide
3 are located both in the vector and in the HIV-1 enhancer
sequence (lane 13 and see Ref. 6). The Ets-1 footprint is
characterized by two DNase I-hypersensitive sites appearing in the
center and at the 5' boundary of the footprint (lanes 6 and
14). These hypersensitive sites disappear with the addition
of 20-100 nM polyamide 1 (Fig. 2C, lanes
7-9) but remain unchanged with the addition of polyamide 3 (lanes 15-17). The simultaneous presence of
polyamide 3 and
N331 results in a broadening of the
footprint, corresponding to a combined footprint created by
N331 and
polyamide 3 (lanes 15-17), whereas the Ets-1
footprint is replaced by the polyamide 1 footprint
(lanes 7-9).
Order of Addition Determines the Inhibitory Activity of Polyamide
1--
An order of addition experiment was performed to
determine whether polyamide 1 could disrupt a preformed
Ets-1·DNA complex (Fig. 3). Various
concentrations of polyamide 1 were added to the DNA probe,
in separate reactions, either 20 min before
N331, simultaneously
with
N331, or after incubation of the probe with
N331 for 30 min.
After an additional 20 min incubation, the reactions were subjected to
nondenaturing gel electrophoresis, and the fraction of DNA in the
Ets-1·DNA complex was determined by phosphorimage analysis. Fig. 3
shows the results of these experiments in graphical form. Polyamide
1 is substantially more inhibitory when added to the DNA
prior to
N331 than when added either simultaneously with
N331 or
after formation of the
N331·DNA complex. These results likely
reflect the similar affinities of
N331 and polyamide 1 for their respective target sites in the HIV-1 enhancer (see
"Discussion").

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Fig. 3.
Order of polyamide 1 addition determines the
inhibitory activity of the polyamide on N331
DNA binding. Gel mobility shift assays were carried out as
described in the legend to Fig. 2, where the indicated concentrations
of polyamide 1 were added to the DNA 20 min prior to adding
N331 (closed squares), or added simultaneously with
N331 (closed triangles), or N331 was incubated with
the DNA for 30 min prior to the addition of polyamide (open
squares). After a subsequent 20-min incubation, the reaction
mixtures were subjected to nondenaturing gel electrophoresis. The dried
gel was subjected to phosphorimage analysis, and the fraction of DNA in
the Ets-1·DNA complex in the presence of polyamide was plotted as a
function of polyamide concentration, normalized to the amount of DNA in
complex in the absence of polyamide (a value of 1.0).
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Full-length Ets-1 Binds DNA Cooperatively with the p50 Subunit of
NF-
B--
The previous binding experiments were done with the high
affinity deletion peptide
N331, which consists of the Ets-1
DNA-binding domain. Full-length Ets-1 binds to the HIV-1 probe with
extremely low affinity, due to the presence of autoinhibitory regions
in the protein (25). Ets-1 has been shown to form partnerships with a
number of different proteins, which appears to counteract these
negative auto-inhibitory effects (reviewed in Refs. 18 and 19). A
recent report showed that Ets-1 physically interacts with NF-
B/NFAT
proteins (13), and other studies have shown that the HIV-1 NF-
B
sites are bound by heterodimers of p50/p65 and p50/RelB, and by p50
homodimers (reviewed in Ref. 7). This prompted us to analyze whether
Ets-1 can bind cooperatively with the p50 subunit of NF-
B and form a
ternary protein-protein-DNA complex. To this end we constructed a
synthetic double-stranded oligonucleotide that contained the binding
sites for Ets-1 and polyamides 1 and 3 in the
natural sequence context and, in addition, a binding site for p50 in
close proximity to the Ets-1-binding site (Fig.
4A). In a gel mobility shift
assay, this probe was bound by full-length Ets-1 with very low
affinity; at the highest protein concentration used (272 nM), only 6% of the probe was bound (Fig. 4B,
lanes 10-15). To examine whether p50 and Ets-1 bind the
HIV-1 enhancer in a cooperative fashion, increasing concentrations of
Ets-1 were added to a constant but limiting amount of p50 (6 nM). Coincubation of p50 and Ets-1 yielded a complex that
migrated at a slower mobility than that formed with either p50 or Ets-1
alone (Fig. 4B, lanes 3-8). Significantly, ternary complex formation was detectable at the lowest concentration of
Ets-1 used, which did not yield a complex with Ets-1 alone (compare
lanes 3 and 10 in Fig. 4B).
Furthermore, at the maximal concentration of Ets-1 used in the presence
of p50, approximately 90% of the probe was shifted into the ternary
complex (lane 8), whereas less than 10% of the probe was
shifted when Ets-1 was used in the absence of p50 at the same
concentration (lane 15), and only 50% of the probe was
shifted when p50 was used alone (lane 2). When this same
experiment was repeated at a lower concentration of p50 (3 nM), only 13% of the probe was converted into the
p50·DNA complex in the absence of Ets-1 (Fig.
5, lane 3). The addition of
full-length Ets-1 had an even more dramatic effect on DNA binding under
these conditions (Fig. 5, lane 4). These data indicate that DNA binding by Ets-1 and p50 is highly cooperative.

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Fig. 4.
Full-length Ets-1 and
NF- B p50 bind DNA cooperatively and form a
ternary complex. Gel mobility shift experiment with a 38-bp
double-stranded oligonucleotide derived from the HIV-1 enhancer and
containing the recognition sites for polyamide 1, Ets-1, and
NF- B. The p50-binding site was moved close to the Ets-1-binding site
to facilitate interaction in vitro. A, sequence of the top
strand of the synthetic 38-bp probe with Ets-1, NF- B, and
polyamide-binding sites are indicated. B, gel mobility shift
assay with a constant amount of p50 (6 nM, 5.8 ng/20 µl)
and increasing concentrations of full-length Ets-1, compared with
full-length Ets-1 in the absence of p50. A plus or
minus sign at the top of the lanes indicates the
presence or absence of protein. The Ets-1 concentrations were 8.5, 17, 34, 68, 136, and 272 nM in lanes 3-8 and
10-15, respectively. The positions of the protein-DNA
complexes and the free probe (F) are indicated.
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Fig. 5.
Cooperative interaction of Ets-1 and p50 at
low concentrations of p50. A gel mobility shift assay with the
38-bp oligonucleotide probe was carried out as described in the legend
to Fig. 4, at a p50 concentration of 3 nM and at an Ets-1
concentration of 136 nM, where indicated. The positions of
protein-DNA complexes and free probe (F) are
indicated.
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Inhibition of Ternary Complex Formation by Polyamide
1--
We next examined whether the cooperative DNA binding
activity by combined p50 and Ets-1 proteins would still be susceptible to polyamide inhibition. When the DNA probe was preincubated with polyamides prior to incubation with proteins, the DNA binding activity
of p50 was not inhibited by either polyamide 1 or the
mismatch polyamide 2 (Fig. 6,
lanes 3-6). The small degree of DNA binding by full-length
Ets-1 was inhibited by polyamide 1 but not by polyamide
2 (Fig. 6, lanes 13-16). When p50 and Ets-1 were
added to the DNA probe after preincubation with polyamide 1, ternary complex formation was prevented, whereas p50 was still able to
bind the probe (Fig. 6, lanes 8 and
9). The mismatch polyamide 2 failed to inhibit
ternary complex formation (lanes 10 and 11).
These results confirm that Ets-1 is an integral part of the ternary
complex, and ternary complex formation can be prevented by blocking
Ets-1 access to its recognition site.

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Fig. 6.
Specific inhibition of ternary complex
formation by polyamide 1. A gel mobility shift assay with the
38-bp oligonucleotide probe was carried out as in Fig. 4. Lane
1 contained no added protein; lanes 2-6 contained p50;
lanes 7-11 contained both p50 and Ets-1; and lanes
12-16 contained Ets-1. The protein concentrations were
136 nM full-length Ets-1 and 6 nM p50.
Polyamide 1 was preincubated with the probe for 20 min at
ambient temperature at a concentration of 100 nM
(lanes 3, 8, and 13) and 200 nM
(lanes 4, 9 and 14); mismatch polyamide
2 was added at 100 and 200 nM in lanes 5, 10, 15 and 6, 11, 16, respectively. The positions of
protein-DNA complexes and the free probe (F) are
indicated.
|
|
A titration experiment showed that the polyamide concentration required
for 50% inhibition of the ternary complex was approximately 3-6
nM. Complete inhibition was achieved with a polyamide
concentration of 100-200 nM (data not shown). This
concentration range is similar to the concentration of polyamide
1 required for preventing the high affinity deletion peptide
N331 from binding to its recognition site (see Fig. 2B).
We also tested whether ternary complex formation could be prevented by
blocking p50 binding with polyamide 3. Polyamide 3 binds immediately upstream of the p50-binding site in the
38-mer DNA probe (Fig. 4A), and preincubation of the probe with polyamide 3 blocks binding of p50 and, hence, ternary complex formation (Fig. 7). However, a
much higher concentration of polyamide 3 than polyamide
1 is required to block ternary complex formation. As before,
Ets-1 binding is not affected by polyamide 3, and curiously,
the DNA binding affinity of full-length Ets-1 appears to be modestly
enhanced (approximately 2-3-fold) in the presence of polyamide
3. We do not have an explanation for this small level of
enhanced binding.

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Fig. 7.
Inhibition of ternary complex formation with
polyamides 1 and 3 on the 38-bp probe. Gel mobility shift assays
were performed as in Fig. 6 with increasing concentrations of
polyamides (Polyam.). Lanes 3-10 contained
polyamide 1, and lanes 13-20 contained polyamide
3 at the following final concentrations: 1.6, 3.1, 6.3, 12.5, 25, 50, 100, 200 nM, respectively. The positions of
protein-DNA complexes and the free probe (F) are
indicated.
|
|
Cooperative Binding and Polyamide Inhibition of Ets-1 and NF-
B
to the Native HIV-1 Enhancer Sequence--
Since the DNA probe used in
the previous gel mobility shift experiments was an artificial
construct, we next tested whether full-length Ets-1 and the p50 subunit
of NF-
B could bind cooperatively to the native HIV-1 enhancer
sequence and form a ternary complex. A double-stranded 73-bp
oligonucleotide, corresponding to positions
160 to
92 of the HIV-1
enhancer and containing the binding sites for polyamides 1 and 3, Ets-1, LEF-1, and NF-
B, was used as a probe in gel
mobility shift experiments. As before, in the absence of p50, Ets-1
bound to the 73-mer with very low affinity (Fig.
8A, lanes 2-5). In contrast,
coincubation of a constant concentration of p50 and increasing
concentrations of Ets-1 yielded a complex that migrated with a slower
mobility than that formed with p50 alone, and the intensity of this
ternary complex increased with increasing Ets-1 concentrations (Fig.
8A, lanes 6-10). These data indicate that full-length Ets-1
can bind the native HIV-1 enhancer cooperatively with NF-
B even
though the NF-
B and Ets-1 sites are separated by 35 bp in the native HIV-1 sequence. Additionally, polyamide 1 inhibits ternary complex formation by preventing Ets-1 binding without affecting p50
binding (Fig. 8B, lanes 5-7). Polyamide
3, which binds downstream of the Ets-1 recognition site and
30-36 bp upstream of NF-
B in the native HIV-1 sequence, does not
significantly inhibit ternary complex formation (Fig. 8B,
lanes 8-10). The
160 to
92 probe contained only one
binding site for NF-
B, whereas two sites are present in the native
HIV-1 enhancer sequence (Fig. 1). To assess whether polyamide
1 could also inhibit ternary complex formation on the full
native enhancer sequence, we used a 143-bp DNA probe corresponding to
nucleotide positions
175 to
33 of the HIV-1 enhancer/promoter
sequence. Similar to the results for the 73-bp probe (Fig.
8B), we find that polyamide 1 can inhibit
formation of the Ets-1·NF-
B·DNA complex with this longer probe
containing two NF-
B sites (Fig. 9). As before, in the absence of p50, full-length Ets-1 binds very weakly to
this DNA probe (lanes 2-4). In the absence of Ets-1, p50
forms two complexes on this probe (lane 5). The addition of
a 100-fold molar excess of unlabeled 143-bp HIV-1 DNA to the
p50-labeled DNA complex reveals that the faster migrating species is
nonspecific, whereas the slower migrating species is the specific
p50-DNA complex (lane 6). Interestingly, the nonspecific
complex is not observed in the presence of Ets-1 (lanes
7-9) and reappears when Ets-1 binding is inhibited by polyamide
1 (lane 12). It seems likely that Ets-1 can
recruit p50 from the nonspecific complex into the specific ternary
Ets-1·p50·DNA complex.

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Fig. 8.
Full-length Ets-1 and
NF- B p50 form a ternary complex with the
native HIV-1 enhancer sequence that is inhibited by polyamide 1. A, sequence of the top strand of the 73-bp DNA probe
(positions 160 to 92). B, gel mobility shift experiment
with increasing concentrations of full-length Ets-1 in the absence of
p50 (lanes 2-5) or in the presence of p50 (lanes
7-10). p50 was used at a constant concentration of 6 nM. The concentrations of Ets-1 were 17, 34, 68, and 136 nM in lanes 2-5 and 7-10,
respectively. C, inhibition of ternary complex formation
between Ets-1, p50, and the native HIV-1 enhancer sequence. The
proteins were added at constant concentrations of 136 nM
for Ets-1 and 11 nM for p50. The polyamide
(Polyam.) concentrations were 1 nM (lanes
5 and 8), 10 nM (lanes 6 and
9), and 100 nM (lanes 7 and
10). Free probe (F) and protein-DNA complexes are
indicated.
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Fig. 9.
Ternary complex formation and polyamide 1 inhibition of Ets-1 and p50 on the native HIV-1 enhancer sequence.
Gel mobility shift experiment with the 144-bp probe, corresponding to
nucleotide positions 175 to 33 of the HIV-1 LTR, and increasing
concentrations of full-length Ets-1 in the absence of p50 (lanes
2-4) or in the presence of p50 (lanes 7-9). p50 was
used at a constant concentration of 6 nM, where indicated
(+). The concentrations of Ets-1 were 68, 136, 272 nM in
lanes 2-4 and 7-9, respectively. The
concentration of Ets-1 in lanes 10-12 was 272 nM. The polyamide (Polyam.) concentrations were
50, 100, 200 nM in lanes 10-12, respectively.
In lane 6, a 100-fold molar excess of the unlabeled probe
was added as a specific competitor (Comp.). Free probe
(F) and protein-DNA complexes are indicated. The complex
with one p50 site occupied is denoted (p50)1 and
the complex with two occupied sites is denoted
(p50)2. The nonspecific p50-DNA complex observed
in the absence of Ets-1 is indicated by an asterisk.
|
|
 |
DISCUSSION |
Differential Inhibition of Ets-1 Binding by Two Polyamides Flanking
the Ets-1 Recognition Site in the HIV-1 Enhancer--
Ets-1 is an
important regulator of many promoters in lymphoid cells, including the
HIV-1 long terminal repeat. We show here that Ets-1 can be inhibited
from binding to its recognition sequence in the HIV-1 enhancer by a
small Py-Im DNA-binding ligand. Polyamides bind in the minor groove of
DNA and, significantly, can inhibit minor groove binding proteins by
binding to a recognition site that is adjacent to but does not coincide
with the recognition site of the DNA-binding protein (6). This is a
crucial requirement for achieving specificity in inhibiting binding of
ubiquitous transcription factors to certain promoters. We speculate
that polyamides, which bind B-type DNA, can inhibit minor groove
binding proteins such as TBP and LEF-1 because polyamide-bound DNA is likely resistant to the extensive bending and unwinding that
accompanies DNA binding by these transcription factors (12, 27-29). In
contrast, we document here that a Py-Im polyamide can also inhibit the
DNA binding activity of Ets-1, a winged helix-turn-helix protein that primarily contacts DNA in the major groove with additional adjacent phosphate contacts.
The Ets-1-binding site in the HIV-1 enhancer is flanked on both sides
by polyamide-binding sites, upstream by polyamide 1 and
downstream by polyamide 3. Both polyamide-binding sites partially overlap with the protein-binding site. However, only polyamide 1 blocks Ets-1 binding, whereas polyamide
3 has no effect, despite the fact that polyamide
3 binds even closer to the GGA recognition core than does
polyamide 1 (Fig. 1). This differential polyamide inhibition
of Ets-1 DNA binding can be explained within the context of the high
resolution model of ETS domain-DNA interactions. The ETS domain
displays a structural motif known as a winged helix-turn-helix which
includes three
helices and a
sheet (14-16, 30, 31).
Significantly, DNA contacts are confined to one face of the DNA helix.
Helix 3 of the helix-turn-helix motif contacts the 5'-GGA(A/T)-3' core within the major groove. A
sheet "wing" and a turn separating helices 2 and 3 are tethered to the phosphate backbone on both sides of
the major groove contacts made by the recognition helix (16) (Fig. 1).
Polyamide 3, which does not disrupt Ets-1 binding, interacts
in the minor groove just beyond the phosphate contacts made by the
wing. Polyamide 3 binds the symmetrical DNA sequence
5'-AGTACT-3' and can conceivably bind in either orientation, that is
either with its terminal
-alanine proximal to the Ets-1 site or its
"hairpin turn"
-aminobutyric acid proximal to the Ets-1 site.
Depending on the orientation of the polyamide, the
-alanine amide
will form a hydrogen bond to the O-2 of T
3 or the
-aminobutyric acid amide will form a hydrogen bond to the N-3 of
A
3' (32) (see Fig. 1). Although ets proteins
make a phosphate contact at T
3, neither of these
potential polyamide contacts is sufficient to block Ets-1 from binding.
On the other hand, an Im/Py pair and a Py/Im pair of polyamide
1 interact with G6'/C6 and
C5'/G5, thus interfering with the phosphate
contacts made by the ETS domain to nucleotides G6' and
C5' (Fig. 1). Since the polyamides do not make phosphate
contacts with DNA, it is likely that inhibition arises from a steric
blockage of the minor groove by the polyamide. These results indicate
that targeting sites adjacent to protein-binding sites may not be
sufficient for polyamide inhibition of certain DNA-binding proteins.
This approach was successful with TBP and LEF-1, likely because these factors extensively bend and unwind the DNA upon binding, whereas polyamides bound to DNA likely prevent this type of distortion. For
Ets-1, however, interference with phosphate contacts was necessary to
inhibit binding. These results demonstrate the importance of taking the
three-dimensional structure of the protein-DNA complex into
consideration when designing polyamides to disrupt a transcription factor-DNA interaction.
We find that the order of addition of polyamide 1 and Ets-1
(
N331) has a dramatic effect on the inhibitory action of the
polyamide (Fig. 3). More pronounced inhibition is observed if the
polyamide is allowed access to its DNA target prior to the addition of
N331 protein than if the protein-DNA complex is allowed to form
prior to the addition of polyamide. An intermediate result is obtained
if both polyamide and
N331 are added to the DNA simultaneously. This
dependence on order of addition likely reflects the similar affinities
of the two DNA ligands for their recognition sites:
N331 binds a
selected high affinity site with an equilibrium dissociation constant
of 8.5 pM (25) and binds the HIV-1 site with approximately
10-fold lower affinity. Similarly, polyamide 1 binds its
target site with a Kd of 50 pM. Given
these comparable binding affinities, which ever ligand has initial
access to its target site will be the primary bound species. In the
context of the living cell, it is likely that transcription factors are
displaced from promoter sequences during both DNA replication and
during mitotic chromosome condensation (33, 34); thus, polyamides would
have access to their target sites during these two phases of the cell
cycle providing a window of opportunity for binding.
Ternary Complex Formation of Full-length Ets-1 and NF-
B p50 on
Adjacent Binding Sites--
Full-length Ets-1 binds to the HIV-1
enhancer with very low affinity. This result is in agreement with
published reports showing that the DNA binding activity of many members
of the ets family is negatively regulated by autoinhibitory
regions present in the protein. It has been suggested that cooperation
with other factors counteracts the autoinhibitory effect of Ets-1
(reviewed in Refs. 18 and 19). Indeed, we also show that when
full-length Ets-1 is coincubated with the p50 subunit of NF-
B and a
DNA probe containing binding sites for both proteins, Ets-1 binds
cooperatively with p50 and forms a ternary complex that migrates with
slower mobility than either protein-DNA complex alone.
The cooperativity observed between Ets-1 and p50 is most likely the
result of direct physical interaction between the two proteins that are
brought to close vicinity by binding to adjacent sites on the same DNA
fragment. A recent report showed that Ets-1 physically interacts with
NF-
B/NFAT proteins in vitro and in vivo and
that this interaction requires the presence of DNA for both binding
sites (13). We also found that cooperative binding between Ets-1 and
p50 required a DNA probe containing a binding site for both proteins on
the same fragment. We could not detect cooperative binding with
oligomers that contained either the Ets-1 or p50 recognition site alone
nor with an equimolar mixture of both oligomers (data not shown).
Significantly, ternary complex formation is inhibited by polyamide
1, which prevents Ets-1 from binding to its recognition site. It is noteworthy that inhibition of Ets-1 DNA binding in the
ternary complex is achieved with the same concentrations of polyamide
1 as required for inhibition of DNA binding by the high
affinity deletion peptide
N331. This observation suggests that
full-length Ets-1 in the ternary complex binds to its DNA recognition
site with a similar affinity as the isolated high affinity deletion
peptide
N331.
The artificial probe that was used in these experiments contained a
polyamide 3-binding site upstream of the p50-binding site
(Fig. 4A). Polyamide 3 prevented ternary complex
formation as well, by preventing p50 from binding to its recognition
sequence. However, the concentration of polyamide 3 required
for inhibition of p50 was significantly higher than that required for
polyamide 1 inhibition of Ets-1. It is likely that a higher
concentration of polyamide 3 is required because of the
different modes of DNA recognition and binding utilized by these two
proteins (15, 35, 36). Ets-1 binds DNA as a monomer, whereas p50 binds
as a dimer; furthermore, the polyamide 3-binding site may be
too distant from the minor groove contacts made by p50, which are
located toward the center of the 10-base pair recognition site. The p50
dimer contacts DNA predominantly in the major groove, where the dimer
grips the DNA duplex like a set of jaws, whereas minor groove contacts
are limited to only two amino acid residues (36). It is therefore
likely that the location of the polyamide 3-binding site is
not optimal for inhibition of the minor groove contacts made by p50.
Cooperative Binding of Ets-1 and NF-
B p50 on Non-adjacent Sites
in the HIV-1 Enhancer--
We show that Ets-1 binds cooperatively with
NF-
B p50 not only on adjacent binding sites but also on the natural
HIV-1 enhancer sequence. In the HIV-1 enhancer sequence the binding
sites for Ets-1 and NF-
B are separated by approximately 35 base
pairs, with an intervening LEF-1-binding site. LEF-1 is considered an architectural protein that coordinates interactions between multiple proteins. For example, LEF-1 induces a sharp bend in the TCR
-gene enhancer, which facilitates interactions between Ets-1, CBF
2, and
ATF/CREB transcription factors bound at distant sites flanking the
LEF-1-binding site (11). However, we found that cooperativity between
Ets-1 and p50 on the HIV-1 enhancer did not require the presence of
LEF-1. Since both the ETS domain and the p50 dimer induce a moderate
bend in the DNA upon binding (15, 36), it is conceivable that the
combined effect of the two proteins on the DNA configuration is
sufficient to bring their binding sites in close proximity to allow
protein-protein interaction.
Numerous physical interactions between ets family members
and other transcription factors, including p50, have been described (reviewed in Ref. 19). However, very few studies have shown cooperative
DNA binding and ternary protein-protein-DNA complex formation between
Ets proteins and other transcription factors (11). Although direct
physical association between Ets-1 and p50 has been demonstrated (13),
this is the first report providing evidence for cooperative DNA binding
and ternary complex formation by Ets-1 and p50 on the HIV-1 enhancer sequence.
Cooperative interactions between transcription factors appear to be
important for regulation of gene expression in general, and it is
thought that these interactions may represent targets for novel
antiviral therapies. The in vitro experiments described in
this report, together with in vivo assays described
elsewhere (6), suggest that small Py-Im polyamides may represent the basis for developing novel therapeutic agents that target transcription.
 |
FOOTNOTES |
*
This work was supported by Grant GM-57148 from the National
Institutes of Health (to J. M. G. and P. B. D.), the National Science Foundation, the Ralph M. Parsons Foundation for predoctoral fellowships (to J. W. T.), and the Howard Hughes Medical Institute for a predoctoral fellowship (to E. E. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd.,
La Jolla, CA 92037. Tel.: 619-784-8913; Fax: 619-784-8965; E-mail:
joelg{at}scripps.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
Py, pyrrole;
HIV-1, human immunodeficiency type 1;
Im, imidazole;
Hp, hydroxypyrrole;
LTR, long terminal repeat;
USF, upstream stimulatory factor;
LEF-1, lymphoid enhancer-binding factor-1;
TBP, TATA box-binding protein;
Dp, dimethylaminopropylamide;
bp, base pair.
 |
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