ETS-1 Transcription Factor Binds Cooperatively to the Palindromic
Head to Head ETS-binding Sites of the Stromelysin-1 Promoter by
Counteracting Autoinhibition*
David
Baillat
,
Agnès
Bègue,
Dominique
Stéhelin, and
Marc
Aumercier§
From the CNRS Unité Mixte de Recherche 8526, Institut de
Biologie de Lille, Institut Pasteur de Lille, B.P. 447, 1 Rue
Calmette, 59021 Lille Cedex, France
Received for publication, January 4, 2002, and in revised form, April 22, 2002
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ABSTRACT |
Stromelysin-1 (matrix metalloproteinase-3) is a
member of the matrix metalloproteinase family. Regulation of its gene
expression is critical for tissue homeostasis. Patterns of increased
co-expression of stromelysin-1 and ETS-1 genes have been observed in
pathological processes. Stromelysin-1 promoter is transactivated by ETS
proteins through two palindromic head to head ETS-binding sites, an
unusual configuration among metalloproteinase promoters. By
using surface plasmon resonance, electrophoretic mobility shift assay,
and photo-cross-linking, we showed that full-length human ETS-1 (p51)
binds cooperatively to the ETS-binding site palindrome of the human
stromelysin-1 promoter, with facilitated binding of the second ETS-1
molecule to form an ETS-1·DNA·ETS-1 ternary complex. The
study of N-terminal deletion mutants allowed us to conclude that
cooperative binding implied autoinhibition counteraction, requiring the
245-330-residue region of the protein that is encoded by exon VII of
the gene. This region was deleted in the natural p42 isoform of ETS-1,
which was unable to bind cooperatively to the palindrome. Transient transfection experiments showed a good correlation between DNA binding
and promoter transactivation for p51. In contrast, p42 showed a poorer
transactivation, reinforcing the significance of cooperative binding
for full transactivation. It is the first time that ETS-1 was shown to
be able to counteract its own autoinhibition.
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INTRODUCTION |
Stromelysin-1 (matrix metalloproteinase-3) is a member of the
matrix metalloproteinase family with a wide spectrum of substrates. It
plays a crucial role in extracellular matrix remodeling during normal
processes such as tissue morphogenesis, growth, and wound repair (1,
2). A tight regulation of its gene expression is critical for tissue
homeostasis. Indeed, its misregulation is associated with pathologic
conditions such as rheumatoid and osteoarthritis (3, 4), Alzheimer's
disease (5), tumor invasiveness, and metastasis (6-8). In addition, it
was recently reported (9, 10) that stromelysin-1 by itself promoted
mammary carcinogenesis in a mouse model system. Stromelysin-1
expression is mainly controlled at the transcription level. A number of
specific DNA elements in the human stromelysin-1 promoter have been
shown to be important in the regulation of its transcription (11, 12).
Among them, two palindromic ETS-binding sites
(EBS)1 with a 5'-GGA(A/T)-3'
core motif at 
216 to 209 and at 208 to 201 bind ETS
transcription factors. This EBS palindrome is required for basal
expression and 12-O-tetradecanoylphorbol-13-acetate tumor
promoter response (13) and is highly conserved through species.
The ETS-1 oncoprotein, the founding member of the ETS family,
transactivates the rat stromelysin-1 promoter through this palindrome (14). These family members are important transcription factors involved
in development (15-18). They have also been implicated in several
types of cancer and other human diseases (19). All ETS proteins share a
conserved ~85-amino acid DNA binding domain (ETS domain) organized
into a winged helix-turn-helix motif (20).
Their specificity for target promoters is controlled at several levels.
Nevertheless, DNA binding modulation can be viewed as the first
transcriptional control. Binding specificity to a given EBS can be
partially provided by the flanking sequences surrounding the EBS core.
Further specificity occurs through combinatorial interactions of ETS
proteins with co-factors at adjacent DNA elements (21, 22). Additional
control is provided by autoinhibition (23). This phenomenon, mediated
by cis-acting inhibitory modules, negatively regulates DNA binding
through intramolecular interactions. ETS-1 represents the best studied
example (24-26). Its autoinhibition can be counteracted by interaction
with protein partners (27-31) resulting in a cooperative binding of
each molecule.
Given that on one hand the same pro-inflammatory factors as
interleukin-1
and tumor necrosis factor 
are able to activate stromelysin-1 and ETS-1 (32, 33) and on the other hand that patterns of
increased co-expression of these both genes have been shown in
processes such as rheumatoid arthritis (34), glomerulonephritis (35),
angiogenesis (36), and tumor invasion (37), there is a strong
presumption that misregulation of stromelysin-1 could be mediated by
ETS-1.
Previous work (13, 14) showed that both EBS of the palindrome acted
synergistically to transactivate stromelysin-1 gene promoter.
Nevertheless, in a heterologous system ETS-1 and ETS-2 showed no
cooperative binding to the palindrome (14). In contrast, other studies
proved that the functional EBS palindromes present in GATA-1 and p53
promoter (38, 39) cooperatively bound ETS-1 and ETS-2.
Given the growing importance of the EBS palindrome for the
transcriptional regulation of stromelysin-1 by ETS proteins (40-42), we reinvestigated the binding of ETS-1 to the stromelysin-1 promoter.
In this study, by using surface plasmon resonance (SPR),
electrophoretic mobility shift assay (EMSA), and photo-cross-linking experiments, we show that the full-length human ETS-1 protein (p51)
binds with a positive cooperativity to the EBS palindrome of the human
stromelysin-1 promoter. This cooperativity is seen exclusively under a
palindromic topology and leads to the formation of a ternary
ETS-1·DNA·ETS-1 complex. By using kinetic analysis and
N-terminal deletion mutants, we conclude that the observed cooperativity is because of a facilitated binding of the second ETS-1
molecule to form the ternary ETS-1·DNA·ETS-1 complex. Furthermore, this mechanism implies autoinhibition counteracting of ETS-1 through the 245-330-residue region of the protein encoded by the exon VII of
the gene. The p42 isoform of ETS-1, lacking only this region, is unable
to bind cooperatively to the palindrome, despite a better binding to
each EBS. Transient transfection experiments show for p51 a good
correlation between DNA binding and promoter transactivation. In
contrast, p42 shows a poorer transactivation reinforcing the significance of cooperative binding for a full ETS-1-mediated transactivation of the promoter. To our knowledge, this is the first
time that ETS-1 is shown to be able to counteract its own autoinhibition.
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MATERIALS AND METHODS |
Site-directed Mutagenesis--
Mutant forms of the human
stromelysin-1 promoter corresponding to M1, M2, M1M2, DR, and IP were
generated by site-directed mutagenesis using appropriate
oligonucleotides (Table I) and the QuickChange Site-directed Mutagenesis kit (Stratagene®) with a
pØGH plasmid (HGH-TGES, Nichols Institute Diagnostics) containing the
1303/+4 fragment of the wild type human stromelysin-1 promoter as a
template (13).
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Table I
Sequences of stromelysin-1 promoter, wild type, and mutants
The EBS core sequences are represented as boldface letters in the
sequence.  and  represent the EBS in both possible orientations,
and × designates a mutated EBS.
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Luciferase Reporter Gene Constructions--
Fragments 478/+4
of the wild type and mutated human stromelysin-1 promoters were excised
from the pØGH vector by XbaI and BamHI
endonuclease digestion. Fragments were blunted with T4-DNA polymerase
and cloned into the SmaI site of the pGL3 basic vector (Promega). Correct insertion and orientation were confirmed by sequencing.
Bacterial Expression Vector Constructions--
Human full-length
ETS-1 and 5' deletion mutant constructions were obtained by PCR
amplification using appropriate primers (Table
II) and cloning into a pTyb2 vector (T7
Impact System, New England Biolabs). The human ETS-1 cDNA inserted
in a pSG5 vector was used as a template. The 5' primers were always
designed in-frame with the NdeI site. The amplified
fragments were submitted to NdeI digestion and cloned into a
pTyb2 vector, previously digested by NdeI and
SmaI endonucleases. The ETS-1 p42 isoform construct was
obtained by PCR site-directed mutagenesis (43), resulting in the large
deletion of the exon VII. All constructs were checked for nucleotide
sequence before expression.
Eukaryotic Expression Vector Construction--
The human ETS-1
cDNA inserted in a pSG5 vector already existed (42). The ETS-1 p42
isoform cDNA cloned in pSG5 vector was obtained by the same way as
for the pTyb2 cloning.
Generation of Biotinylated Oligonucleotides for SPR
Experiments--
The 99-bp biotinylated double-stranded DNA fragments
were obtained by PCR amplification using
5'-biotinyl-GAATTCCAGTCAATTTTTCCAG-3' as a forward primer and
5'-CAAGGCAACACAGTGATTAATC-3' as a reverse primer. Amplified fragments
were purified on QIAquick columns (Qiagen) and checked for sequence
before immobilization on the Sensor Chip.
Expression and Purification of ETS-1 Proteins--
Proteins were
purified using the T7-Impact System (New England Biolabs).
Escherichia coli (ER2566) was transformed with the appropriate recombinant plasmid. Fresh overnight cultures were diluted
1:25 in 250 ml of Luria-Bertani medium and incubated at 37 °C with
shaking at 230 rpm. When culture density reached
A595nm = 0.7, isopropyl--D-thiogalactopyranoside was added to a final concentration of 0.3 mM. The culture was incubated at
30 °C with shaking for 3-4 h. Cultures were harvested, and pellets
were washed with phosphate-buffered saline and suspended in 10 ml of
lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA, 0.1% (v/v) Triton X-100, complete protease
inhibitors mixture (CompleteTM, Roche Molecular
Biochemicals)). Bacteria were lysed with a French press using a 1000 pounds/square inch pressure. Lysates were clarified by a 5-min
centrifugation at 20,000 × g at 4 °C followed by a subsequent 15-min centrifugation at 20,000 × g at
4 °C. Each clarified lysate was applied to a 5-ml chitin bead column
(New England Biolabs). Columns were washed with 20 volumes of column
buffer (lysis buffer without protease inhibitors) and rapidly flushed
with 3 volumes of elution buffer (column buffer without Triton X-100)
containing 50 mM dithiothreitol (DTT) (Roche Molecular
Biochemicals). Columns were then stored 16 h at 4 °C for
peptidic cleavage. Proteins were eluted by 15 ml of elution buffer in
1-ml fractions. Fractions with proteins were pooled, and for further
purification were diluted and chromatographed on a Mono S HR 5/5 column
(Amersham Biosciences) equilibrated with 10 mM Tris, pH 8, 50 mM NaCl, 1 mM DTT, and 0.5 mM
phenylmethylsulfonyl fluoride using a Bio-Rad Biologic Chromatography
System (Bio-Rad). Proteins were eluted by a NaCl gradient (0.05-1
M). Fractions containing the protein were quick-frozen by
immersion in liquid nitrogen and stored at 80 °C after dialysis against HBS-EP (0.01 M Hepes, pH 7.4, 0.15 M
NaCl, 3 mM EDTA, 0.005% (v/v) polysorbate 20). Yields were
measured by colorimetry (Bio-Rad protein assay) and corrected by
comparison against known protein standards (bovine serum albumin) on
SDS-PAGE after Coomassie Blue staining.
Electrophoretic Mobility Shift Assay--
Double-stranded
synthetic oligonucleotides corresponding to the WT, M1, M2, WT + 4, and
M1M2 mutants of the stromelysin-1 (223/194) promoter region (Table
I) were end-labeled using T4 polynucleotide kinase and
[
-32P]ATP and were subsequently purified by
electrophoresis on a 20% polyacrylamide gel in TBE buffer (90 mM Tris borate, 1 mM EDTA). Recombinant
proteins (4 pmol) were incubated with 0.5 ng of probe in 20 µl of
binding reaction buffer (20 mM Tris, pH 7.9, 80 mM NaCl, 1 mM EDTA, 2 mM DTT, 10%
glycerol) for 30 min on ice. Complexes formed were resolved on a 5%
polyacrylamide (acrylamide/bisacrylamide 29:1, Euromedex)
non-denaturing gel in 0.25× TBE buffer at room temperature. Gels were
dried and autoradiographed at 80 °C.
Kination Assay--
Recombinant human ETS-1 p51 (4 pmol)
was phosphorylated by incubation with 150 units of
calmodulin-dependent protein kinase II (CaMKII, rat
truncated recombinant protein, New England Biolabs) at 30 °C for
various times in 20 µl of kination buffer (20 mM Tris, pH
7.5, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA, 2 mM CaCl2, 2.4 µM calmodulin, 100 µM ATP) in the presence
of [-32P]ATP. The reaction was stopped by
addition of 20 µl of 2× gel loading buffer (0.1 M Tris,
4% SDS, 1.44 M -mercaptoethanol, 20% glycerol, 0.2%
xylene cyanol, and 0.2% bromphenol blue) and subsequent boiling for 2 min. Samples were resolved by SDS-PAGE, and gels were dried and
quantified using a PhosphorImager (Amersham Biosciences). For gel shift
assays, the recombinant proteins (4 pmol) were phosphorylated under the
same conditions for 90 min in absence of radioactive ATP. Subsequent
binding reactions were performed with 0.5 ng of 32P-labeled
probe. The volume was adjusted to 30 µl with binding reaction buffer.
Cross-linking Experiments--
Cross-linking reactions were
carried out in a total volume of 20 µl in phosphate-buffered saline
solution (Invitrogen). 4 pmol of ETS-1 were incubated with various
amounts of 32P-labeled oligonucleotides (0.125 to 2 ng) for
30 min on ice. Ru(bpy)3Cl2 (Aldrich) and
ammonium persulfate were added to 150 µM and 2.5 mM, respectively, just before illumination by standard flashlight at a distance of 10 cm. Then the samples were immediately quenched with 20 µl of 2× gel loading buffer and heated to 100 °C
for 2 min. Complexes were resolved by electrophoresis through a 10%
SDS-polyacrylamide gel. Gel were dried and autoradiographed at
80 °C.
SPR Binding Assay--
SPR measurements were carried out using a
BIAcore 2000 apparatus (Biacore®). Double-stranded biotinylated
oligonucleotides (99 bp long) were immobilized on a
streptavidin-coupled CM5 Sensor Chip using standard protocol as
indicated in the Amine Coupling Kit (Biacore®). HBS-EP BIA certified
buffer (0.01 M Hepes, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% (v/v) polysorbate 20, Biacore®) was used
as a running buffer. Briefly, flow rate was fixed at 10 µl/min.
Streptavidin was injected at 500 ng/µl in 10 mM sodium acetate, pH 3.5, for 12 min. Biotinylated double-stranded
oligonucleotides were injected through each flow cell at 200 ng/ml onto
the Sensor Chip until a suitable stable RU signal was obtained. The
integrity and quantity of fixed DNA was checked by a 120-s injection of 1.6 µM calf histone H1 (Sigma) at 10 µl/min.
Equilibrium binding experiments were carried out at 25 °C using a
flow rate of 10 µl/min. Proteins were injected at the desired
concentration in HBS-EP for 120 s. The Sensor Chip was regenerated
by a 60-s injection of 0.03% SDS. Final curves were obtained by
subtraction of the signal corresponding to a flow cell functionalized
with M1M2 oligonucleotide. Resonance units (RU) for calculation of the
binding ratios were measured at the end of the injection when binding
equilibrium was reached.
SPR Kinetic Assay--
Kinetic experiments were carried out at
15 °C at a flow rate of 30 µl/min on a CM5 Sensor Chip prepared as
described above with a 100-RU stable fixation of each biotinylated
double-stranded oligonucleotide. ETS-1 p51 isoform was injected using
the KINJECT procedure for 90 s at 7.25, 14.5, 29, and 58 nM concentrations. Dissociation of the complex was then
monitored for 300 s before regeneration by a 60-s injection of
0.03% SDS in distilled water. Each injection was repeated three
times to obtain a complete data set of 12 curves. Raw data were
corrected by subtraction of the blank curve corresponding to M1M2
oligonucleotide. Each data set was globally fitted with Biaeval® 3.1 software. The schematic representation of the models that we edited and
used for the data analysis and their related set of differential rate
equations are listed in Table III. For
each model, the kinetic parameters as well as the maximum binding
capacity of the immobilized ligand were considered as global parameters
for a given data set. Moreover, two local parameters were added for
each curve to take into account the refractive index changes at the
beginning of the wash-on and wash-off phase.
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Table III
Schematic representation of the kinetic models and the related
differential equations
A corresponds to the analyte (ETS-1) and B to the ligand (DNA).
A2B stands for a ternary complex composed of one ligand and two
analyte molecules. A2B* depicts the same ternary complex but in
a rearranged form. The concentration of X is represented by
[X].
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Transfection and Reporter Gene Assay--
HEK293 cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum in 12-well plates (2 ml per well) to reach 60-80%
confluence at the time of transfection. Efficiency of transfection was
tested with a -galactosidase encoding expression vector under the
control of the cytomegalovirus promoter after coloration of the
transfected cells with
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal)
(Roche Molecular Biochemicals). Before transfection, Exgen 500 Transfection Reagent (1.5 µl per well, Euromedex) was incubated with
250 ng of each respective reporter (pGL3) and expression vector (pSG5)
for 10 min at room temperature in a volume of 50 µl of a 150 mM NaCl solution. Cell medium was changed for 500 µl of
Opti-MEM (Invitrogen), and DNA-Exgen 500 mixture was added. After
16 h, medium was changed for 2 ml of Dulbecco's modified Eagle's
medium. Cells were harvested 48 h after transfection with 250 µl
of cell lysis buffer (1% Triton X-100, 25 mM
glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT). 20-µl aliquots of each supernatant were tested for luciferase activity (luciferase assay kit,
Promega) using a Lumat LB 9501 (Berthold). The expression of the
proteins of interest (ETS-1 p51 or p42) was tested by Western blot
analysis on total cell lysates using a primary antibody directed against the ETS-1 DBD (C-20, Santa Cruz Biotechnology).
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RESULTS |
SPR Measurement of the Cooperative Binding of ETS-1 to the EBS
Palindrome of the Stromelysin-1 Promoter--
To investigate the
characteristics of the interaction between ETS-1 and the head to head
EBS palindrome located 216/201 in the stromelysin-1 promoter
(referred to as wild type (WT) in the following), several mutants of
the EBS repeat were produced. Either one (M1 or M2) or both (M1M2) EBS
were inactivated by directed mutagenesis exchanging the 5'-GGAA-3' core
consensus with 5'-AAAA-3' (Table I). Biotinylated PCR fragments of the
276/177 region were used as a ligand for SPR measurements of the
interaction between ETS-1 and the EBS palindrome (Fig.
1A). We
observed a positive cooperative binding to the WT site in comparison to
M1 or M2 mutants. Indeed, the measured RU signal corresponding to the
ETS-1 binding to the WT site was higher than the sum of the RU signal
for M1 and M2 mutants (dotted line on Fig. 1A).
To determine whether the cooperative binding was dependent on the
mutual orientation of the EBS, we produced mutants in which one (direct
repeat, referred to as DR) or both (inverted palindrome, referred to as
IP) EBS orientations were changed, taking care to preserve their
flanking sequence (Table I). SPR measurements showed a complete
disappearance of the cooperative binding to the IP and DR sites as
binding to these mutants was approximately twice the binding to the M1
site (Fig. 1B).
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Fig. 1.
ETS-1 specifically binds to the head to head
EBS palindrome in a cooperative way. A, sensorgrams of
a 200 nM ETS-1 injection over a Sensor Chip functionalized
with 280 RU of WT, M1, and M2 oligonucleotides. The dotted
line represents the sum of M1 and M2 sensorgrams. B,
sensorgrams of a 200 nM ETS-1 injection over a Sensor Chip
functionalized with 260 RU of WT, DR, IP, and M1 oligonucleotides (IP
sensorgram comes from another Sensor Chip with the same amount of
immobilized DNA). A and B, a flow cell
functionalized with M1M2 oligonucleotides was used as a reference for
nonspecific binding. C, gel shift assay. ETS-1 (4 pmol) was
incubated with WT (lanes 1-4) or M1 (lane 5)
32P-labeled DNA probe (0.5 ng) in the absence (lane
1) or in presence of WT (200×, lane 2), M1M2 (200×,
lane 3), and (M1 400×, lane 4) unlabeled
competitors, WT (lane 6), and M1 (lane 7) free
probes were loaded. D, photo-cross-linking assay resolved by
SDS-PAGE. The same amount of ETS-1 (4 pmol) was incubated with
increasing amounts (0.125, 0.25, 0.5, 1, and 2 ng) of WT (lanes
2-6) or M1 (lanes 8-12) 32P-labeled DNA
probe prior addition of photo-cross-linker
(Ru(bpy)3Cl2, ammonium persulfate) and
illumination. Lanes 1 and 7 are controls
containing 2 ng of WT or M1 probe, respectively, cross-linked in the
absence of ETS-1. E, same experiment as D,
realized with an equimolar mixture of 32P-labeled 15-mer
oligonucleotides (referred to as 15-mer) corresponding to 223/209
and 208/194 sequences of the stromelysin-1 promoter as a probe
(0.5, 1, and 2 ng, lanes 1-3). Lane 4 is a
control containing 2 ng of probe in absence of ETS-1. The arrow
1 indicates binary ETS-1·DNA complex. The arrow 2 indicates ternary ETS-1·DNA·ETS-1 complex.
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This quantitatively confirms the observation that ETS-1 or ETS-2 tends
to bind head to head EBS palindromes, such as those encountered in
GATA-1 (38) or p53 (39) promoters, better than a single EBS or other
topology of EBS repeats (44). The fact that ETS-1 could be able to
discriminate between different topologies of EBS repetitions tends to
prove that this effect could not be driven by structural effects like
cooperative bending of the DNA facilitated by the proximity of the EBS.
Indeed structural studies showed that ETS DNA binding domain (DBD)
binding to DNA induces moderate bending (45, 46). If such a phenomenon
was the unique strength of the cooperativity, binding to IP and DR
mutant sites would also be enhanced by the DNA bending generated by the
first ETS-1 molecule. Then other mechanisms, like protein-protein
interactions, should be envisaged.
Visualization of ETS-1-DNA Complexes by EMSA and
Photo-cross-linking--
The major drawback of BIAcore® technology
is its inability to identify the complexes formed between the ligand
and the analyte during the injection time. So we confirmed our
observations by visualizing the protein-DNA complexes at the
equilibrium by EMSA and photo-cross-linking.
EMSA showed the formation of a single major complex between ETS-1 and
the WT probe (Fig. 1C, lane 1). Whereas a 200×
molar excess of WT non-labeled probe was able to disrupt this complex (Fig. 1C, lane 2), a 400× molar excess of M1
probe, corresponding to the same amount of binding sites, was unable to
do so (Fig. 1C, lane 4). Moreover, the
association of ETS-1 with M1 probe only led to a weak fading signal
(Fig. 1C, lane 5) in comparison to the WT
complex, and thus with the same protein and probe amounts (compare
lanes 1 and 5 on Fig. 1C). Similar
results were obtained with M2 probe (data not shown). This experiment
confirms the cooperative binding of ETS-1 to the head to head EBS
palindrome present in the stromelysin-1 promoter.
In order to visualize the complex formed with M1 probe, which did not
seem to resist migration through the polyacrylamide gel used under our
experimental conditions, we performed cross-linking with
Ru(bpy)3Cl2. This reagent enabled instant high
yield cross-linking with no bridging agent (47). At low probe
concentrations, only a weak signal was visible with WT probe (Fig.
1D, lane 3), and no signal was observed with M1
(Fig. 1D, lanes 8-10). This signal, migrating at
~120 kDa, corresponds to two ETS-1 molecules linked to the WT probe.
Increasing the probe concentration results for the M1 probe (Fig.
1D, lanes 11 and 12) in the formation
of a weak complex at about 60 kDa corresponded to a single ETS-1
molecule linked to the probe. This signal appeared as the concentration of WT probe increased (Fig. 1D, lane 6) but
remained less intense than the 120-kDa signal. Interestingly, no signal
was observed at 120 kDa in an experience using an equimolar mixture of
32P-labeled oligonucleotides corresponding to 223/209
and 208/194 sequences of the stromelysin-1 promoter as a probe
(referred to as EBS 15-mer in Fig. 1E). We obtained a
pattern similar to M1, although we could have hoped to restore a
trimeric complex by protein-protein interactions. This suggests that if
such contacts exist, they are dependent on the strict vicinity and
positioning provided by the topology of the palindromic binding sites.
Kinetic Study of the Interaction between ETS-1 and the
Stromelysin-1 Promoter--
We characterized the binding cooperativity
by modeling the interaction between ETS-1 and the EBS palindrome of the
stromelysin-1 promoter. New data sets were produced under conditions
more favorable for kinetic studies as follows: (i) lower binding
capacity (100 RU of immobilized DNA) and higher flow rate (30 µl/min)
to reduce mass transfer effects at the surface of the Sensor Chip; (ii) lower temperature to reduce kinetic rates; and (iii) addition of bovine
serum albumin to 100 µg/ml in the running buffer to prevent excessive
refractive index changes during injections. A typical quadruplet of
curves for each oligonucleotide (M1, M2, and WT) is shown in Fig.
2. The kinetic models used to fit the data sets and the related differential rate equations are represented in Table III. Kinetic and equilibrium parameters obtained for each model are listed in Table IV.
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Fig. 2.
Surface plasmon resonance kinetic analysis of
ETS-1 interaction with the EBS palindrome of the stromelysin-1
promoter. Representative set of sensorgrams illustrating the real
time binding of various concentrations (7.25, 14.5, 29, and 58 nM) of ETS-1 to the M1 (A), M2 (B),
and WT (C) stromelysin-1 promoter. M1M2 mutant was used as a
reference for nonspecific binding. Analysis was performed as described
under "Materials and Methods." Kinetic models used and results are
summarized in Tables III and IV, respectively.
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Table IV
Kinetic and equilibrium parameters for ETS-1 interaction with the EBS
palindrome of the stromelysin-1 promoter, as determined by surface
plasmon resonance.
All the notations for the parameters are consistent with the ones used
in Table III.
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ETS-1 binding to M1 or M2 oligonucleotides was fitted according to the
simplest kinetic model provided, a one to one interaction. ETS-1
binding to the WT oligonucleotide was fitted with our own edited models
of increasing complexity, representing the possible interactions of two
ETS-1 molecules with DNA. We tried to keep to a minimum the number of
kinetic parameters in order to preserve the significance of the model.
We considered models in which binding of the two ETS-1 molecules could
be independent (models A and B) or sequential (models C and D). We also
introduced the possibility of a conformational change of the ternary
complex after binding to the DNA (models B and D). In addition to the
kinetic parameters, for each of these models we calculated in Table IV
an apparent binding affinity corresponding to the affinity of two
independent and equivalent EBS, referred to as
Kd(app), giving the same global
affinity. This apparent affinity enabled us to determine, in each case,
the binding cooperativity in comparison to the M1 and M2
oligonucleotides (see Table IV for results and equations).
The fitting data show that, in the presence of two binding sites,
kinetics seem to be faster (compare ka1
and kd1 with M1 or M2 and the WT models
in general) but without considerably altering the overall equilibrium
constants (compare Kd1 values). This can
be due to a difference of accessibility to the DNA or more likely to
the difference in binding mechanisms between both DNA configurations
(M1 or M2 in comparison to WT). Considering the 
2 values
of the models, it is obvious that simple model depicting independent
binding sites (model A) is insufficient to describe the overall binding
mechanism. Nevertheless, adding a conformational change after DNA
binding (model B) improves fitting (2 from 3.4 to 2.77).
In that case, Kd values
(Kd1 and
Kd2) are similar to the
Kd1 values obtained for M1 and M2, and
the conformational change provides the 6.7-fold cooperativity for the
ternary complex formation.
In order to describe better a cooperative mechanism, a sequential
binding (models C and D), where the binding of the first molecule is
able to influence the binding of the second one, seems to be more
accurate. Evidence of this is the fact that the calculated cooperativity fold increases according to the 2 value. A
simple sequential model (model C) provides a better 2
value than model A (2.65 compared with 3.4), which uses the same number
of parameters but with independent binding sites. It is noteworthy that
Kd1 between model A and C is not greatly affected, but Kd2 is 3-fold lower, in
accordance with the definition of a cooperative binding mechanism.
Indeed the reduction of Kd2 is mainly
due to an increase of ka2, reflecting a
better association of the second protein to the complex. The last model
(model D), comprising a sequential binding followed by a conformational
change, provides the better 2 of the series (0.996) and
so the best description of the cooperative phenomenon. As for model C,
but to a greater extent, the Kd value of the second
step (Kd2) is reduced compared with the
first one (about 50 times lower than
Kd1) or to the second step of model B
(about 20 times lower) which uses the same number of parameters but
with independent binding sites. In both cases this reduction is due to
the increase in ka1 values. The
Kd3 value of 0.95 indicates that at the
equilibrium the ternary complex exchanges freely between two
conformations. The cooperativity fold is then maximum for this model
reaching 19.7-fold for a Kd(app) of 18.7 nM.
This kinetic study confirms the cooperative binding of two ETS-1
molecules to the EBS palindrome. The cooperativity observed seems to be
driven by the facilitated binding of the second ETS-1 molecule to form
the ternary complex rather than by the formation of a ternary complex
in a locked conformation after a conformational change as
kd2, ka3,
and kd3 gave no evidence of it. Indeed,
in that case we would have encountered slower dissociation steps for
the ternary complex, with lower kd2 and
kd3 values.
Mapping of the Protein Region Responsible for the Cooperative
Binding by N-terminal Deletion Mutants of ETS-1 Protein--
In order
to determine whether a particular protein region was responsible for
the observed cooperative behavior, we produced N-terminal deletion
mutants of ETS-1 by PCR amplification and cloning into pTyb2 vector for
bacterial expression (Fig.
3A). Recombinant proteins were
purified as under "Materials and Methods." A SDS-PAGE of the
various mutants was silver-stained to assess purity (Fig.
3C).
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Fig. 3.
Organization of ETS-1 and its N-terminal
deletion mutants. A, map of ETS-1 and its deletion
mutants produced with T7 Impact System. B, secondary
structure of the ETS-1 DBD (-helices H1-H3 and -sheets S1-S4) and
inhibitory regions (HI-1, HI-2, and H4). C, 15% acrylamide
SDS-PAGE of ETS-1 and its various deletion mutants, silver-stained
according to Ref. 59.
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The ability of the different deletion mutants to cooperate for binding
to the EBS palindrome was evaluated by SPR measurement at the
equilibrium (Fig. 4A). The
same concentration (200 nM) of each protein was injected
over a Sensor Chip functionalized with WT, M1, and M2 oligonucleotides.
At the end of the injection, when binding equilibrium was reached, and
after blank correction using M1M2 oligonucleotide, RU signal for each
flow cell was measured, and the following ratio was calculated:
WT/M1 = RU(WT)/RU(M1). Results are displayed in Fig.
4B. The WT/M2 and WT/(M1 + M2) ratios, also representative
of the cooperative binding of the proteins to DNA, showed similar
variations (data not shown) and so will not be mentioned in this study.
We observed a rapid increase of the WT/M1 ratio from 
N331, which
represents a DBD without its N-terminal inhibitory region, to N245.
For deletion mutants beyond amino acid 245, it stabilized at a value
corresponding to the ratio obtained with the full-length protein.
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Fig. 4.
Localization of the ETS-1 peptidic region
implicated in the stromelysin-1 promoter binding cooperativity. As
for Fig. 1 and Fig. 2, M1M2 oligonucleotide was used as a reference for
nonspecific DNA binding. A, amount of protein bound to WT
and M1 oligonucleotides at the equilibrium phase during a 200 nM injection of ETS-1 or its N-terminal deletion mutants,
expressed in RU. B, representation of the ratio between the
different proteins bound to WT oligonucleotide and M1 oligonucleotide
for a 200 nM protein injection. RU for calculation were
measured at the end of the injection at equilibrium phase.
C, protein binding to WT and M1 oligonucleotides, expressed
in fmol/mm2. D, gel shift assay. Same amount of
ETS-1 or its N-terminal deletion mutants (4 pmol) was incubated
with the WT or M1 32P-labeled probe (0.5 ng).
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RU variation is representative of a mass variation at the surface of
the Sensor Chip with the correlation that 1000 RU correspond to a
variation of 1 ng/mm2 at the surface. Thus, we were able to
estimate the quantity of protein bound per surface unit at the
equilibrium for each oligonucleotide (Fig. 4C). The binding
pattern of the M1 oligonucleotide was consistent with the known
mechanism of binding regulation of ETS-1 by autoinhibition (21).
Indeed, the amount of protein bound decreased from N301 to N245,
as long as the inhibitory module was restored to the protein. But at
the same time, despite the complete inhibitory module restoration, the
quantity of bound protein to WT oligonucleotide increased and was
maximum for N245, resulting in the observed cooperativity. Beyond
amino acid 245, the quantity of bound protein to M1 oligonucleotide
remained low, and at the opposite end, the quantity of bound protein to
WT oligonucleotide was still high but lower than for N245 (this
could be due to the presence of the N-terminal part of the protein that
may influence DNA binding by sterical or more complex mechanisms).
Nevertheless, WT/M1 ratio remains constant from N245 to the
full-length protein.
To confirm these results and have evidence of the molecular species
involved, we performed EMSA with the different mutants using WT and M1
probes (Fig. 4D). We observed the complete reversion of the
binding pattern from N331 to N245 (Fig. 4D,
lanes 1-8). N331 forms the same strong binary complex with one
protein molecule bound to the probe with M1 or WT oligonucleotides
(lanes 1 and 2), whereas the ternary complex is
hardly visible with the WT probe (Fig. 4D, lane
2). This remains consistent with the SPR results where the WT/M1
ratio was close to 1 suggesting that at equilibrium a ternary complex
was not present with the WT oligonucleotide. N301 was also able to
form a binary complex with the M1 probe (Fig. 4D, lane
3) but also a strong slower migrating ternary complex made of two
proteins bound to the WT probe (lane 4). The binary complex,
which was observed with M1, was also present with the WT probe but
weaker. N280 binds weakly to the M1 probe (Fig. 4D,
lane 5) and only formed with the WT probe a slower migrating ternary complex (lane 6). Binding patterns for N245,
N144, N45, and ETS-1 were identical, e.g. no distinct
complex formed with M1 probe (Fig. 4D, lanes
7, 9, 11, and 13) and only a
strong slower migrating ternary complex formed with WT probe
(lanes 8, 10, 12, and
14).
These observations tend to designate the region located between amino
acid 245 and 331 as responsible for the cooperative binding of two
ETS-1 molecules to the EBS palindrome. The mechanism implied is the
suppression of the DNA-binding autoinhibition. It seems possible that
two ETS-1 molecules overcome DNA binding autoinhibition by a
protein-protein interaction in the 245-331-residue region or another
mechanism implying this region, during the ternary complex formation,
that would favor an unrepressed form with an HI-1 inhibitory helix
unwound (see Fig. 3B for schematic representation of the
structural elements of the ETS-1 DBD and inhibitory regions). Such
mechanism has already been described for ETS-1, which was able to
interact with a binding partner in order to stabilize its unrepressed
conformation (29, 31). It would account for the easier and faster
formation of the ternary complex emphasized by the kinetic study.
Binding Pattern of the p42 Isoform of ETS-1 to the EBS Palindrome
of the Stromelysin-1 Promoter--
ETS-1 physiologically exists under
two different isoforms: p51, the full-length protein, and p42, a
shorter isoform that lacks the 245-331-residue region encoded by exon
VII of the gene (Fig. 5A).
These two proteins, although sharing similar characteristics due to
their common DBD and pointed domain, also have distinct properties (48,
49). It was particularly interesting to study the binding of the ETS-1
p42 isoform to the EBS palindrome of the stromelysin-1 promoter. Its
cDNA was cloned in a pTyb2 vector and the recombinant protein was
expressed and purified (see "Material and Methods").
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Fig. 5.
Comparison between binding of ETS-1 p51 and
p42 isoforms to the EBS palindrome of the stromelysin-1 promoter.
A, schematic organization of p51 and p42 isoforms of ETS-1.
P represents a serine susceptible to be phosphorylated by
CaMKII. B, gel shift assay. Equal amounts (4 pmol) of ETS-1
p51 (lanes 1-6) and ETS-1 p42 (lanes 7-12)
isoforms are incubated with WT (lanes 1-3 and
7-9) or M1 (lanes 4-6 and 10-12)
32P-labeled oligonucleotides (0.5 ng) in the presence of
increasing amounts of unlabeled competitor (50× lanes
2, 5, 8, and 11, and 200×
lanes 3, 6, 9, and 12).
C, gel shift assay. The same amount (4 pmol) of ETS-1 p42
was incubated with WT (lane 1), M1 (lane 2), M2
(lane 3), and WT + 4 (lane 4)
32P-labeled probes (0.5 ng). D, SPR experiment.
Ratio of protein bound to WT and M1 or WT and M2 oligonucleotides
during the equilibrium phase of a 100 nM p51 or p42
injection. E, variation of the previous ratios as a function
of the analyte ( , p51, or  , p42) concentration (75, 100, 150, and
200 nM). The arrow 1 corresponds to the binary
p42·DNA complex, and arrow 2 to the ternary
p42·DNA·p42 complex, and the arrow * to a complex formed
with a minor contaminant.
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By using EMSA, we compared DNA binding to WT and M1 probes for the
ETS-1 p51 and p42 isoforms. Surprisingly, p42 showed no binding
difference between WT and M1 oligonucleotides (Fig.
5B, lanes 7 and 10). Because only one
EBS is present within M1 probe and no ETS-1-ETS-1 interaction is
reported in the literature, this suggests that only a binary complex
formed with both probes. The minor faster migrating band observed (Fig.
5B, arrow *, lanes 7 and
10, and also Fig.
6B) could be due to a minor
contaminant present in the p42 protein preparation and cannot be
considered as the binary complex, whereas the upper band would be the
ternary complex. Indeed, both complexes were observed with the two
probes, whereas M1 cannot bind more than one ETS-1 molecule (Fig.
1D) because only one EBS is present. Moreover, increasing
the spacing between both EBS of 4 bp (see WT + 4 in Table I) results in
the formation of a slower migrating complex corresponding to the
expected ternary complex (Fig. 5C, lane 4),
whereas only a binary complex is observed with WT, M1, and M2 probes
(Fig. 5C, lanes 1-3). Finally, the formation of
the same complex with M1 and WT probes was previously encountered with
N331 (Fig. 4D), which also lacks the 245-331-residue region. It reinforces the crucial role of this region for ETS-1 ability
to discriminate the EBS topology on DNA.
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Fig. 6.
Effect of ETS-1 phosphorylation by CaMKII on
the cooperative binding to the head to head EBS palindrome of the
stromelysin-1 promoter. A, phosphorylation kinetics of
ETS-1 p51 by recombinant rat CaMKII in the presence of
[-32P]ATP. AU, arbitrary units.
B, gel shift assay. Equal amounts (4 pmol) of ETS-1 p51
isoform (lanes 1-4), N245 (lanes 5-8), ETS-1
p42 isoform (lanes 9-12), and N331 (lanes
13-16) were incubated with WT 32P-labeled
oligonucleotides (0.5 ng, all lanes except
2, 6, 10, and 14), M1
32P-labeled oligonucleotide (0.5 ng, lanes
2, 6, 10, and 14) after
phosphorylation by CaMKII (lanes 3, 7,
11, and 15), or after incubation with phosphorylation
buffer without kinase (lanes 4, 8, 12,
and 16).
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The same SPR equilibrium measurements as for the ETS-1 deletion mutants
were carried out with ETS-1 p42 isoform on WT, M1, and M2
oligonucleotides (Fig. 5D). The WT/M1 and WT/M2 ratios calculated for p42 isoform are close to that of N331, the shortest deletion mutant lacking the 245-331-residue region, reflecting the
loss of cooperativity and its inability to form a ternary complex with
the WT oligonucleotide. The influence of protein concentration on the
cooperative binding mechanism was evaluated by measuring the previous
ratios for different concentrations (Fig. 5E). Their
variation for p51, increasing for low concentrations where the
cooperative factor is high, is in agreement with a cooperative behavior. On the contrary, for p42 isoform, the near 1 ratio value is
independent on the protein concentration. All together, these data
demonstrate the implication of the 245-331-residue region encoded by
exon VII in the human ETS-1 cooperative binding to the head to head EBS
palindrome present in the stromelysin-1 promoter.
Effects of ETS-1 Phosphorylation by CaMKII on Cooperative
Binding to the EBS Palindrome of the Stromelysin-1
Promoter--
Calcium-dependent phosphorylation of ETS-1 on
residues Ser-251, Ser-257, Ser-282, and Ser-285 was reported to
negatively regulate ETS-1 DNA binding in vitro and in
vivo (50, 51). The proposed mechanism is a reinforced
autoinhibition by stabilization of the inhibitory conformation through
electrostatic interactions between phosphoserines and basic residues of
the inhibitory module.
In order to extend our model and to determine the influence
of calcium-dependent phosphorylation on the ETS-1
cooperative binding, we realized in vitro phosphorylation
assays using CaMKII. First, we realized phosphorylation kinetics using
ETS-1 as a substrate to determine time conditions under which
phosphorylation was maximum for the amount of substrate and enzyme
considered (Fig. 6A). We further used a 90-min incubation
corresponding to the stationary phase of the reaction.
Phosphorylation effects were tested by EMSA, comparing binding to the
WT probe of ETS-1 p51 and p42 isoforms incubated in the presence or
absence of CaMKII. We also tested phosphorylation effects on N331,
which lacks calcium-dependent phosphorylation sites, and
N245, which both have a similar binding behavior to p42 and p51,
respectively. For ETS-1 p51 isoform and N245, hardly any binding of
the phosphorylated proteins was observed with the WT probe (Fig.
6B, lanes 3 and 7). For N331 or
ETS-1 p42 isoform no significant difference in amounts of bound protein
between the CaMKII-treated sample and the control (Fig.
6B, lanes 11 and 15) was observed.
These results confirm the role of autoinhibition and the implication of
the 245-331-residue region in the cooperative binding to the EBS
palindrome. It seems that reinforcing autoinhibition by phosphorylation
of the serine residues reduces dramatically the cooperative binding.
This can be explained either by the stabilization of the inhibitory
conformation of ETS-1, which reduces affinity for DNA, or by a possible
electrostatic repulsion of the negatively charged phosphoserines that
would prevent protein-protein interaction in the 245-331-residue region.
Activation of the Stromelysin-1 Promoter by ETS-1 p51 and p42
Isoforms--
It was interesting to investigate whether the difference
of DNA binding of the p51 and p42 isoforms was correlated with their transcriptional activation. The 478/+4 region of the WT, M1, M2, and
M1M2 stromelysin-1 promoters was cloned in the pGL3 basic vector (Fig.
7A). These vectors were
co-transfected with either p51 or p42 eukaryotic expression vectors in
HEK293 cells. Culture lysates were tested for luciferase activity. For
p51, mutation of one of both EBS sites had dramatic effect on
transactivation, resulting in an ~90% loss of activity. Double
mutation readily abolished activation by p51. These results, obtained
with ETS-1, were similar to those obtained with ETS-2 protein on the
human stromelysin-1 promoter (13). This experiment provides a direct link between cooperative DNA binding and functional transactivation. Interestingly, the ETS-1 p42 isoform did not activate the stromelysin-1 promoter in the same way as the p51 isoform did (only 28%). Western blot analysis of the ETS-1 proteins in cell lysates after transfection (Fig. 7C) showed no significant difference between p51 and
p42 expression. Thus, differential expression of the proteins cannot be
responsible for the observed difference in transactivation. We can
infer that either p42 does not share the same transcriptional activity
as p51 or that the presence of two ETS-1 molecules and their proper
spatial orientation are required for maximum activation of the
stromelysin-1 promoter.
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Fig. 7.
Activation of the stromelysin-1 promoter by
ETS-1 p51 and p42 isoforms. A, reporter gene constructs
used for the transient transactivation assay. The 478/+4 region of
the WT and mutant stromelysin-1 promoters was cloned into a pGL3basic
vector, and LUC represents the luciferase reporter gene.
B, transient transfection assays are realized using HEK293
cells at 60-80% confluence. 250 ng of each luciferase reporter vector
were co-transfected with 250 ng of either p51 or p42 eukaryotic
expression vector. Luciferase activities obtained were expressed as a
percentage of the WT promoter activity co-transfected with the p51
expression vector (3.9-fold). Exact percentages are indicated on
top of the bars. Results are the average of two
representative experiments done in triplicate. C, Western
blot analysis of HEK293 cell lysates after transfection with pSG5
(lane 1), pSG5-p51 (lane 2), and pSG5-p42
(lane 3). The primary antibody used is directed against
ETS-1 DNA binding domain (C-20, Santa Cruz Biotechnology).
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DISCUSSION |
The human stromelysin-1 promoter has been shown to be differently
regulated by various members of the ETS family through the EBS
palindrome located 216/201. ETS-1, ETS-2 (13, 14), and PEA-3 (52)
are transcriptional activators, whereas TEL is reported to repress the
gene (53). In addition, ETS-related gene (ERG) is able to
inhibit the transcriptional activation mediated by ETS-2 (40). In this
study, we investigated the mechanism of ETS-1 binding to this
palindrome. We confirmed that DNA binding is cooperative, requiring the
head to head orientation of the EBS and their strict vicinity (Fig. 1),
which is fully consistent with results obtained with other EBS
palindromes (39, 44). An anterior report showed cooperative
transactivation, whereas no cooperative binding to the EBS palindrome
was observed (14). By working with purified proteins and a homologous
system, we showed cooperative binding with no ambiguity in comparison
to this former study realized in a heterologous system. Kinetic
analysis of ETS-1 binding to the WT promoter (Fig. 2 and Tables III and IV) confirms this hypothesis and enables us to give a plausible mechanism for the cooperativity. The binary complex formed between one
ETS-1 molecule and an EBS facilitates the fast binding of the second
ETS-1 molecule to form the ternary complex. The high rate of this step
could be explained by a protein-protein contact between ETS-1 molecules
that would give the right orientation, positioning, and conformation
for the second ETS-1 molecule to recognize the EBS. By using deletion
analysis of the ETS-1 protein, we further demonstrated that the
245-331-residue region, encoded by exon VII of the gene, was
responsible for the observed cooperativity (Figs. 3 and 4), and this
strongly suggests that the alleviation of the DNA binding
autoinhibition is implicated in the cooperative binding. The sum of the
kinetic and equilibrium SPR analyses causes us to propose the following
binding model (Fig. 8) in which the first
ETS-1 molecule bound to DNA facilitates the binding of a second ETS-1
molecule through an interaction implying the exon VII-encoded region.
The second ETS-1 molecule would be presented in the right orientation
and in an uninhibited conformation, explaining the binding
cooperativity observed. This molecular event is represented in Fig. 8
with square brackets. The ETS-1 molecules contact through their exon VII-encoded region, and the DNA-bound ETS-1 molecule helps
the second one to acquire its open uninhibited conformation in
the right orientation to bind DNA, according to the palindromic topology of the binding sites. After formation of the ternary complex,
the ETS-1 molecules flip between two conformations on DNA corresponding
to the last equilibrium of the figure. According to the model, we
proposed it to be a conformational change between the exon VII-encoded
regions which were implicated in the previous step of the mechanism and
could then adopt a relaxed conformation after the ternary complex was
formed.
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Fig. 8.
Proposed model for ETS-1 cooperative binding
to the stromelysin-1 promoter. It was based on kinetic and
equilibrium analyses of ETS-1 interaction with the EBS palindrome of
the stromelysin-1 promoter and on ETS-1 known structure and binding
mechanism. N and C represent the N-terminal and
C-terminal part of ETS-1, respectively. We propose that after binding
to DNA (first step corresponding to ka1
and kd1 parameters), the first ETS-1
molecule facilitates the binding of the second one by positioning and
helping it to reach its uninhibited state through a contact involving
the exon VII-encoded region (second step corresponding to
ka2 and
kd2). The intermediary state where the
first ETS-1 molecule contacts the second one before the complete
formation of the ternary complex is represented by square
brackets. After formation of the ternary complex, ETS-1 molecules
are able to adopt a relaxed conformational state (last step
corresponding to ka3 and
kd3). We propose it to be a
conformational change between the exon VII-encoded regions that were
implicated in the previous step and could rearrange once the ternary
complex is formed.
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This may have biological significance because the natural ETS-1 p42
isoform, lacking the exon VII-encoded region, fails to bind
cooperatively to the EBS palindrome and allows the binding of only one
p42 molecule (Fig. 5). The absence of the inhibitory helices HI-1 and
HI-2, preventing the formation of the postulated compact inhibitory
bundle, could generate sterical hindrance and account for this result
(see Figs. 3, A and B, and 5A and Ref. 21 for structural elements of ETS-1). This phenomenon was also encountered with the shorter N331 deletion mutant. Therefore, the
C-terminal region of p42 or N331, spanning amino acids 416-441 and
comprising the H4 inhibitory helix, could be responsible for the
observed binding inhibition of a second p42 or N331 molecule to the
DNA because, except for the DBD, it is the only region they share in
common. Such a mechanism remains hypothetical because recent
co-crystallization of ETS-1 amino acids 331-440 with Pax5 (54) shows
no evidence of a C-terminal region (comprising H4) extending away from
the DBD. Other possibilities could be envisaged independently or in
conjunction. Indeed, differential DNA bending between p51 and p42,
which we rejected to explain the binding cooperativity of p51 because
it was dependent on the EBS orientation, could contribute in this case
to prevent a second p42 molecule from entering into the major groove of
the DNA. A way to link destructuring of the inhibitory module with the
differential DNA bending is the recently elucidated mechanism of
interaction between the N-terminal end of helix H1 and the phosphate
backbone of the DNA (55). A difference of H1 conformation between p42
and p51, linked to the absence of an inhibitory module, would influence DNA bending through a different neutralization of the phosphate backbone charge by H1.
Our results appear of interest in the context of the
complex regulation of stromelysin-1 expression by factors of the ETS family. Previous studies showed that on the one hand, in given experimental conditions, the AP-1 complex was required for ETS-1 transcriptional activity (56) and on the other hand that the recruitment by direct protein-protein interactions of the AP-1 complex
by ETS-2 was increased in the presence of the EBS palindrome of the
stromelysin-1 promoter (41). Hence, the observed binding cooperativity,
mediated by a structural rearrangement implying the exon VII-encoded
region, could play a crucial role in the optimum presentation of the
molecule to its partners. These spatial and molecular events could lead
to the unmasking or the formation of a specific interaction surface.
This does not only concern transcriptional partners like AP-1 or
CBF2 but also general transcriptional coactivators. Indeed, CBP/p300
was shown to interact directly with ETS-1 and ETS-2 and particularly in
the case of transactivation of the stromelysin-1 promoter (42, 57).
More recently, Sp100 was also shown to be able to interact with ETS-1
and stimulate its transcriptional activity (58). It is also noteworthy
that not only the domain comprising HI-1 and HI-2 inhibitory helices is
implicated but rather the entire exon VII-encoded region. The latter
includes the four serines responsible for the
CaMKII-dependent phosphorylation, regulating DNA binding
(50, 51), which we proved to also play a role in the cooperative
binding to the EBS palindrome (Fig. 6). It also contains an SCRLTQS
motif located 261-267 in the protein sequence. This motif, according
to computer-predicted secondary structure of ETS-1, forms an -helix
and also contains an arginine that is resistant to tryptic digestion
(60). This suggests a stable secondary structure that could be involved
in the autoinhibition regulation or in protein-protein contacts.
The observed cooperative binding can be easily linked to the strategy
of combinatorial transcription regulation by ETS proteins. Various
works studied cooperative binding of the ETS-1 protein with
transcriptional partners such as CBF2 (30, 31), USF-1 (27), or TFE3
(28) where intermolecular contacts raise autoinhibition. In the
particular topology of the head to head EBS palindrome, we propose that
the two ETS-1 molecules play the role of reciprocal transcriptional
partners. In addition, this view can account for the observation that
ERG inhibits the ETS-2 transcriptional activation of the
stromelysin-1 promoter in transient co-transfection (40). ETS-2, like
ETS-1, requires the presence of both vacant EBS to overcome
autoinhibition and bind efficiently to the DNA. Thus, an ERG
molecule would be able to occupy one of the two EBS long enough to
prevent the cooperative fixation of two ETS-2 molecules. This also
suggests a differential regulation of the promoter by the two isoforms
of ETS-1, p42 and p51. The latter binds cooperatively to the palindrome
and achieves maximum transactivation (Fig. 7) but binds poorly to M1 or
M2 mutant due to DNA binding autoinhibition. In contrast, the p42
isoform can bind efficiently to a single EBS and thus prevent access to
the second EBS of the palindrome, acting as a dominant negative
for the activation of the stromelysin-1 promoter or other promoters
where EBS are organized in the palindrome.
We postulated that ETS-1 overcomes its autoinhibition via
intermolecular contacts involving the exon VII-encoding region which would favor the rapid formation of a ternary complex. The organization of two head to head EBS distant by 4 bp on a regular B-shaped double-stranded DNA in conjunction with the spatial structure of an ETS
DBD bound to DNA indicates that both ETS-1 molecules are located on the
same side of the DNA, and that the inhibitory modules are facing each
other. However, we were not able to evidence a direct protein-protein
contact. Various techniques including cross-linking, in
vitro GST fusion pull-down assays, or SPR experiments failed to
reveal a direct protein-protein interaction (data not shown). These
difficulties may have two major causes. First, the supposed interaction
is DNA-dependent, requiring strict proximity and proper
arrangement of the molecules. Second, the inhibitory module does not
exist on its own, resulting from the packing of four distinct partners:
inhibitory helices HI-1 and HI-2 located at the N terminus of the DBD,
inhibitory helix H4 at the C terminus of the DBD, and helix H1 in the
DBD (see Fig. 3B). To use an isolated exon VII peptide for
in vitro interaction assays appears useless because its
proper structure would not be obtained in absence of the C-terminal
part of the protein. In this context, it is noteworthy that such an
involvement of the exon VII-encoded region of ETS-1 in a cooperative
binding to a transcriptional partner was already encountered with
CBF2 and resulted with the same difficulty to prove direct
protein-protein interaction even with such a sensitive tool as SPR
(31).
The EBS palindrome with 4-bp interval between ETS-binding cores present
in the stromelysin-1 promoter is not found in other known promoters of
matrix metalloproteinases. Its ability to recruit, in a specific way,
two ETS-1 p51 molecules to form a ETS-1·DNA·ETS-1 ternary complex
via the exon VII-encoded region is inherent to the autoinhibition
binding mechanism of p51. To our knowledge, it is the first time that
ETS-1 was shown to be able to counteract its own autoinhibition through
such a complex. The existence of such a ternary complex remains to be
checked in cells expressing both stromelysin-1 and ETS-1. Given that
such palindromes are found in other promoters regulated by ETS
proteins, these EBS palindromes might represent a hot spot to recruit
autoinhibited ETS proteins and lead to specific transcriptional complexes.
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ACKNOWLEDGEMENTS |
We thank Drs. J. Coll and A. Verger for
stimulating discussions; Dr. D. Régnier for critical reading of
the manuscript; Dr. M. Dutreix for help in using Biacore® apparatus;
and N. Spruyt and S. Dequiedt for technical assistance. We are grateful
to Dr. G. Buttice for providing the human stromelysin-1 promoter.
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FOOTNOTES |
*
This work was supported in part by Grant ARC 5601 from the
Association pour la Recherche sur le Cancer (to M. A.).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.
Recipient of a BDI (Bouvse de Docteur Ingénieur)
fellowship from the CNRS and Région Nord-Pas de Calais.
§
To whom correspondence should be addressed. Tel.: 33-3-20-87-10-97;
Fax: 33-3-20-87-11-11; E-mail: marc.aumercier@ibl.fr.
Published, JBC Papers in Press, May 28, 2002, DOI 10.1074/jbc.M200088200
| |
ABBREVIATIONS |
The abbreviations used are:
EBS, ETS-binding
site;
CBF, core binding factor;
SPR, surface plasmon resonance;
M1, mutant site 1;
M2, mutant site 2;
M1M2, mutant sites 1 and 2;
DR, direct repeat;
IP, inverted palindrome;
EMSA, electrophoretic mobility
shift assay;
DTT, DL-dithiothreitol;
CaMKII, calmodulin-dependent protein kinase II;
Ru(bpy)3Cl2, ruthenium(II) tris-bipyridyl
dichloride;
RU, resonance unit;
DBD, DNA binding domain;
WT, wild type;
DR, direct repeat;
ERG, ETS-related gene.
| |
REFERENCES |
| 1.
|
Sternlicht, M., D.,
and Werb, Z.
(1999)
in
Guidebook to the Extracellular Matrix and Adhesion Proteins
(Kreis, T.
, and Vale, R., eds)
, pp. 505-563, Oxford University Press, New York
|
| 2.
|
Vu, T. H.,
and Werb, Z.
(2000)
Genes Dev.
14,
2123-2133[ |
TOP
ABSTRACT
INTRODUCTION
