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INTRODUCTION |
In eukaryotes, gene expression appears to be regulated by the
assembly of various combinations of transcription factors at promoters
and enhancers. The ability of transcription factors to interact
specifically with one another, resulting in the formation of
hetero-oligomeric complexes, enables the generation of diverse inducible and developmentally regulated programs of gene expression. Biochemical and functional characterization of transcription factor complexes has shown that the structural information necessary for their
assembly is provided by both protein-protein and protein-DNA contacts
(1). Particularly, Ets and Jun/Fos family members commonly function as
part of the integrated regulatory complexes. For example, complex
formation of the Ets protein Elk-1/SAP-1 with the serum
responsive factor is critical for the regulation of the
c-fos promoter (2); the Ets-related protein GAPB
forms heterotetramers with GABP
to activate the immediate early
promoters of HSV-1 (3-5); the association of Pu-1 with NF-EM5/Pip is
important for the regulation of immunoglobulin light chain enhancers
(6, 7); and the binding of a Jun/Fos heterodimer along with the nuclear
factor of activated T cells to composite elements is critical for the
regulation of genes involved in T cell activation (8).
The Ets family proteins share a highly conserved 85-amino acid winged
helix-turn-helix DNA-binding domain (ETS domain) that is able to bind
the consensus DNA core sequence 5'-GGA(A/T)-3' and to engage in
protein-protein interactions (9-11). The Jun and Fos families
consist of related bZIP1
proteins that are able to heterodimerize and bind the consensus DNA
sequence 5'-TGACTCA-3' (12). Ets proteins act synergistically with a
variety of other transcription factors to regulate many cellular and
viral promoters and enhancers (9, 10, 13). The Jun/Fos heterodimer is
one of the well characterized partners of Ets proteins (10, 13).
In vitro binding studies have demonstrated a direct
interaction of various Ets proteins like Ets-1, Elf-1, Pu-1, Fli-1,
Ets-2, and Erg with the Jun protein moiety but never with Fos alone
(14-17). These direct protein interactions have been shown to involve
the DNA-binding domain of the two partners. However, the amino acid
residues critical for these highly intricate interactions have not been
precisely mapped so far.
Here we studied the molecular contacts between the human Erg protein
and the Jun/Fos heterodimer to gain insights into the general
mechanisms by which these two families of transcription factors
interact to regulate gene expression. In this respect, we used
molecular modeling techniques and the available crystal structures of
the ETS domain and the Jun/Fos heterodimer complexed to DNA to predict
individual residues involved in the Erg-Jun/Fos-DNA ternary complex
formation. The selected ETS domain residues were thus mutated and then
tested for their ability to support Jun/Fos recruitment in
vitro and in vivo. Interestingly two conserved amino
acids in the ETS domain (residues Arg367 and
Tyr371 in Erg) are required for efficient recruitment of
the Jun/Fos heterodimer. Whereas the R367K substitution abolished DNA
binding, interaction with Jun, and consequently transcriptional
cooperation, the Y371V mutation abrogated interaction with Jun and
synergy without abolishing DNA binding. Therefore the structural
determinants of Erg that are important for ternary complex assembly are
also required for transcriptional synergy. We thus propose that
interdependent protein-protein and protein-DNA contacts regulate
Erg-Jun/Fos-DNA complex assembly. The functional changes induced by
these mutations define the location of a putative conserved Jun binding
interface in the ETS domain.
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EXPERIMENTAL PROCEDURES |
Molecular Modeling--
The proposed ETS-Jun/Fos-DNA complex
model assumes that the recognition consensus DNA sequence 5'-GGAA-3' of
the ETS domain is located upstream from the DNA binding site
5'-TGACTCA-3' of the Jun/Fos heterodimer, as observed in the
polyomavirus enhancer. The aim of this model is to predict
protein-protein interactions when the Jun/Fos heterodimer and the ETS
domain of Erg are simultaneously bound to their respective DNA targets,
as observed on individual crystal structures. This model is based on
the x-ray crystallographic coordinates of the Jun/Fos-DNA (Ref. 18;
Protein Data Bank code 1fos) and Elk-1-DNA (Ref. 19; Protein Data Bank
code 1dux) complexes and was built using the molecular modeling
software Insight II (Molecular Simulations Inc.). The ETS domains of
the Elk-1 and Erg proteins are indeed highly similar, with an overall sequence identity of 55%. The DNA fragment in the Elk-1-DNA complex x-ray structure (19) displays an important bending centered on
7G; only the part of DNA extending from G at position 7 to
T at position 13 (5'-GAAGTGT-3') and its complementary strand were then
considered. Because important conformational rearrangements can occur
upon ternary complex formation that remain unpredictable by molecular
modeling means, we deliberately used a simple rigid docking approach.
First the Jun/Fos heterodimer was replaced at the center of a
30-nucleotide DNA structure adopting a standard double-stranded
B-conformation. Then to investigate individual residues that could be
involved at the protein-protein interface when Elk-1 and Jun/Fos are
simultaneously bound to their DNA targets, we computed all possible
overlapping superimpositions of the selected Elk-1-DNA fragment
(5'-GAAGTGT-3') along the 30-nucleotide standard DNA structure with the
Jun/Fos heterodimer remaining fixed at its center. The set of atoms
used for DNA structure superimpositions were deoxyribose atoms C1',
C2', C3', C4', and O4'. The root mean square values provided were
around 1.5 Å. The ETS domain of the Elk-1-DNA complex was thus
progressively advanced toward the Jun/Fos heterodimer. From all of the
built ETS-Jun/Fos-DNA complexes, only one displays intermolecular
contacts without steric clashes between protein backbones (see Fig. 2).
Finally, facing the simplicity of the followed strategy, the structures
of DNA and proteins being kept rigid, energy minimization was not
appropriate in this context.
Cell Culture--
ROS 17.2/8 (rat osteosarcoma) cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum.
Plasmid Constructions--
The Erg deletion mutant expression
vectors (constructs Erg (1) and Erg (307)) have been
described previously (17). The amino acid substitutions and disrupted
ETS domain derivatives were constructed by polymerase chain reaction
using appropriate primers flanked by convenient restriction sites. Full
details and primer sequences are available on request. Briefly,
deletion mutants were obtained by polymerase chain reaction
amplification and subcloned into pCRII (Invitrogen). Constructs were
then cloned via EcoRI/BglII sites into pSG5
vector for further transcription and translation in vitro.
To construct recombinant pGEX-2TK (Amersham Pharmacia Biotech), the DNA
fragments encoding Jun, DBD, DBD-R367K, DBD-Y371V, and DBD-D374V were
first generated by using polymerase chain reaction amplification of
pSG5 hu-Jun and pSG5 hu-Ergp55, respectively, and subcloned into pCRII
vector. The BamHI/EcoRI (Jun) or EcoRI (DBD) fragments were cloned in-frame into the cloning sites present within the pGEX-2TK polylinker. The amino acid junctions of the GST
vector with the Jun or Erg sequences (in bold type) are: GST-Jun, SVGSMTAK; GST-DBD, GRPVYLGSGQ; GST-DBD-R367K,
IRPVYLGSGQ; GST-DBD-Y371V, GRPVYLGSGQ; and
GST-DBD-D374V, GRPVYLGSGQ. All constructs were verified by
DNA sequencing.
Transfection and Luciferase Assays--
The day before
transfection, ROS 17 2/8 cells were plated at 50-60% confluence in
6-well plates. For transfection, cells were incubated with 1.0 µg of
plasmid DNA and 4 µl of polyethyleneimine (Euromedex,
Souffelweyersheim, France) for 6 h in 1 ml of OptiMEM and then in
fresh complete medium. When necessary, pSG5 plasmid was used as a
carrier. For reporter assays, detailed transfection conditions are
indicated in the relevant figure legend. Cells were lysed 24 h
after transfection and assayed for luciferase activity with a Berthold
(Nashua, NH) chemioluminometer. Results presented are the means of at
least five transfections.
In Vitro Protein Synthesis and Pull-down Assays--
In
vitro translated proteins were generated with a rabbit
reticulocyte in vitro transcription/translation system
(TNT/Promega) and labeling with 50 µCi of
[35S]methionine/50 µl of reticulocyte lysate. The
translation products were visualized by SDS-polyacrylamide gel
electrophoresis and quantified by PhosphorImager (Molecular Dynamics).
The bacterial expression of GST constructs and the purification of GST
fusion proteins were performed as described (17). Expression levels of
the various GST fusion proteins were confirmed by Coomassie staining
(data not shown) and by Western blot (see Fig. 5B).
For pull-down assays, 50 µl of glutathione-Sepharose beads (Amersham
Pharmacia Biotech) were incubated with 1 µg of GST, GST-Jun, GST-DBD,
GST-DBD-R367K, GST-DBD-Y371V, or GST-DBD-D374V fusion proteins in NETN
(20 mM Tris-HCl, pH 8, 200 mM NaCl, 1 mM EDTA, 0, 5% Nonidet P-40) for 1 h at 4 °C.
Beads were washed three times with incubation buffer (12 mM
HEPES, pH 7.9, 4 mM Tris-HCl, pH 7.9, 50 mM
NaCl, 10 mM KCl, 1 mM EDTA) and resuspended in
30 µl of a mixture containing 35S-labeled protein
expressed in reticulocyte lysate and incubation buffer. After 1 h
at 4 °C, beads were washed six times with NETN and mixed with SDS
sample buffer. The bound proteins were analyzed by SDS-polyacrylamide
gel electrophoresis followed by autoradiography.
Electrophoretic Mobility Shift Assays and Western
Blotting--
The DNA binding reaction was performed at room
temperature for 30 min in a total volume of 20 µl containing 20 mM Tris, pH 7.5, 80 mM NaCl, 2 mM dithiothreitol, 0.1% Triton X-100, 5% glycerol, and 5 µg/ml poly(dI-dC). As a probe, we used a double-stranded oligonucleotide corresponding to the polyomavirus enhancer (Py) 5'-GATCTTTAAGCAGGAAGTGACTAACTGACCGCAGGTGGATC-3' or to an ETS consensus (EBS) 5'-GATCTTCGAAACGGAAGTTCGAG-3' and labeled with [
-32P]dATP at a concentration of
10,000 cpm/reaction. Protein-DNA complexes were resolved by 5-10%
polyacrylamide gel containing 2% glycerol in TBE buffer.
Autoradiography was performed on dry gels using an extra film to quench
radioactivity arising from the 35S-labeled proteins.
Before electrophoretic mobility shift assay, GST fusion proteins were
visualized and quantified by Western blotting. Proteins were separated
by SDS-polyacrylamide gel electrophoresis and then transferred to a
Hybond-C Extra membrane (Amersham Pharmacia Biotech) with a Bio-Rad dry
blotter using 25 mM Tris-base, 192 mM glycine, and 20% (v/v) methanol as the transfer buffer. Membrane was blocked in
PBS with 5% (w/v) dry milk for 1.5 h and stained with a rabbit antibody against the ETS domain of the human Erg protein (20) and a
secondary goat anti rabbit/peroxidase antibody (Amersham Pharmacia
Biotech). Antibody incubations were performed for 1.5 h in PBS
with 5% (w/v) dry milk followed by four 15-min washes in PBS with
0.1% Nonidet P-40. For detection we used the ECL chemiluminescent peroxidase substrate kit from Amersham Pharmacia Biotech. For transient
transfection experiments, cell extracts were prepared from confluent
6-well plates. Cells were washed in cold PBS and harvested with Laemmli
SDS sample buffer. Extracts were boiled for 5 min, and samples were
resolved on 12% SDS-polyacrylamide gels and transferred to a Hybond-C
Extra membrane (Amersham Pharmacia Biotech) as for GST fusion detection.
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RESULTS |
Requirements for Erg-Jun/Fos-DNA Ternary Complex
Assembly--
In our previous study (17), we defined functional
domains of the Erg transcription factor (Fig.
1A) and showed that Erg sequences 253-472, including the ETS domain, are necessary for Jun/Fos
recruitment. To better characterize the Erg sequences important for Jun
recruitment and Erg-Jun/Fos-DNA ternary complex assembly, we prepared a
recombinant protein, named GST-DBD, comprising the ETS domain of Erg
(amino acids 307-392 fused to GST) and Jun/Fos proteins
(full-length proteins translated in vitro using rabbit reticulocyte lysates) to perform GST pull-down assay. As shown in Fig.
1B, 35S-labeled Jun (lane 5) and the
Jun/Fos heterodimer (lane 6), but not Fos alone (lane
4), bind specifically to immobilized intact GST-DBD but not to GST
alone (lanes 7-9). Thus these results suggest that the ETS
domain of Erg alone includes minimal sequences for recruiting the
Jun/Fos heterodimer in vitro.

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Fig. 1.
The ETS domain of Erg is involved in
Erg-Jun/Fos-DNA complex formation. A, functional
domains of Erg. The diagram depicts the principal structure/function
domains of Erg as previously described in Ref. 17. PTN,
pointed domain; CAE, central alternative exons;
CD, central domain; ETS domain, ETS domain
represented with its three helices and four -sheets;
CTD, C-terminal transactivator domain. Numbers
indicate the amino acid sequence number. B, interaction of
Jun/Fos with the ETS domain of Erg fused to GST. GST-DBD (lanes
4-6) or GST (lanes 7-9) was tested for association
with in vitro translated 35S-labeled Jun and Fos
alone or both (lanes 1-3). C, cooperative DNA
binding of Erg and Jun/Fos heterodimer on the Py. Band shift analyses
of Jun and Fos proteins prepared by in vitro translation
were coincubated with the GST fusion GST-DBD in the absence
(lanes 1-4) or presence (lanes 5 and
6) of the indicated antibodies ( IgG). All lanes contained
the Py probe. Bands corresponding to the Erg-DBD-Py complex, the
Jun/Fos-Py complex, ternary complex Erg-DBD-Jun/Fos-Py, and the
supershift with GST-IgG are indicated by arrows.
F, free probe; S, supershift. D,
synergistic interaction between Erg and Jun/Fos on the polyomavirus
enhancer. Transient transfection assays were performed in ROS cells by
using the reporter and expression plasmids shown. ROS cells were
transfected with 400 ng of the Py reporter gene and 200 ng of the
indicated expression plasmids. Empty pSG5 vector was used as a carrier
to complement at 1 µg total. Data are expressed as -fold luciferase
activity. Representative assays are shown with bars
indicating the error between triplicates.
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We then performed electrophoretic mobility shift assays with the Py,
known to contain adjacent EBS and AP-1 binding sites and to bind Ets
and Jun/Fos proteins (21). Thus we incubated a 32P-labeled
Py probe with purified GST recombinant proteins, corresponding to the
Erg DNA-binding domain (GST-DBD), and with Jun/Fos proteins cotranslated in vitro. As shown in Fig. 1C, the
Py probe formed, in the presence of both Erg-DBD and Jun/Fos proteins,
a specific ternary complex that migrated more slowly than the secondary
Jun/Fos-Py complex alone (compare lanes 2 and 4).
A similar ternary complex was observed with full-length Erg (data not
shown). Assembly of the ternary complex required both intact EBS and
AP-1 binding sites because mutation of these sequences prevented
ternary complex formation (data not shown). To verify that the ternary
complex contained both Erg-DBD and the Jun/Fos heterodimer, we
incubated the binding reactions with specific antibodies against GST
fusion protein or Jun (
GST-IgG and
Jun-IgG, respectively). The
addition of
Jun-IgG abolished formation of the Jun/Fos-Py and
Erg-DBD-Jun/Fos-Py complexes but not the Erg-DBD-Py complex (Fig.
1C, lane 5). Conversely
GST-IgG hindered
formation of the Erg-DBD-Py and Erg-DBD-Jun/Fos-Py complexes but not
the Jun/Fos-Py complex (lane 6). The functional consequences
of these physical interactions were then analyzed by transient
transfections with a reporter plasmid containing four copies of the
polyomavirus enhancer (Fig. 1D). As expected, cotransfection
of Erg and Jun/Fos proteins resulted in a 10-fold transactivation,
which revealed a cooperative effect between the two partners. Together
these results indicate that Erg and the Jun/Fos heterodimer can form a
ternary complex on the adjacent binding sites of the polyomavirus
enhancer and that physical interactions between the two proteins
contribute to ternary complex assembly.
A Structural Model for the ETS-Jun/Fos-DNA Ternary
Complex--
Although we identified the ETS DNA-binding domain as an
instrumental scaffold for interaction with the Jun/Fos heterodimer and
for transcriptional cooperation, we did not identify amino acids that
participate in this mechanism. To make predictions about residues that
could be involved in interactions between Erg and the Jun/Fos
heterodimer, we modeled an ETS-Jun/Fos-DNA ternary complex (Fig.
2) using the available crystal
coordinates of the Jun/Fos-DNA (18) and Elk-1-DNA complexes (19). The use of Elk-1-DNA instead of Erg-DNA complexes for which no
crystallographic data are as yet available seemed appropriate; there is
close similarity in the overall scaffold of the ETS domains because the
winged helix-turn-helix motif and DNA-contacting residues are strongly conserved (19, 22). The rigid docking strategy followed to build the
ETS-Jun/Fos-DNA ternary complex model is described under "Experimental Procedures."

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Fig. 2.
Model of the ternary complex ETS-Jun/Fos-DNA
highlighting the putative protein interface between the two
partners. This model was built on the basis of x-ray
crystallographic coordinates of the Jun/Fos-DNA (18) and Elk-1-DNA
complexes (19) using the molecular modeling program Insight II
(Molecular Simulations Inc.). Residues located at the interface are
depicted in sticks: Lys267 in Jun
(K267), Arg62, Tyr66, and
Asp69 in Elk-1 conserved in Erg (respectively,
Arg367, Tyr371, and Asp374).
The backbones of macromolecules are represented by ribbons,
Fos is in yellow, Jun is in red, the ETS domain
of Elk-1 is in blue, and standard B-DNA is in
pink. In addition, the LXXLL motif of the ETS
domain is shown in green.
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In our ETS-Jun/Fos-DNA model (Fig. 2), the two protein partners bind to
the major groove on opposite sides of the DNA helix, positioning the
helix
3 of the ETS domain and the N-terminal part of the
Jun basic domain in close proximity. Principally we located Jun residue
Lys267 and Elk-1 residues Arg62,
Tyr66, and Asp69 conserved in Erg
(Arg367, Tyr371, and Asp374) at the
protein-protein interface. As observed in the different available
crystal structures of ETS-DNA complexes (5, 19, 23, 24), the arginine
residue Arg62 (Arg367 in Erg) is involved in
hydrogen bonds with the DNA core sequence 5'-GGAA-3' and seems to be
unavailable for supplementary interactions. Moreover the positive
charge of its guanidinium group should not allow favorable interactions
with the N-terminal basic domain of Jun, which is also positively
charged. On the other hand, the phenol ring of Tyr66
(Tyr371 in Erg) involved in DNA contacts in the SAP-1-DNA
complex structure (24) but not in Elk-1-DNA (19) and the carboxylate
group of Asp69 (Asp374 in Erg), well accessible
at the protein surface, could interact with the basic residue
Lys267 of Jun (Fig. 2). However, if Tyr66 is
highly conserved within the sequences of the ETS family and may thus be
instrumental, Asp69 is frequently mutated (22) and thus may
not play a major role in ETS-Jun/Fos complex formation (see below).
The Helix
3 of the ETS Domain Is Required for
Interaction with Both DNA and Jun--
Structural studies have
revealed that the ETS domain forms a winged helix-turn-helix motif
described to fold into a four-stranded anti-parallel
-sheet with
three
-helices (Fig. 3A),
where helix
3 is the major DNA recognition component
(19, 22, 24). Our model (Fig. 2) suggests that this helix
3 should also include sequences allowing the recruitment
of the Jun/Fos heterodimer. To test this hypothesis, we prepared a set
of differentially truncated polypeptides in the ETS domain and
expressed in rabbit reticulocyte lysates. We then generated different
Erg protein mutants in which either the first helix
1,
both helices
1 and
2, or both
3 and
4
-sheets of the ETS domain were
deleted (Fig. 4A). As
previously described (25), the integrity of the 85-amino acid ETS
domain is necessary to bind an EBS, whereas Erg proteins lacking
N-terminal helix
1, helices
1 and
2, or
3 and
4
-sheet
failed to bind DNA (Fig. 4B). Strikingly our pull-down
assays using glutathione S-transferase (GST) fusion proteins
indicated that the ETS domain retained the ability to interact with
GST-Jun (Fig. 4C, lanes 3, 6,
9, 12, 15, and 18), even
when the
1 and
2 helices or the
3 and
4
-sheets had been deleted and
the DNA binding had been abolished (Fig. 4, B and
C). Interestingly, these results revealed that deletion of
the helix
1 did not affect Erg-Jun/Fos interaction, excluding the possibility that the conserved LXXLL motif, a
potential protein-protein interface (26, 27), participates in the
binding of Jun/Fos. Moreover Erg and Jun proteins could form a complex independently of DNA binding because specific interactions between the
Jun/Fos heterodimer and the ETS domain of Erg were also observed in the
presence of ethidium bromide (data not shown), which is known to
disrupt DNA-protein interactions (28). Thus although the ETS domain of
Erg is the minimal Jun/Fos interaction domain, altering structural
features such as the secondary structures can be without effect on the
interaction with Jun. We have previously shown that deletion of the
whole ETS domain abolishes Jun binding (17). Consequently all these
results point out the DNA recognition helix
3 as the
major ETS domain component for interaction with Jun protein.

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Fig. 3.
The Erg ETS domain and its relationship to
other ETS domain-DNA structures. A, amino acid
sequences of the ETS domain of Erg and other members of the Ets family.
The secondary structures are indicated above the sequence;
helices are represented by boxes, and strands are
indicated by arrows. Strictly conserved residues that make
DNA contacts are indicated with black squares (19, 24), the
conserved Arg367, Tyr371, and
Asp374 residues are boxed, and the amino acid
substitutions used in this study are indicated with arrows.
B, amino acid sequences of the basic domain of Jun and Fos
families (44). The variant amino acid Lys267 of Jun singled
out by our model and the corresponding Arg143 of Fos are
boxed.
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Fig. 4.
Identification of Erg ETS domain regions
essential for interaction with Jun/Fos. A, structure of
the Erg deletion mutants. Amino acid numbers of encoded proteins are
indicated for each construct. Interactions observed in B and
C are summarized on the right. The minimal domain
(helix 3) that is involved in interaction with Jun is
indicated. B, band shift analysis of wild type and Erg
protein mutants. 5-10 µl of in vitro translated proteins
were incubated with the labeled ETS consensus oligonucleotide
5'-GATCTTCGAAACGGAAGTTCGAG-3'. The empty vector pSG5
utilized as a control is indicated. F, free probe. C,
GST pull-down assays showing the interaction between GST-Jun and Erg,
using the deletion constructs shown in A.
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Point Mutations in the Erg ETS Domain Impair Its Ability to
Interact with Jun/Fos Heterodimer--
We then attempted to locate in
the helix
3 the amino acids more specifically involved
in Erg-Jun/Fos interactions. Based on our molecular model of the
ETS-Jun/Fos-DNA ternary complex, we identified essentially two Erg
residues Tyr371 and Asp374 (Tyr66
and Asp69 in Elk-1) (Fig. 2). In the x-ray structure of the
SAP-1-DNA complex (24), the tyrosine residue (Tyr371 in
Erg) clearly participates in DNA recognition by contacting the thymine
located on the complementary strand to the 5'-GGAA-3' core sequence.
However this residue seems not strictly required for DNA binding
because its substitution does not always abolish DNA recognition (22,
29). Note also that in the Elk-1-DNA complex crystal structure, no
direct contacts of this residues are observed with DNA (19). Aspartic
acid 374 of Erg (Asp69 of Elk-1 and Val68 of
SAP-1) does not interfere with DNA binding but was recently described
to be a key residue for Pax-Ets-DNA ternary complex formation
(30, 31). Finally, with respect to the arginine residue
Arg367 of Erg also present at the putative protein
interface region, no direct contact with Jun is expected. However in
absence of DNA, the helix
3 has been shown to be
involved in interactions with Jun, making this residue still an
instrumental candidate (Fig. 4). Therefore, we used
oligonucleotide-directed mutagenesis to convert Tyr371 to
valine, Asp374 to valine, and Arg367 to
lysine in the context of the intact ETS domain of Erg (307) fused to GST (Fig. 5A).
Substitution of Arg367 with lysine should affect both DNA
binding and Jun interaction, whereas substitution of Tyr371
with valine and Asp374 with valine would be expected to
impair only the binding of Erg to Jun.

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Fig. 5.
The R367K and Y371V mutations in the Erg ETS
domain strongly influence the interaction of Erg with Jun/Fos.
A, structure of the Erg mutants in the context of the ETS
domain fused to GST. Interactions observed in B-E are
summarized on the right. The substitutions are indicated by
arrows. B, band shift analyses of wild type and
Erg ETS mutants. Top panel, purified GST fusion (500 ng,
lanes 1-5) were incubated as in Fig. 4B.
Bottom panel, Western blot analysis of GST fusion
(lanes 1-5). Molecular mass markers (in kDa) are
indicated. C, interaction of Jun/Fos with the GST-DBD.
GST-DBD (lanes 4-6), GST-DBD-R367K (lanes 7-9),
GST alone (lanes 10-12) was tested for association with
in vitro translated Jun and Fos alone or together
(lanes 1-3). D, ability of wild type Erg DBD and
two mutants, Y371V and D374V, to bind with the Jun/Fos heterodimer.
Experiments were performed as in C. E, mutations
that impair the ability of Erg to interact with Jun/Fos or to bind DNA
have no effect on their capacity to homodimerize.
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As expected (22, 29), the mutation R367K abolished DNA binding (Fig.
5B, lane 3). On the other hand, the Y371V mutant exhibited a lower DNA binding in comparison with the wild type ETS
domain, whereas D374V binds DNA with a stronger affinity (Fig. 5B, lanes 4 and 5, respectively). By
in vitro competition binding assays, we estimated that the
Y371V mutant showed approximately a 5-fold decrease in DNA binding
(data not shown). However, in the context of the full-length protein,
this Y371V mutant entirely retained the transcriptional activity (Fig.
6B).

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Fig. 6.
Functional cooperation between Jun/Fos and
various Erg mutants. A, structure of the constructs
used in transient transfections. The indicated point mutations in the
ETS domain of Erg were introduced in the context of the full-length
protein and cloned into pSG5. B, ROS cells were
cotransfected with the polyomavirus enhancer-Luc reporter and different
Erg mutants as indicated in the absence (B) or in presence
(C) of Jun/Fos. Results are expressed as -fold activation
relative to basal promoter activity. D, analysis of the Erg
mutants expression by Western blotting. ROS cells were transfected with
1 µg of different Erg mutants as indicated. Cells were harvested as
described under "Experimental Procedures," and extracts were
analyzed by SDS-gel electrophoresis and probed with anti-Erg antibody
(1:1000) and developed by ECL (Amersham Pharmacia Biotech) according to
the manufacturer's protocol.
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In GST pull-down assay, equivalent amounts of beads and bound Erg ETS
domain fusion proteins based upon protein determination and staining of
samples resolved by SDS-polyacrylamide gel electrophoresis were used,
and expression levels of intact fusion proteins were monitored by
Western blotting with an antibody against the ETS domain of the human
Erg protein (Fig. 5B). Strikingly the R367K mutant, selected
for noninteraction with DNA, scarcely interacted with Jun or the
Jun/Fos heterodimer (Fig. 5C, compare lanes 5 and
6 with lanes 8 and 9). Conversely,
although the mutation Y371V is not deleterious for DNA binding
by Erg (Fig. 5B), it strongly decreases recruitment of the
Jun moiety. On the other hand, the D374V substitution had no effect
(Fig. 5D, compare lanes 2-4).
To exclude the possibility that these functional changes are due to an
alteration of the overall folding of the mutants, we have taken
advantage of the ability of Erg proteins to homodimerize (17). Indeed,
we recently showed that the Erg proteins could form homodimers in
vitro and that sequences required for this purpose are included in
the ETS domain (17). Thus mutant proteins that fail to interact with
Jun/Fos (Fig. 5, C and D) are still able to
homodimerize (Fig. 5E). Taken together, these data indicate that amino acids Arg367 and Tyr371 of the ETS
domain, but not Asp374, play an important role in the
recruitment of the Jun/Fos heterodimer by Erg, whereas the Erg
homodimerization is independent of the mutated residues.
Erg-Jun/Fos Interaction Mutants Fail to Cooperate from Ets-AP-1
Composite Sites--
The experiments described above demonstrated that
critical amino acids in the DNA recognition helix
3 of
the ETS domain could interact with the Jun/Fos heterodimer but did not
address whether it functioned in Ets-AP-1-regulated gene expression.
Cotransfection assays were used to determine whether mutations that
abrogated physical interaction with Jun would influence transcriptional synergy. Briefly ROS cells were transfected with a reporter gene containing four copies of the polyomavirus enhancer (Py), with expression vectors for Erg mutants (Fig. 6A) and Jun/Fos.
Expression of intact wild type Erg activated the Py enhancer ~4-fold
over vector control (Fig. 6B). As expected, the mutant
R367K, which is defective for DNA binding, is also inactive for
transactivation (Fig. 6B). Despite the difference of their
in vitro DNA binding capacities observed with respect to the
wild type protein (Fig. 5B), the Y371V and D374V mutations
did not affect Erg transactivation of the Py enhancer. This apparent
discrepancy cannot be a consequence of overexpression because all the
mutant proteins were expressed in transfected cells at the same levels
as the wild type Erg protein (Fig. 6D).
We next examined the effect of these Erg mutants on their functional
interaction with Jun/Fos (Fig. 6C). Transfection of Jun/Fos alone activated the Py enhancer about 3-fold (Fig. 6C),
whereas Erg alone activated the Py enhancer 4-fold (Fig.
6B). However in the presence of both Erg and Jun/Fos, Py
enhancer is synergistically enhanced to 10-fold. Significant functional
synergism between Erg and Jun/Fos is also revealed by the Erg-D374V
mutant, whereas Erg-R367K abolished cooperation (Fig. 6C).
Interestingly, the Erg-Y371V mutant failed to achieve synergy with
Jun/Fos because it cooperated only 6-fold.
To evaluate and quantitate more accurately the effects of the Erg point
mutations on the Ets/AP-1 synergistic response, we calculated a true
synergy fold. The Ets/AP-1 synergy fold is defined as Py enhancer
activity in the presence of a combination of Erg plus Jun/Fos
constructs divided by the sum of the individual fold activations
induced by Jun/Fos alone and each Erg mutant construct alone. Thus
cotransfection of wild type Erg and Jun/Fos resulted in levels of Py
enhancer activity about 2-fold greater than that predicted based on the
sum of their individual responses. By contrast a synergy fold value of
1 would reveal an additive response, thereby indicating a loss of
functional interaction. Indeed the data presented in Fig. 6C
show that the Erg mutant D374V retained synergistic activation of the
Py enhancer in combination with Jun/Fos, with a synergy fold value of
about 2, as the Erg wild type. In contrast R367K or Y371V substitutions
abrogated Ets/AP-1 synergy, reflected by synergy fold values lower than
1. Thus physical interactions between the ETS domain of Erg and Jun are
necessary for proper Erg-Jun/Fos synergistic activation of the Py enhancer.
 |
DISCUSSION |
In this report we have explored the role of specific residues
located at the protein interface of the Erg-Jun/Fos-DNA complex, focusing on their functional importance in regulating gene expression. Previously we and others showed that the ETS domain was critical for
interaction with Jun/Fos (14-17). By molecular modeling and mutational
studies, we demonstrated here that the helix
3 of the
ETS domain, known to be involved in DNA recognition, is also involved
in Jun/Fos recruitment. Two substitutions in this helix (Y371V
and D374V in Erg) preserve DNA binding in vitro, whereas a
third (R367K) abolishes both DNA binding and Jun/Fos recruitment. However when coexpressed with the Jun/Fos heterodimer, the Y371V mutant
did not display a transactivation cooperation similar to that observed
with the D374V mutant or the wild type. Thus Tyr371 of Erg
is clearly required for the interaction with Jun/Fos and also for the
synergistic activation of the Erg-Jun/Fos-DNA complex.
Overall Conserved Structure of the Winged Helix-turn-Helix-bZIP-DNA
Complex?--
Our initial goal was to distinguish between ETS domain
functions involved in DNA binding and protein-protein interaction. We
firstly reasoned that the structural determinants required for
Erg-Jun/Fos complex formation are located away from the DNA recognition
helix
3. Therefore we investigated the role of the conserved LXXLL motif located in helix
1
(Figs. 2 and 3A) as a potential protein-protein interface
because this motif has been described to be sufficient for
ligand-dependent interaction between nuclear receptors and
coactivators (26, 27). We thus introduced the L320A mutation in this
motif of the Erg protein. However this substitution together with the
deletion of the helix
1 (Fig. 4C) has no
effect on interaction with Jun or on DNA binding (data not shown).
Surprisingly our experimental data coupled with our molecular model
rather suggest that helix
3 defines the location of a
Jun binding interface in the ETS domain. Interestingly the recent
crystal structure of the winged helix-turn-helix IRF-2 protein (32)
lead to the proposal of a model of bZIP-IRF-DNA complex formation at
the interferon-
enhancer (32, 33); both cases are reminiscent of
ours (Fig. 2). It is noteworthy that the regions of closest approach
between ATF-2/Jun and IRF-3 are the bZIP N terminus and the IRF DNA
recognition helix (32, 33). Differences in amino acid sequence between
ATF-2, Jun, and Fos and between IRF-1, IRF-3, and Ets may underlie the
specific structural and transcriptional properties of the distinct complexes.
In good agreement with this idea, we note that the Jun family members
have been shown to interact with Ets proteins, whereas the Fos family
members do not share this property (14). This suggests that amino acids
shared by Jun family members but not present in Fos family members
could interact with the Ets proteins. A mapping of the minimal Jun
domain (14) involved in Ets-Jun interactions located the interaction
within residues 263-282 in the basic domain of Jun. The
Lys267 Jun residue singled out by our model is precisely
located within this domain and thus represents a suitable candidate for
the bridging with Erg through Tyr371 (Fig. 2). Intriguingly
alignment of this restricted domain from different Jun and Fos proteins
revealed that residue Lys267 is conserved in Jun proteins
but not in Fos proteins where it is replaced by Arg143
(Fig. 3B). We thus attempted to create a Fos compensatory
mutant R143K that could restore interaction with the ETS domain.
However the mutation R143K alone is not sufficient (data not shown),
suggesting that residues at the periphery of Lys267 of Jun
are also instrumental (Fig. 3B).
Identification of Mutations That Disrupt the Erg-Jun/Fos
Interaction without Abolishing DNA Binding by Erg--
A key amino
acid for Jun/Fos recruitment by Erg is an almost invariant tyrosine
residue present in the DNA recognition helix
3 (Figs. 2
and 3A). In our ternary complex model, this residue is
located close to the basic domain of Jun, suggesting that it could play
a direct role in interactions between these two proteins. The
substitution Y371V in the ETS domain alters the affinity of Erg for an
EBS probe (Fig. 5B), suggesting that the aromatic residue could interact directly with DNA, as observed in the SAP-1-DNA complex
with the corresponding residue Tyr65 (24). However this
mutation drastically hinders the Erg-Jun/Fos complex formation (Fig.
5D). Hence because of a reorientation of its side chain, the
tyrosine residue could prefer to interact with the Jun protein in the
Erg-Jun/Fos-DNA ternary complex. Recently, a certain flexibility was
indeed reported for this residue because its side chain clearly adopted
two different conformations in Elk-1-DNA (19) and SAP-1-DNA complexes
(24). In this context, Tyr371 of Erg could act as a
discriminator of protein interactions/coactivation and DNA binding.
According to the available crystal structures of ETS domains (5, 19,
23, 24), the absolutely conserved residue arginine 367 of Erg is
clearly crucial for DNA binding. It is thus not surprising that
mutation R367K abolishes DNA binding in vitro and
consequently showed no transcriptional activation. Initially we
envisioned an indirect role for this residue in Erg-Jun/Fos complex
assembly. Indeed, in SAP-1-DNA complex structure (24), the guanidinium
group of Arg61 (Arg367 in Erg) is engaged in
hydrogen bonds with the DNA core sequence 5'-GGAA-3' but also makes
close contacts with the phenol ring of Tyr65
(Tyr371 in Erg). The mutation of the conserved arginine
residue by a lysine could induce a rearrangement of the contiguous
aromatic side chain and then modify interactions of Erg protein with
Jun/Fos heterodimer.
The ETS DNA-binding Domain: One Domain, Multiple
Functions--
Partnerships between transcription factors are required
for specific binding and control of gene expression. Although these interactions are quite common, the structural basis for complex formation and identification of instrumental amino acids have only been
determined for a small number of examples (5, 8, 30, 31, 34-36). For
instance, the isolated DNA-binding domain of GATA proteins mediates
physical interactions with FOG (34, 36). However all the mutant GATA-1
proteins that impaired FOG interaction retained the ability to bind DNA
(35, 36). In our case, unlike GATA-1, mutants of Erg, such as R367K,
selected for noninteraction with DNA, also prevent association with the Jun/Fos heterodimer. Similarly the ability of Ets proteins to be
recruited by Pax-5 coincides with the presence of a specific aspartic
acid (Asp374 of Erg in this study) within the ETS
DNA-binding domain (30, 31). However, Ets protein (SAP-1, which has a
valine residue instead of an aspartic acid), which impaired Pax-5
binding, retained the ability to bind DNA (30, 31). Importantly
mutation of this position had no effect on Erg-Jun/Fos complex
formation (Fig. 5D), suggesting that the structural
determinants required for this complex overlap with but can be
uncoupled from those required for Pax-5/Ets complex formation.
The idea of considering the ETS DNA-binding domains as protein-protein
interaction domains has begun to emerge recently. In particular,
a growing number of protein-protein interactions have been shown to be
mediated by the Pu-1 ETS domain. Indeed, Pu-1 has previously been found
to interact with Jun family members (14, 37), proteins of the C/EBP
family (38, 39), and erythroid zinc finger transcription factor GATA
(40-42). Strikingly the binding surfaces for Erg and Pu-1 on Jun/Fos
appear to differ significantly (41). In our study (Fig. 4C),
unlike Pu-1, the
3/
4 domain does not seem
to be involved in the recruitment of Jun. A functional significance of
this apparent discrepancy could be overcome by the unconserved position
Tyr371 in Erg versus Asn236 in Pu-1
(Fig. 3A). As we strongly demonstrated the instrumental role
of this Tyr371 residue in Erg for Jun recruitment as well
as transcriptional synergy (Figs. 5D and 6C), we
concluded that the adaptation of different mechanisms for
protein-protein interactions reflects the structural and functional
diversity of Ets proteins. Interestingly, recent work also suggests
that the binding surfaces for Fli-1 and Elk-1 on serum response factor
are different (43). Further detailed mutagenesis is required to define
precisely the mechanism of complex formation involving Ets proteins. In
addition, we presently also do not exclude the possibility that
although mutating the Tyr371 residue abrogates Erg/Jun
interaction in vitro, in vivo this mutation may
not be sufficient to fully inhibit the formation of a complex
comprising full-length Erg-Jun/Fos, which may be stabilized by other interactions.
Interestingly, Erg mutant proteins that fail to interact with Jun/Fos
are still able to homodimerize (Fig. 5E), suggesting that
homodimerization is independent of the mutated residues. Because the
homodimerization of Erg proteins seems to be incompatible with DNA
binding and gene regulation (17), this raises the intriguing possibility that formation of the Erg-Jun/Fos ternary complex leads to
"derepression" of Erg homodimers by protein-protein interactions. This hypothesis is currently under investigation. In conclusion, our
studies provide a framework for understanding the molecular mechanism
of the physical interactions between Jun/Fos, Erg proteins, and DNA and
are compatible with the following observations: (i) helix
3 defines the location of a putative conserved Jun
binding interface in the ETS domain, (ii) the Tyr371
residue is not critically involved in efficient DNA binding, but (iii)
its integrity is required for an efficient Erg-Jun/Fos transcriptional
cooperation. Finally, whether these particular transcriptional
conformations could be extended to all the transcription complexes
involving the ETS and bZIP family members remains to be assessed.