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INTRODUCTION |
Eukaryotic transcription factors are grouped into families based
on the type of DNA-binding domains that they possess. Due to high
sequence conservation within these DNA-binding domains, members of
particular families bind to similar DNA sequences. Thus, mechanisms
must be in place to prevent promiscuous binding to inappropriate sites
and provide specificity to ensure binding to the correct sites. One
such mechanism is autoinhibition, whereby proteins block their own DNA
binding by employing cis-acting inhibitory modules (reviewed in Ref.
1). These modules also often act to block other activities of
transcription factors such as transcriptional activation or nuclear
localization. Conversely, mechanisms exist to reverse these inhibitory
interactions and allow recruitment of proteins to the correct
promoter/enhancer sites at appropriate times. Activating triggers can
include post-translational modifications such as phosphorylation or the
binding to other coregulatory proteins or transcription factors.
The transcription factor Ets-1 represents a paradigm for this type of
regulatory mechanism. Ets-1 is part of the larger ETS-domain family of
proteins that bind to sequences containing the central core
trinucleotide GGA (2, 3). Sequences flanking this motif permit subtle
variations in binding specificities for individual family members. Due
to this relatively low stringency of binding, many ETS-domain
transcription factors possess autoinhibitory domains, including Elk-1
(4), PU.1 (5), and members of the PEA3 subfamily (6, 7, 8). DNA binding
by Ets-1 is tightly controlled by an inhibitory module composed of
-helices lying N- and C-terminal to the ETS-domain (9, 10).
Disruption of one of the N-terminal
-helices results in relief of
autoinhibition and permits high affinity DNA binding (11, 12). Ets-1
has been shown to bind to DNA cooperatively with several transcription
factors including USF-1 (13) and AML-1/CBF
2 (14-16). In the case of
the Ets-1·AML-1 complex, this cooperativity appears to result
from interaction of the two proteins and subsequent mutual disruption
of inhibitory modules in each protein, thereby leading to a loss of
autoinhibition (13, 17, 18). Alternative routes for relief of
autoinhibition have not yet been demonstrated in Ets-1, but in a
different ETS-domain transcription factor Elk-1, phosphorylation has
been shown to act as a trigger to relieve autoinhibition and thereby
stimulate DNA binding (14, 19). Furthermore, in the case of Elk-1, DNA binding is also subject to further control and is negatively regulated in trans, by members of the Id subfamily of helix-loop-helix
(HLH)1 proteins (20).
In this study we have investigated how DNA binding is regulated in a
member of a different subfamily of ETS-domain transcription factors,
PEA3. The PEA3 group of ETS-domain transcription factors contains three
different proteins, PEA3/E1AF, ERM, and ER81 (reviewed in Ref. 21).
Members of this group of proteins are conserved at both the sequence
and functional levels in vertebrates as diverse as humans and zebrafish
(6, 22). Biological roles for PEA3 family members include promoting
muscle cell differentiation (23) and the definition of connecting
muscle motor and sensory neurones (24). Embryonic expression patterns
and aberrant expression in metastatic tumors, suggest a role for PEA3
proteins in regulating cell motility during organogenesis/development
and during tumor progression (reviewed in Ref. 21).
We recently demonstrated that in common with the other ETS-domain
transcription factors, murine PEA3 and human ERM (7, 8), DNA binding by
zebrafish PEA3 is subject to stringent regulation by autoinhibitory
mechanisms (6). Here we investigate the molecular basis for this
inhibitory process in zebrafish PEA3 and demonstrate one route by which
this autoinhibition can be relieved. Our results point to an intriguing
model in which the regions that autoregulate DNA binding by PEA3 also
participate in cooperative DNA binding with the partner protein USF-1,
thereby coordinately regulating the loss of autoinhibition, with the
stimulation of nucleoprotein complex formation.
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MATERIALS AND METHODS |
Plasmid Constructs--
The following plasmids were used for
making proteins by in vitro transcription/translation.
pcDNA3Id3 (encoding full-length Id3) (25), pAS477 (encoding
full-length zebrafish PEA3; amino acids 1-494), pAS283 (encoding PEA3
amino acids 341-432), pAS330 (encoding PEA3 amino acids 1-432), and
pAS331 (encoding PEA3 amino acids 341-494) have been described
previously (6). pAS530 (encoding PEA3 amino acids 321-494), pAS531
(encoding PEA3 amino acids 277-494), pAS532 (encoding PEA3 amino acids
258-494), pAS533 (encoding PEA3 amino acids 258-452), pAS604
(encoding PEA3 amino acids 321-452), pAS605 (encoding PEA3 amino acids
229-494), pAS774 (encoding PEA3 amino acids 321-474), and pAS1153
(encoding PEA3 amino acids 321-432) are deletion derivatives of pAS477
and were subcloned as NcoI-SacI-cleaved PCR
products into the same sites in pAS37 (26). The following plasmids
encode the indicated mutant derivatives of PEA3. pAS619 (PEA3-(258-452)-R339P) and pAS1165 (PEA3-(258-452)-V330P) were constructed from the template pAS533. pAS1155 (PEA3-(321-474)-E460P), pAS1156 (PEA3-(321-474)-L468P), pAS1163
(PEA3-(321-474)-K450P), pAS1164 (PEA3-(321-474)-F453P), and pAS1166
(PEA3-(321-474)-I437P) were constructed from the template
pAS774. Site-directed mutations were introduced by a two-step PCR
protocol using a mutagenic primer and two flanking primers as described
previously (27). Details of mutagenic primers can be supplied upon request.
The following plasmids were used for expressing PEA3 derivatives as
C-terminal hexahistidine/FLAG-tagged proteins in
Escherichia coli. pAS609, pAS610, pAS1161, and pAS1162
(encoding PEA3 derivatives; PEA3-(321-452), PEA3-(341-432),
PEA3-(341-452), and PEA3-(321-432)) were constructed by inserting the
NcoI-XhoI-digested PCR-derived fragments into the
same sites in pET-Hnef-PFH (28). C-terminal hexahistidine-tagged USF-1
derivatives were expressed in E. coli from the plasmids
pET3his-USFII (encoding full-length USF-1), pEC12 (encoding
USF-1-(144-310)) and pEC14 (encoding USF-1-(197-310)) (29). The
following plasmids were used for expressing GST fusion proteins in
E. coli: pGEX-Id2 (encoding full-length Id2) (30) and pAS462
(encoding PEA3-(341-432)) (6). pAS992, pAS998, and pAS999, encode GST
fusions to PEA3-(1-494), PEA3-(1-337), and PEA3-(430-494),
respectively, and were constructed by inserting either an
NcoI/XhoI fragment from pAS501 or
BamHI/XbaI- cleaved PCR products (primer pairs
ADS730/753 and ADS754/755, respectively) into the same sites in pGEX-KG
(31).
The following plasmids were constructed and used for use in mammalian
cell transfections. pCH110 encodes a SV40 promoter-driven
-galactosidase gene (Amersham Pharmacia Biotech). pAS501 (encoding full-length zebrafish PEA3) (6), pSG5-USF-1 (pAS849; encoding full-length human USF-1), and pSG5-USF-2 (pAS850; encoding full-length human USF-2) (32) have been described previously. pAS849 and pAS850
were also used for in vitro transcription-translation
purposes. pAS1123 encodes a luciferase gene driven by two copies of the E-box/ets motif derived from the HIV-1 LTR and was constructed by
inserting the annealed oligonucleotides ADS671/ADS672 into the
SalI site of pAS819 (pT81, kindly provided by E. Oettgen). Both copies are inserted in the inverted orientation in comparison to
the natural HIV-1 LTR.
The following plasmids were constructed for use in yeast one-hybrid
assays. pAS468 encoding PEA3-(341-494) was constructed by ligating a
BamHI/SpeI-cleaved PCR product (primer pairs
ADS288/262 on template pAS333) into the same sites in pMSe4 (33).
pAS497 and pAS613, encoding PEA3-(1-494) and PEA3-(258-494),
respectively, were constructed by ligating
SpeI/NcoI fragments from pAS360 and pAS343 into
the same sites in pAS468.
The sequences of all plasmids constructed from PCR-derived products
were verified by automated dideoxy sequencing.
Protein Expression and Purification--
In vitro
transcription and translations were carried out as described previously
(34). Hexahistidine-tagged and GST fusion proteins were expressed in
the E. coli strains BL21(DE3) and JM101, respectively, and
purified as described previously (29, 35, 36).
Gel Retardation Assays--
Gel retardation assays were
performed in 12-µl reaction volumes as described previously (37). The
binding sites used were the E74 site (38), the E-box/ets site found in
the HIV-1 LTR (oligonucleotide pair ADS671/ADS672; top strand,
5'-TCGATCATCACGTGGCCCGAGAGCTGCATCCGGAGTAC-3') and the mutated version of the E-box/ets site (oligonucleotide pair
ADS881/ADS882; top strand,
5'-TCGATCATCACGTGGCCCGAGAGCTGCATGGGGAGTAC-3'). The ets-motif and E-box are underlined and
italicized, respectively. The mutated bases are indicated in
boldface. In competition assays with Id3, the Id3 and PEA3
proteins were allowed to bind for 15 min at room temperature before the
addition of 32P-labeled DNA probe. Antibody supershifts
were carried out by adding 1-2.5 µl of anti-FLAG M2 antibody (Sigma)
together with PEA3 derivatives prior to incubation with the DNA.
Protein·DNA complexes were resolved on nondenaturing polyacrylamide
gels cast in either 0.5× or 1× TBE (Tris-borate (90 mM), EDTA (2 mM)).
In Vitro Protein·Protein Interaction Assays--
Interactions
between PEA3 and Id or USF proteins were investigated using GST
pull-down assays as previously described (38).
Cell Culture, Transfection, and Reporter Gene Assays--
293
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Life Technologies, Inc.).
Duplicate transfection experiments were carried out in six-well plates
using Superfect reagent according to the manufacturer's recommendations (Qiagen). Following transfection, cells were incubated in 0.5% fetal calf serum (Life Technologies, Inc.) for 24 h prior to cell extract preparation and luciferase assays as described previously (36). Transfection efficiencies were standardized by
normalizing for the
-galactosidase activity from cotransfected pCH110 as described previously (36).
Yeast One-hybrid Screening--
Yeast one-hybrid screening was
carried out by an adaptation of the protocol described previously (33).
The yeast reporter strain Ets-Lacrep
was constructed by integrating the pMSv1 reporter (33) into the
his3 locus of the Saccharomyces cerevisiae strain
W3031A (MAT
, ho, his3-11,15;
trp1-1; ade2-1; leu2-3,112;
ura3; can1-100). Untransformed Ets-Lacrep was grown in YEPD medium
(2% Bactopeptone, 1% yeast extract, 2% glucose) at 30 °C with
shaking. Full-length PEA3 (pAS497) was used as a bait in a yeast
one-hybrid screen, using the reporter strain
Ets-Lacrep and a mouse 11-day embryonic library
cloned in pGAD10 (CLONTECH). The PEA3 expression
vector pAS497 contains a galactose-inducible promoter. However, because
induction with galactose resulted in a block in cell growth, presumably
due to the toxicity of the PEA3 protein, no induction was used in the
screen and we relied upon leaky expression of the bait protein.
Transformations were carried out essentially as described previously
(39) and potential positives were identified by filter assays for
-galactosidase activity. Filter and liquid assays for
-galactosidase activity were carried out essentially as described
previously (33). Positive clones were verified by retransforming the
Ets-Lacrep reporter strain with DNA recovered
from potential "positives" in the presence and absence of the bait
protein. Positive interacting clones that activated the reporter only
in the presence of the bait protein were sequenced, and the identity of
sequences elucidated using NCBI BLAST searches. Two screens (total of
157,000 colonies screened) resulted in the identification of 13 clones
that scored positive in the above tests. One of these, pAS956, encodes
the C-terminal part of mouse USF-1.
Figure Generation and Data Quantification--
All figures were
generated electronically from scanned images of autoradiographic images
using Picture Publisher (Micrografix) or Adobe PhotoDeluxe and
PowerPoint version 7.0 (Microsoft) software. Final images are
representative of the original autoradiographic images.
Phosphorimaging data were quantified using Tina software (version 2.08e).
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RESULTS |
Mapping and Characterization of the Autoinhibitory Cis-acting
Modules in PEA3--
DNA binding by members of the PEA3 subfamily of
ETS-domain transcription factors has previously been shown to be
regulated by autoinhibitory mechanisms (6-8). In the case of zebrafish PEA3, three regions have been shown to be involved in regulating DNA
binding (Fig. 1A) (6). Deletion of region
I led to a small increase in DNA binding, whereas more dramatic
increases in DNA binding were observed upon deletion of regions II and
III. Combined deletion of regions II and III, to leave just the ETS
DNA-binding domain, led to further enhancement of DNA binding by
PEA3.

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Fig. 1.
Mapping the DNA binding inhibitory domains in
PEA3. A, schematic representation of a series of
truncated PEA3 constructs. The ETS DNA-binding domain is indicated by a
white box and the N-terminal acidic domain by a gray
box. The regions previously determined to inhibit DNA binding by
PEA3 (6) are indicated (I-III). B, gel
retardation analysis of N-terminally and C-terminally truncated PEA3
proteins. Equal molar quantities of the indicated in vitro
translated PEA3 derivatives were bound to the E74 site.
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To further define the extents of the inhibitory domains II and III, a
series of finer N- and C-terminal deleted proteins were constructed
(Fig. 1A). Sequential deletion of N-terminal sequences up to
amino acid 321 did not result in significant increases in DNA binding
(Fig. 1B, lanes 1-5). However, a large increase
in the efficiency of DNA binding was observed upon deletion to amino acid 341 (corresponding to the start of the ETS-domain), which was
further enhanced upon simultaneous deletion of the sequences located
C-terminally to the ETS-domain (Fig. 1B; lanes 6 and 7). Thus, the N-terminal extent of inhibitory domain II
is between amino acids 321 and 341. C-terminally truncated proteins
were tested in the context of the protein PEA3-(321-494) that contains the intact inhibitory domain II. In this case, enhanced DNA binding was
observed upon deletion to amino acid 452 (Fig. 1B,
lane 9). Because PEA3-(321-474) was still inhibited (Fig.
1B, lane 10), the C-terminal extent of inhibitory
domain III is located between amino acids 452 and 474. Deletion to
amino acid 432 did not lead to any further increases in DNA binding,
although simultaneous deletion of inhibitory domain II led to a further
enhancement of DNA-binding activity (Fig. 1B, lanes
7 and 8). Thus, the inhibitory domains II and III that
lie adjacent to the ETS-domain have been mapped to short 20-amino acid
motifs. Deletion of either one of these inhibitory motifs results in a
decrease of autoinhibition, whereas their simultaneous deletion leads
to a loss of autoinhibition.
Work on the autoinhibitory mechanism used by a different ETS-domain
transcription factor, Ets-1, has implicated the involvement of an
inhibitory module composed of
-helices lying N- and C-terminal to
the ETS-domain (9, 10). Disruption of one of the N-terminal
-helices
results in relief of autoinhibition (11, 12). Little sequence
conservation exists between Ets-1 and PEA3 in the regions immediately
outside the ETS-domain. However, to test whether a similar mechanism
involving interacting
-helices might operate in PEA3,
proline-scanning mutagenesis was used to probe for the presence of
functionally important
-helices in inhibitory motifs II and III.
Because proline residues are incompatible with regular
-helices, the
insertion of proline residues is expected to disrupt these secondary
structure elements. Several conserved proline residues exist in the
regions flanking the ETS-domain (Fig.
2A). Further proline residues
were introduced in the sequences between these residues (Fig.
2A), and their effect on DNA binding by PEA3 was monitored
(Fig. 2, B and C). The position of proline
insertions was chosen so that the spacing between any two proline
residues was less than a full helical turn. First, the effect of
inserting proline residues into inhibitory motif II in the context of
PEA3-(258-452) was investigated. This protein has lost the C-terminal
inhibitory domain III. However, neither of the two mutations tested
caused an increase in DNA binding by PEA3 (Fig. 2B).
Similarly, the insertion of proline residues at several positions into
inhibitory motif III in PEA3-(321-474) (containing inhibitory domains
II and III), caused little stimulation of DNA binding activity. These
data therefore demonstrate that it is unlikely that the formation of
-helices within the inhibitory motifs is a functionally important requirement for autoinhibition of DNA binding by PEA3.

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Fig. 2.
Proline scanning of the autoinhibitory
motifs. A, schematic representation of full-length
PEA3, the truncated protein PEA3-(258-452), and the minimal
"autoinhibited" form, PEA3-(321-474). Annotation is as in Fig. 1.
The sequence of regions flanking the ETS DNA-binding domain of
zebrafish (z) PEA3-(321-474), its human (h) and
murine (m) homologues, and the related family members ERM
and ER81 is shown below. The numbers above the
sequences refer to zebrafish PEA3, whereas the numbers at
the ends refer to the N- and C-terminal amino acids in each
sequence. Identical (shaded) and similar
(asterisks) amino acids conserved among these proteins are
indicated. The locations of proline residues that are either conserved
(white arrows) or mutated (black arrows) are also
indicated. B and C, gel retardation analysis of
the indicated wild-type and proline mutants of PEA3-(258-452)
(B) and PEA3-(321-474) (C) relative to
PEA3-(321-452). Equal molar quantities of each in vitro
translated PEA3 derivative were bound to the E74 site.
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Id Proteins Act in Trans to Inhibit DNA Binding by PEA3--
It
was recently demonstrated that the members of the Id family of
helix-loop-helix proteins can bind to members of the TCF subfamily of
ETS-domain transcription factors and subsequently inhibit their DNA
binding (20). Because interaction with Id proteins is via the ETS
DNA-binding domain of the TCFs, we tested whether Id proteins could
also inhibit DNA binding by PEA3 (see Figs.
3A and 5A for PEA3
derivatives tested). First, we used GST pull-down assays to test
whether Id2 can bind to a series of GST-PEA3 fusion proteins. Id2 could
bind to both full-length PEA3 (PEA3-(1-494)) and the isolated PEA3
ETS-domain (PEA3-(341-432)) (Fig. 3B, lanes 3 and 4) but not to PEA3 derivatives that lacked the
ETS-domain (Fig. 3B, lanes 5 and 6).
Reciprocal experiments were carried out in which GST-Id2 fusion
proteins were used to pull-down truncated PEA3 proteins. Consistent
with the above results, full-length PEA3, PEA3-(1-432), and the
isolated PEA3 ETS-domain (PEA3-(341-432)) could bind to Id2 (Fig.
3C). Collectively, these data demonstrate that Ids interact
with PEA3 and that the ETS-domain of PEA3 is their point of
interaction.

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Fig. 3.
Id proteins bind to and inhibit DNA binding
by PEA3. A, schematic representation of a series of
truncated PEA3 constructs. Annotation is as in Fig. 1. B and
C, GST pull-down analysis of (B)
35S-labeled Id2 bound to the indicated GST-PEA3 derivatives
and (C) the indicated 35S-labeled PEA3 proteins
bound to GST-Id2. D, gel retardation analysis of the
indicated PEA3 derivatives in the absence (lanes 1, 5, and 9) or presence of increasing molar
equivalents (1-fold, lanes 2, 6, and
10; 4-fold, lanes 3, 7, and
11; 9-fold, lanes 4, 8, and
12) of Id3. Both Id3 and the PEA3 derivatives were
synthesized by in vitro translation, and bound to the E74
site. The amount of binding in the presence of each concentration of
Id3 relative to each PEA3 derivative in the absence of Id3 was
quantified and shown graphically below each panel.
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To study the functional consequences of this interaction, the effect of
Id proteins on DNA binding by PEA3 was investigated. Several PEA3
derivatives (Fig. 3A) were tested that lack one of the
inhibitory motifs and, therefore, exhibit detectable DNA binding. DNA
binding by all the PEA3 derivatives tested was inhibited upon titration
of increasing concentrations of Id3 (Fig. 3D). Moreover, the
degree of inhibition was similar in all cases and because the minimal
DNA-binding region tested is the ETS-domain (in PEA3-(341-432)), this
part of the protein therefore represents the target for inhibition. Thus, binding of Id proteins to the ETS-domain of PEA3 results in
inhibition of DNA binding in trans, in an analogous manner to the
cis-acting inhibition mediated by the autoinhibitory module.
Isolation of USF-1 as an Interaction Partner for PEA3--
Because
DNA binding by PEA3 is tightly inhibited by intramolecular
autoinhibitory motifs, this inhibition must be relieved to permit its
recruitment to DNA. One such mechanism might involve interaction with a
second transcription factor or coregulatory protein (reviewed in Ref.
1). We therefore employed a yeast one-hybrid assay to identify proteins
that might act to potentiate the activity of PEA3, either via providing
linkages to the basal machinery, recruitment to the DNA or a
combination of both. The system we used was adopted from a previous
study (33) and is depicted in Fig.
4A. A stable yeast strain was
constructed that contains an integrated LacZ reporter
construct driven by 5 ets binding sites,
Ets-Lacrep. Subsequently, expression vectors for
PEA3 were introduced into the Ets-Lacrep strain,
in combination with a cDNA library encoding GAL4 activation domain
fusion proteins derived from day-11 mouse embryos. This stage was
selected because mouse PEA3 is strongly expressed at this time point
(40) and zebrafish PEA3 is strongly expressed at an equivalent
developmental stage (6). Several positive clones were isolated; one of
which contains the C-terminal half of the basic
helix-loop-helix-leucine zipper (bHLHZip) transcription factor USF-1
(Fig. 4B). Upon retransformation, USF-1 was only able to
efficiently activate the LacZ reporter in the presence of a
cotransformed vector encoding PEA3 (Fig. 4C). Liquid
-galactosidase assays were subsequently performed to demonstrate the
requirement of both proteins and to map the interaction determinants in
PEA3 needed for maximal activation of the LacZ reporter
gene. In the presence of the empty expression vectors, little reporter
gene activity was observed (Fig. 4D), whereas in the
presence of either vectors encoding USF-1 alone or one of a series of
truncated PEA3 proteins, modest activation of the reporter was observed
(black and white bars; Fig. 4D).
However, in the presence of USF-1 and full-length PEA3 (PEA3-(1-494)),
a large increase in reporter gene activity was observed. Similarly,
large increases in reporter gene activity were observed in the presence
of USF-1 and either of the truncated PEA3 proteins PEA3-(258-494) and
PEA3-(341-494) (blue bars; Fig. 4D). These data
therefore demonstrate that USF-1 represents a true interaction partner
for PEA3 and that a region(s) between amino acids 341-494 is
sufficient for binding to USF-1 in this assay.

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Fig. 4.
Identification of USF-1 as a PEA3 interaction
partner by yeast one-hybrid screening. A,
schematic representation of the -galactosidase reporter gene
used in the yeast one-hybrid screen. The reporter contains a basal cycP
promoter driven by 5× ets DNA-binding sites (33). PEA3 binds to these
sites in vivo and recruits proteins such as USF-1 from the
library to activate transcription of the LacZ gene. The GAL4
activation domain in the USF-1 fusion protein is indicated by a
red box. B, schematic representation of the
domain structure of full-length USF-1 and the USF-1 fusion protein
isolated from the yeast two-hybrid screen. The bHLHZip DNA-binding
domain and GAL4 activation domain in the fusion protein are indicated
by white and red boxes, respectively.
C, -galactosidase filter assay of yeast strain
Ets-Lacrep, containing the USF-1 expression
vector and either the empty expression vector pMSe4 or expression
vector containing full-length PEA3. D, quantitative liquid
-galactosidase assays of yeast strain
Ets-Lacrep, containing the indicated
combinations of the empty expression vectors pGAD10 and pMSe4 or the
same vectors containing the indicated PEA3 derivatives (in pMSe4) or
USF-1-(160-310)* (in pGAD10). Data are presented (average of duplicate
samples) relative to PEA3 expression vector alone (taken as 1).
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PEA3 and USF-1 Interact in Vitro and Synergistically Activate
Transcription in Vivo--
GST pull-down assays were used to verify
the interactions between PEA3 and USF-1. A series of truncated PEA3
proteins fused to GST were constructed (Fig.
5A) and tested for their
ability to interact with in vitro translated full-length
USF-1 (Fig. 5B). Of the constructs tested, full-length PEA3
and the isolated ETS DNA-binding domain (PEA3-(341-432)) were able to
bind to USF-1 (Fig. 5B, lanes 2 and
3). Neither the region N-terminal (PEA3-(1-337)) nor the
region C-terminal (PEA3-(430-494)) to the ETS-domain could bind to
USF-1 (Fig. 5B, lanes 4 and 5).
Deletion of the bHLHZIP domain of USF-1 resulted in a loss of binding
to PEA3, indicating that the N-terminal region of USF-1 is insufficient
for binding in solution. Therefore, the DNA-binding domain of
USF-1 is required for the formation of such complexes (data not
shown).

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Fig. 5.
PEA3 interacts with USF-1 in vitro
and synergistically activates transcription with USF-1 in
vivo. A, schematic representation of a
series of truncated PEA3 constructs fused to GST. Annotation is as in
Fig. 1. B, GST pull-down analysis of 35S-labeled
full-length USF-1 bound to the indicated GST-PEA3 derivatives.
C, schematic representation of the luciferase reporter gene
used in transfection experiments. The reporter contains a basal TK
promoter driven by 2× E-box-ets DNA-binding sites from the HIV-1 LTR
(Fig. 6A) (13). PEA3 binds to the ets motif, whereas USF
binds to the E-box to activate transcription of the Luc
gene. D, the indicated combinations of USF-1/USF-2 and PEA3
expression vectors (500 and 200 ng, respectively) were transfected into
293 cells in the presence of the ets/E-box-Luc reporter (100 ng). Data
are normalized for the activity of a cotransfected -galactosidase
expression vector and presented (average of duplicate samples) relative
to the ets/E-box-Luc reporter alone (taken as 1) and are representative
of three independent experiments.
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To verify that PEA3 and USF-1 can functionally interact on a
physiologically relevant site in vivo, we analyzed their
ability to regulate the activity of a luciferase reporter gene in
mammalian cells driven by a composite element from the HIV-1 long
terminal repeat (LTR). This element is composed of a relatively high
affinity E-box and a weak ets (PEA3 binding) motif and has previously
been shown to be synergistically regulated by USF-1 and a different ETS-domain transcription factor, Ets-1 (13). When PEA3 or USF-1/USF-2 alone were transfected into 293 cells, moderate levels of reporter gene
activity were observed (2.1- and 4.5-fold, respectively). However, in
the presence of both PEA3 and USF-1/USF-2, synergistic activation was
observed (11.7-fold).
Collectively, these data therefore demonstrate that the interactions
between PEA3 and USF-1 can be verified in vitro and that these proteins work together to activate transcription in
vivo from a physiologically relevant site.
PEA3 and USF-1 Positively Regulate Each Other's DNA Binding
Activity--
USF-1 and Ets-1 have previously been shown to be able to
cooperatively bind to the E-box-ets motif in the HIV-1 LTR (Fig. 6A) (13). Because interactions
between PEA3 and USF-1 have been demonstrated, we tested whether
similar cooperativity could be observed between these two proteins on
this DNA binding site. Two different protein derivatives were tested in
each case, consisting of either the minimal DNA-binding domains of each
protein (PEA3-(341-432) and USF-1-(197-310)), the full-length USF-1
protein (USF-1- (1)), or an extended PEA3 protein encompassing
additional motifs outside the ETS-domain (PEA3-(341-452)) (Fig.
6B). These proteins were purified to near homogeneity from
overexpressing bacterial strains.

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Fig. 6.
PEA3 stimulates the DNA binding activity of
USF-1 and cooperatively binds DNA with USF-1. A,
sequence of the composite E-box/ets element from the HIV-1 LTR. The
cores of the E-box and ets binding sites are boxed. B,
schematic representation of a series of truncated PEA3 and USF-1
constructs. Annotation is as in Figs. 1 and 4. C-E, gel
retardation analysis using the indicated combinations of PEA3 and USF-1
proteins and the E-box/ets binding site. All proteins were expressed
and purified from bacteria. C, DNA binding by USF-1
derivatives (USF-(1-310), 0.5 pmol; USF-(197-310), 1.5 pmol) in the
absence (lanes 1 and 5) or presence of increasing
amounts (6 pmol, lanes 2 and 6; 25 pmol,
lanes 3 and 7; 50 pmol, lanes 4 and
8) of PEA3-(341-432). D, DNA binding by
USF-(1-310) (0.5 pmol) in the absence (lanes 1 and
5) or the presence of increasing amounts (6 pmol,
lanes 2 and 6; 25 pmol, lanes 3 and
7; 60 pmol, lanes 4 and 8) of the
indicated PEA3 derivatives. The DNA·protein complexes alone are
shown, as extended electrophoresis was used to allow maximum resolution
and the free binding site was run off the end of the gels.
E, antibody supershift experiments in the presence of PEA3
(25 pmol) derivatives and USF-(1-310) (0.5 pmol) to detect the
presence of the indicated FLAG-tagged PEA3 derivatives in USF-1
complexes. Complexes containing USF-1 and PEA3 are indicated by
black and gray arrows, respectively. White
arrows refer to a higher order USF-1·PEA3 complex (D)
and antibody induced supershifted complexes (E). The
asterisk refers to a band that is not reproducibly
observed.
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Because the PEA3 ETS-domain was sufficient for interaction with USF-1,
we first tested whether this domain could stimulate DNA binding by
USF-1 (Fig. 6C). PEA3-(341-432) was able to stimulate DNA binding by both full-length USF-1 (USF-1-(1-310)) and the bHLHZip
DNA-binding domain of USF-1 (USF-1-(197-310)). However, at the
concentrations used here, PEA3 binding to the E-box-ets motif was not
visible at the exposure shown, indicating that, in comparison to USF-1,
it has a low affinity for this site (see also Fig. 6E,
lane 2). Next we compared the ability of PEA3-(341-432) and
the longer PEA3 protein PEA3-(321-452), which contains additional regions that flank the ETS-domain (Fig. 6B), to stimulate
DNA binding by full-length USF-1 (Fig. 6D). Both these PEA3
derivatives were able to stimulate the formation of a USF-1 complex.
However, extended electrophoresis permitted resolution of a novel lower mobility complex that formed only in the presence of PEA3-(321-452) (Fig. 6D, lanes 5-8). This complex most likely
contains PEA3, in addition to USF-1. To test this hypothesis, antibody
supershift experiments were carried out using an antibody directed
against the FLAG tag in the PEA3 derivatives. Addition of anti-FLAG
antibody gave a small supershift in the presence of PEA3-(341-432),
but efficiently shifted a significant proportion of the complex formed with PEA3-(321-452) (Fig. 6E, compare lanes 3 and 6). Higher concentrations of antibody had little further
effect on PEA3-(341-432) but resulted in loss of the supershifted
complex formed with PEA3-(321-452) (data not shown; Fig.
7C).

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Fig. 7.
Identification of PEA3 and USF-1 domains
required for ternary complex formation. A-C, gel
retardation analysis using the indicated combinations of PEA3 and USF-1
proteins and the E-box-ets binding site. Schematic representations of
the truncated PEA3 and USF-1 constructs used in each experiment are
shown above or to the left of each set of lanes,
respectively. Annotation is as in Figs. 1 and 4. The DNA·protein
complexes alone are shown as extended electrophoresis was used to allow
maximum resolution and the free binding site was run off the end of the
gels. The USF-1 (5 pmol) and PEA3 (3 pmol) proteins used were expressed
in and purified from bacteria. Anti-FLAG antibodies were added to
detect the presence of FLAG-tagged PEA3 derivatives in protein·DNA
complexes. In this experiment a high concentration of antibody was used
that results in abolition of ternary complex formation rather than a
supershift as in Fig. 6. Complexes containing USF-1 are indicated by
black arrows, whereas white arrows refer to
ternary USF-1·PEA3 complexes. D, the location of the USF
binding and autoinhibitory motifs on the linear PEA3 sequence. The
dotted line indicates that this part of the C-terminal
inhibitory motif, although not sufficient, might be required for
inhibiting DNA binding by PEA3. E, model showing a proposed
mechanism for the relief of autoinhibition by interaction with USF-1.
The inhibitory motifs are depicted as black elipses. Direct
interactions between the ETS-domain of PEA3 and USF-1 and putative
interactions with the autoinhibitory motifs of PEA3 are indicated by
two-way arrows. An alternative route for relief of
autoinhibition other than USF-1 binding is indicated by a
question mark. This might involve interactions with other
proteins or post-translational modification.
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Collectively, these data therefore indicate that, although both
PEA3-(341-432) and PEA3-(321-452) stimulate DNA binding by USF-1,
only the latter forms a stable ternary DNA-bound complex with USF-1.
Therefore, although the ETS DNA-binding domain of PEA3 is sufficient to
stimulate DNA binding by both full-length USF-1 and the USF-1 bHLHZip
DNA-binding domain, additional PEA3 sequences are required for the
formation of stable ternary complexes.
Mapping the Regions of PEA3 Required for Ternary Complex Formation
with USF-1--
To identify the regions in USF-1 and to further define
the regions in PEA3 that are required to permit ternary complex
formation, a series of truncated PEA3 and USF-1 derivatives were tested
for their ability to form ternary DNA-bound complexes (Fig. 7,
A-C). In this series of experiments, the largest PEA3
protein tested was PEA3-(321-452), which was shown previously to be
sufficient to form ternary complexes with full-length USF-1 (Fig.
6D). Two additional PEA3 proteins that lack either the N-
(PEA3-(341-452)) or C-terminal (PEA3-(321-432)) extensions to the
ETS-domain were tested, to determine whether either of these regions
were sufficient to permit ternary complex formation. A concentration of
USF-1 proteins that gave >50% probe binding was used in these
experiments, and the gels were run for extended times to permit complex
resolution. Of the proteins tested, only PEA3-(321-452) was able to
form a ternary complex with full-length USF-1 (USF-1-(1-310)) (Fig.
7A, lane 15). This complex was abolished upon
addition of anti-FLAG antibody (Fig. 7A, lane
16). In contrast, the FLAG antibody had no effect on complexes
formed in the presence of the truncated PEA3 derivatives (Fig.
7A, lanes 4, 8, and 12),
indicating that PEA3 was not part of these complexes. Thus both the N-
and C-terminal extensions to the PEA3 ETS-domain are required in
combination to form ternary complexes with USF-1. Interestingly, these
same regions participate in intramolecular autoinhibition of DNA
binding by PEA3, indicating an intriguing link between stimulation of DNA binding by PEA3 and relief of autoinhibition (Figs. 1 and 7D; see "Discussion")
To map the ternary complex determinants in USF-1, two truncated USF-1
derivatives were tested. Although USF-1-(144-310) could form a complex
with PEA3-(321-452) (Fig. 7B, lane 7), the
isolated bHLHZip domain in USF-1-(197-310) was unable to form a
ternary complex with PEA3-(321-452) (Fig. 7C, lane
3). None of the other truncated PEA3 derivatives tested could form
complexes with either of these truncated USF-1 proteins (Fig.
7B, lane 3; data not shown). Therefore, the
region immediately preceding the bHLHZip domain (amino acids 144-197)
is required for ternary complex formation with PEA3.
Thus in the case of both PEA3 (amino acids 321-341 and 433-452) and
USF-1 (amino acids 144-197), regions flanking their DNA-binding domains are required for the formation of stable DNA-bound ternary complexes.
DNA Binding Sites for PEA3 and USF-1 Are Required for Ternary
Complex Formation--
Results from the yeast one-hybrid system
indicated that PEA3 and USF-1 can interact on DNA in the absence of
USF-1 binding sites (Fig. 4). To test whether the formation of such
complexes could be detected in vitro and whether reciprocal
complexes on a USF-1 site could be detected, we analyzed the binding of
PEA3 and USF-1 to sites that contained only an ets or an E-box binding site (Fig. 8).

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Fig. 8.
Both ets and E-box binding sites are
required for ternary complex formation. Gel retardation analysis
using the indicated combinations of PEA3 and USF-1 proteins and the
indicated binding sites. The ets binding site is the high affinity E74
site, whereas the E-box site is identical to the E-box-ets site used in
Figs. 6 and 7 with two point mutations in the ets binding motif. The
USF-1 and PEA3 proteins were as described in Fig. 7. Complexes
containing USF-1 are indicated by black arrows, whereas
white arrows refer to ternary USF-1·PEA3 complexes.
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Binding of PEA3-(321-452) to the ets site was barely detectable due to
the presence of the autoinhibitory domains (Fig. 8, lane 3).
The addition of USF-1 did not lead to an enhancement of binding or the
formation of a ternary complex (Fig. 8, lane 2). In the
presence of both E-box and ets sites, ternary complex formation could
be readily detected (Fig. 8, lane 5; see Figs. 6 and 7).
However, upon mutation of the ets site, although USF-1 binding to the
E-box was unaffected, ternary complex formation was abolished (Fig. 8,
lanes 7 and 8).
Collectively, these results therefore demonstrate that in
vitro, specific protein·DNA contacts by both PEA3 and USF-1 are required for the formation of ternary complexes.
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DISCUSSION |
Autoinhibition is emerging as a novel mechanism for regulating the
activities of eukaryotic transcription factors. There are numerous
examples where autoinhibition results in a reduction of the DNA binding
activity of the transcription factor (reviewed in Ref. 1).
Subsequently, this autoinhibition is relieved, either in response to
activation of signal transduction pathways or the presence of a
coregulatory partner protein. Indeed, the ETS-domain family provides
multiple additional examples of combinatorial interactions that have
been investigated in detail, where DNA binding by at least one
component of a complex is enhanced (reviewed in Refs. 2 and 3). Here,
we have located the cis-acting autoinhibitory motifs in the ETS-domain
transcription factor PEA3 and identified two proteins that act in trans
to modify DNA binding by PEA3. The Id proteins act to inhibit DNA
binding, whereas USF-1 acts to promote DNA binding by PEA3. Thus a
complex network of intra- and intermolecular protein·protein
interactions serve to modify the DNA binding potential of PEA3.
Mechanisms for Negatively Regulating DNA Binding by PEA3--
DNA
binding by PEA3 subfamily members is regulated by cis-acting
autoinhibitory domains (6-8). Regions in the C- and N-terminal part of
PEA3 are involved in regulating its DNA binding potential. Here we have
further localized the C-terminal inhibitory domains in PEA3 to two
short regions that lie on either side of the ETS DNA-binding domain
(summarized in Fig. 8D). These regions are highly conserved
among all members of the PEA3 subfamily (45% identical; 76% similar)
and between species (79 and 87% identical between human and zebrafish
PEA3) (Fig. 2A). These similarities are particularly
striking considering that the overall identity among all PEA3 subfamily
members is only 20% outside the ETS-domains (6). This sequence
conservation suggests that the autoinhibitory mechanism is
evolutionarily conserved among PEA3 subfamily members and hence further
emphasizes the likely importance of this regulatory mechanism.
The two inhibitory motifs that we have identified act in concert to
inhibit DNA binding by PEA3 as deletion of either one leads to a
substantial relief of autoinhibition. Each domain on its own can only
partially inhibit DNA binding by PEA3 (Fig. 1). These observations
indicate that the two motifs might form a single autoinhibitory module
as observed in Ets-1 (9, 10). However, unlike Ets-1, where this module
is composed of three
-helices, our results indicate that the
formation of
-helices does not appear to be required for the
function of this module in PEA3. Furthermore, there is essentially no
sequence conservation between the autoinhibitory modules of Ets-1 and
PEA3. Thus, although both these modules serve to inhibit DNA binding,
their molecular mechanisms of action are likely to differ. It is
currently unclear how DNA binding is inhibited, however, this module
might either mask the DNA binding surface or, alternatively, inhibit
DNA binding by allosterically affecting the DNA binding surface.
Indeed, a similar model has been proposed for autoinhibition of Ets-1
(11, 12).
In addition to the cis-acting autoinhibitory motifs, we also
demonstrate that trans-acting proteins (the Id proteins) can inhibit
DNA binding by PEA3. The Id proteins were previously demonstrated to
bind to the ETS-domain and inhibit DNA binding by members of the TCF
subfamily (20). Similarly, the Id proteins bind to the ETS DNA-binding
domain in PEA3 (Fig. 3). However, one key difference is that the Id
proteins inhibit PEA3 binding to the high affinity E74 site, whereas
the TCFs are only affected on lower affinity sites. This might reflect
intrinsic differences between these different proteins in the precise
mode of DNA recognition and/or Id protein binding. The mechanism used
by Id proteins to inhibit DNA binding by ETS-domain transcription
factors is unknown, although they may act by analogous mechanisms
proposed for the cis-acting autoinhibitory motifs discussed above.
Interaction with USF-1 Enhances DNA Binding by PEA3--
In this
study, we have identified USF-1 as an interaction partner for PEA3 and
demonstrate that USF-1 recruits PEA3 into DNA-bound complexes. This
recruitment might merely reflect the role of protein·protein interactions in enhancing the binding of PEA3. However, USF-1 might
also lead to relief of autoinhibition. This latter hypothesis is
supported by the observation that the two motifs in PEA3 that are
required for ternary complex formation with USF-1 overlap with the two
motifs that constitute the autoinhibitory module (Fig. 8D).
Again, these two motifs are required in combination to permit ternary
complex formation as deletion of either motif abolishes cooperative
binding with PEA3 (Fig. 8C). Thus an important regulatory
module has been identified that is involved in stimulating the
formation of nucleoprotein complexes and inhibiting DNA binding. USF-1
appears to act as a switch that changes the activity of this module.
It is noteworthy that the ability of USF-1 to form ternary complexes
with PEA-3 is dependent on a region between amino acids 144-196, which
is conserved between USF family members and contains a USF-specific
region (USR) (41, 42 and references therein). The USR plays an
important role in many of the functions of USF, including transcriptional activation, nuclear localization (41, 42), and
regulatory phosphorylation (29) and also been proposed to recruit
either cell-specific coactivators or repressors. Thus in both PEA3 and
USF-1, important regulatory domains play additional roles in promoting
complex formation.
USF-1 was also isolated as an interaction partner for a different
ETS-domain transcription factor, Ets-1, by using the same one-hybrid
system (13). Cooperative DNA binding between USF-1 and Ets-1 and
synergistic transcriptional activation was also observed, although the
mechanisms of cooperativity appear to differ. For example, in the
case of USF-1 and Ets-1, the respective DNA-binding domains of each
protein were sufficient for interaction in solution and to
cooperatively form ternary complexes. However, in the case of USF-1 and
PEA3, such reciprocal cooperativity was not observed unless additional
regions flanking each of the DNA-binding domains were present. Although
the ETS DNA-binding domain of PEA3 was sufficient to stimulate DNA
binding by the bHLHZip DNA-binding domain of USF-1, no stable ternary
complexes were formed. This is consistent with our observation that the
ETS DNA-binding domain was sufficient for interaction with USF-1 in GST
pull-down assays (Fig. 5) and that a minimal PEA3 construct that lacks
the N-terminal region flanking the ETS-domain was sufficient to
interact with USF-1 in the yeast one-hybrid assay (Fig. 4). Indeed, the
cooperativity we see (DNA binding by PEA3 is barely detectable in the
absence of USF-1), appears significantly more than that observed
between Ets-1 and USF-1. In this regard, a key difference between our own and the previous study is, therefore, the quantities of proteins used (we used 30-fold less ETS-domain protein), and it is possible that, at higher concentrations, we may also have detected residual cooperativity between the isolated DNA-binding domains. Thus, with PEA3
and USF-1, although it is clear that it is possible to promote complex
formation in the absence of autoinhibitory modules and E-box binding
sites in vivo by protein overexpression (as observed with
Ets-1/USF-1), other factors likely contribute to DNA·protein complex
formation. Indeed, two DNA binding sites and the autoinhibitory regions
of each protein are absolutely required for the formation of
protein·DNA complexes in vitro. We believe that this is
more indicative of the true physiological situation.
Thus one model for how these proteins interact and cooperate, is that
interactions occur between the ETS- and bHLHZip DNA-binding domains,
which can promote DNA binding but, in the case of PEA3 and USF-1, are
insufficient to permit the formation of ternary complexes. The regions
flanking these domains are required for stable complex formation (Fig.
7E). In light of our results and the study of Ets-1
interactions with AML-1 (13, 15), it will be interesting to re-evaluate
the possible role of additional motifs in Ets-1 in cooperative DNA
binding with USF-1.
It is currently unclear how cooperativity in the USF-1·PEA3 complex
is achieved. It is likely that protein·protein interactions between
USF-1 and PEA3 serve to increase the affinity of PEA3 for the binding
site. In addition, disruption or reorientation of the autoinhibitory
module might transmit signals in an allosteric manner to the ETS
DNA-binding domain of PEA3. Further experimentation is required to
probe these possibilities further.