From the Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario L8S 4K1, Canada
Received for publication, June 22, 2000, and in revised form, November 8, 2000
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ABSTRACT |
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PEA3, a member of the Ets family of transcription
factors, is a nuclear phosphoprotein capable of activating
transcription. Mouse PEA3 comprises 480 amino acids and bears an
~85-amino acid ETS domain near its carboxyl terminus. Whereas
analyses of bacterially expressed PEA3 revealed that the ETS domain is
required for sequence-specific DNA binding, little is known of the
functional domains in the protein required for its activity in
mammalian cells. To this end, we defined the location of the PEA3
functional domains in COS cells. PEA3 bears a strong activation domain
near its amino terminus, which is flanked by two regions that
independently negatively regulate its activity. PEA3 expressed in COS
cells was incapable of binding to DNA in vitro. However,
DNA binding activity could be unmasked by incubation with a
PEA3-specific antibody. Analyses of the DNA binding activity of PEA3
deletion mutants revealed that two regions flanking the ETS domain
independently inhibited DNA binding; deletion of both regions was
required to detect DNA binding in the absence of a PEA3-specific
antibody. Under these conditions, the ETS domain was sufficient for
sequence-specific DNA binding. These findings suggest that the activity
of PEA3 is exquisitely controlled at multiple functional levels.
The Ets family of transcription factors are defined by an
evolutionarily conserved ~85-amino ETS domain (1). These proteins are
found exclusively in multicellular organisms and are thought to play
cardinal roles in development and oncogenesis (2). Multiple
ets genes have been identified in individual organisms; over
20 mammalian genes have been discovered thus far (3). Ets proteins are
sequence-specific DNA-binding proteins that regulate transcription
(reviewed in Ref. 2). Generally, these proteins activate transcription,
but several members of the family are known to repress this process.
DNA binding is achieved by interaction between the ETS domain and an
~10-base pair sequence element termed the Ets binding site
comprising a highly conserved central core sequence, 5'-GGA(A/T)-3'.
Individual Ets proteins demonstrate specificity for sequences flanking
this core, but it is not uncommon for different Ets proteins to bind to
the same Ets binding site. Structural analyses of the ETS domain reveal
a winged-helix-turn-helix structure akin to that of the
Escherichia coli catabolite activator protein and the
HNF3/forkhead and heat shock transcription factors (4-7).
Mouse pea3 (8) (the human gene
is named ETV4 and has also been termed E1A-F) (9,
10) is the founding member of the pea3 subfamily of
ets genes. This subfamily also includes er81 (ETV1) (11-13) and erm (ETV5)
(14, 15). Each of these genes is located on a different chromosome
(16), but all three genes share a common architecture comprising 14 equivalently sized exons that encode similar sequences of the
respective proteins (16-18). The overall amino acid sequence
similarity of the PEA31 subfamily is ~50% (17). The
three longest stretches of greatest sequence similarity include the
85-amino acid ETS domain (95% sequence identity); an acidic region
near the amino terminus composed of 32 amino acids (85% sequence
identity); and a region at the carboxyl terminus comprising ~60 amino
acids (50% sequence identity) (17).
PEA3 is overexpressed in breast tumors both in humans and mice,
suggesting a role for PEA3 in this malignancy (19, 20). 76% of all
human breast tumors contain elevated levels of PEA3 RNA; 93% of the
c-ERB-B2/HER2-positive subclass of these tumors overexpress PEA3 (20).
PEA3 is also overexpressed in mouse mammary tumors arising in
transgenic mice engineered to express murine c-Erb-B2/Her2 in their
mammary glands (19).
Chromosomal translocations involving the PEA3
subfamily genes have been implicated in a minority of Ewing's sarcomas
in humans (12, 21, 22). The same region of EWS is
translocated and juxtaposed to sequences encoding the ETS domain of the
FLI-1 ETS gene, which is the most common translocation in
this disease (23). EWS-FLI-1 chimeric genes encode EWS-FLI-1
fusion proteins that bear the ETS domain. The transcriptional activity
of these chimeric proteins significantly supersedes that of FLI-1, and,
unlike FLI-1, they induce transformation of a mouse 3T3 fibroblast cell
line (24-27). These findings imply that the transcriptional activity of these chimeras is associated with their transforming and oncogenic potential.
Like other Ets proteins that have been studied, PEA3 binds to DNA with
specificity and activates transcription of reporter plasmids bearing
PEA3-responsive promoters (8). Analyses of glutathione
S-transferase-PEA3 chimeras expressed in and purified from
E. coli demonstrate that the ETS domain is required and
sufficient for sequence-specific DNA binding as assessed in
vitro by EMSA (8). However, the location of sequences required for
transactivation and DNA binding in mammalian cells has not been
investigated. To this end we constructed and measured the specific
activity of a series of unidirectional amino and C-terminal deletion
mutants of PEA3 and of GAL4-PEA3 chimeras in mammalian COS cells. Our analyses revealed that PEA3 bears a strong activation domain near its
amino terminus that is flanked by two negative regulatory regions,
which markedly repress its activity. PEA3 expressed in COS cells was
incapable of binding to DNA. However, DNA binding activity could be
uncovered by incubation with PEA3-specific antibodies or by deleting
two regulatory regions that flank the ETS domain. Hence, both the
transactivation and DNA binding functions of PEA3 are negatively
controlled, implying that a mechanism(s) exists to exquisitely regulate
its activity.
Cell Culture--
COS-1 cell were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum and
antibiotics (penicillin, streptomycin, and fungizone). The cells were
grown at 37 °C in a humidified 5% CO2 atmosphere.
Construction of Recombinant Plasmids and Isolation of Deletion
Mutants--
The PEA3 expression vector (pRSV-PEA3) was constructed by
cloning PEA3 cDNA sequences (8) between the HindIII and
XbaI sites of pRc/RSV (Invitrogen). The amino-terminal
deletion mutants were derived from pRSV-PEA3 by polymerase chain
reaction amplification using suitable primers followed by molecular
cloning of the product into HindIII-XbaI-cleaved
pRc/RSV. The primers used to derive the amino-terminal deletion mutants
all comprised a HindIII site and a translation initiation
consensus sequence 5'-ACCATGG-3' (28, 29); the invariant
sequence of all of the primers is 5'-GGAAGCTTACCATGG-3' (the HindIII
recognition site and the translation initiation codon are underlined).
All of the polymerase chain reaction primers used to generate the
C-terminal deletion mutants contained an XbaI restriction
site and a stop codon (5'-TAG-3'); the complementary
sequence of this primer is 5'-TAGAGATCTCC-3' (the stop
codon and XbaI cleavage site are underlined). A naturally occurring PEA3 codon precedes the stop codon in the mRNA of each C-terminal deletion mutant. The variable sequence of each primer used
corresponded to that which flanks the deletions described above.
GAL4-PEA3 chimeras were constructed by cloning polymerase chain
reaction-amplified PEA3 sequences from RSV-PEA3 in frame between the
BamHI and XbaI sites of pSG424 (30). Primers
containing a stop codon were used to derive GAL4-PEA3 chimeras bearing
C-terminal deletion mutants as described above.
The PEA3-responsive unit of the reporter plasmid
(PEA35-luciferase)
comprises five copies of an optimal, high affinity PEA3 binding site,
5'- TGGCCGGAACCG-3' (the core sequence of the Ets binding
site is underlined).2 These sequences were cloned into the
XhoI site of pGL3-Ad MLP. This reporter was derived by
cloning a synthetic double-stranded oligonucleotide
(5'-AGATCTCGAGCTCGGGGGGCTATAAAAGGGGGATCTGAATTCGAGAAGCTT-3') bearing the adenovirus type 2 major late promoter TATA box and flanking
sequences between the BglII and HindIII sites of
the pGL3 basic luciferase reporter plasmid (Promega).
The GAL4 reporter plasmid (GAL45-luciferase) contained five
copies of a GAL4 DNA binding sites cloned into the SmaI site
of the pGL3-Ad MLP reporter (31). The junctions and inserts of all
recombinant plasmids were sequenced on both strands to ensure that
mutations were not introduced during polymerase chain reaction and
molecular cloning.
Assessment of the Transcriptional Activity of PEA3 and of
GAL4-PEA3--
The transcriptional activity of PEA3, GAL4-PEA3, or
their derivatives was assessed indirectly by measuring luciferase
activity in equivalent amounts of lysate protein isolated from COS
cells transfected with the appropriate luciferase reporter plasmid and either a PEA3 or GAL4-PEA3 effector plasmid. This value was normalized to the amount of PEA3 or an active and readily detected GAL4-PEA3 chimera (GAL4-PEA3dlC85) expressed in each experiment as determined by
immunoblot analysis to derive the specific transcriptional activity of
each protein. Because many of the mutant versions of PEA3 and GAL4-PEA3
were expressed at lower levels than the corresponding wild type (WT)
proteins, we measured the specific activity of WT PEA3 or of
GAL4-PEA3dlC85 over a range of protein concentrations. This was
accomplished by transfecting COS cells with varying amounts of the
appropriate effector plasmid and assay of the amount of luciferase
activity present in equal amounts of lysate protein. Normalization of
this value to the amount of PEA3 or GAL4-PEA3dlC85 expressed in the
same experiment revealed a linear increase in luciferase activity that
was directly proportional to the amount of PEA3 or GAL4-PEA3dlC85
present in the lysates. The PEA3 and GAL4-PEA3 deletion mutants assayed
in this study were expressed within the limits of these values.
COS cells were seeded into six-well plates at a density of 1.2 × 105 cells/well, and 24 h later they were transfected
with a total of 2 µg of DNA using 6 µl of LipofectAMINE reagent as
specified by the manufacturer (Life Technologies, Inc.). 48 h
after transfection, the cells were harvested and lysed as described below.
PEA3 transcriptional activity was determined following transfection of
0.5 µg of the PEA3-responsive reporter plasmid
(PEA35-luciferase) with one of several doses (0.2, 0.4, 0.6, and 0.8 µg) of the effector plasmid (pRc/RSV-PEA3) encoding
WT PEA3. The total amount of effector DNA was maintained at 0.8 µg by
adding empty effector DNA (pRc/RSV). The total DNA concentration of
each transfection mixture was adjusted to 2 µg with sheared salmon sperm DNA. The activity of the PEA3 deletion mutants was determined after cotransfection of 0.5 µg of the reporter plasmid with a single
dose (0.8 µg) of the effector plasmid. The specific activity of the
various PEA3 deletion mutants was expressed relative to that of WT
PEA3, which was set at 100%.
The activity of GAL4-PEA3 chimeras was assessed similarly, except that
0.25 µg of the GAL4 reporter plasmid was cotransfected with up to 0.5 µg of effector DNA encoding the various GAL4-PEA3 chimeric proteins.
The total concentration of pSG424 DNA was adjusted to 0.5 µg when the
activity of select GAL4-PEA3 chimeras was assessed by titration.
Because we could not detect the GAL4 DNA binding domain encoded by
pSG424 using immunoblotting, and because GAL4-PEA3 (bearing full-length
PEA3) was transcriptionally inactive, we expressed the specific
activity of the various GAL4-PEA3 chimeras relative to that of
GAL4-PEA3dlN85, an active and readily detected fusion protein. The
magnitude of luciferase expression effected by the chimeras was
calculated by measuring the ratio of luciferase activity obtained from
cells transfected with the reporter to that obtained from cells
transfected with the reporter and effector plasmid encoding a
particular GAL4-PEA3 chimera following normalization of the abundance
of the chimera in equal amounts of lysate protein as described below.
Within an experiment, each DNA preparation was transfected in
quadruplicate; two wells of a six-well dish were independently processed to measure luciferase activity, and two wells were
independently used to determine the abundance of PEA3 or its
derivatives by immunoblot analysis. Cell extracts for luciferase assays
were prepared from each well using 200 µl of reporter lysis buffer (Promega catalog no. E3971) according to the manufacturer's
instructions. Luciferase activity was measured using luciferase assay
reagent (Promega catalog no. E1483). Luciferase activity was expressed in relative light units per µg of total protein after subtracting the
activity of the empty effector plasmid control.
To determine the abundance of PEA3 or its derivatives, the cell
monolayers were washed in the wells with ice-cold phosphate-buffered saline and then scraped from the plates in 1 ml of phosphate-buffered saline. The cells were concentrated by centrifugation, resuspended in
50 µl of lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl,
pH 7.4, 5 mM EDTA, 400 mM NaCl, 10 µg/ml
leupeptin, 10 µg/ml pepstatin, and 2 µg/ml phenylmethylsulfonyl
fluoride), and then incubated on ice for 25 min. The lysate was
centrifuged for 5 min at 13,000 rpm at 4 °C in a microcentrifuge,
and the supernatant was used for immunoblot analysis.
The total protein concentration of each sample was determined by the
Bradford dye method using a kit provided by Bio-Rad with bovine serum
albumin (Sigma) as the standard. Equal amounts of protein (20 µg of
total protein from cell extracts or 8 µg of protein from nuclear
extracts) from the duplicate samples were resolved by 10%
SDS-polyacrylamide gel electrophoresis and transferred to a
polyvinylidene difluoride membrane by electroblotting. The membrane was
washed in distilled water and then blocked for 20 min at room
temperature with phosphate-buffered saline containing 5% nonfat dry milk.
PEA3 and its deleted derivatives were detected on the membrane by use
of one of several antibodies including one monoclonal antibody and two
different polyclonal antibodies. The PEA3-specific monoclonal antibody
is named MP16 and recognizes determinants between residues 226 and
245.3 The rabbit polyclonal
antibodies are termed PN1 and PC2 and recognize epitopes at the extreme
amino and carboxyl terminus of PEA3,
respectively.4 PN1 was
derived by injecting rabbits with a keyhole limpet
hemocyanin-conjugated peptide corresponding to the first 15 amino acids of mouse PEA3, whereas PC2 was raised by injecting rabbits
with a keyhole limpet hemocyanin-conjugated peptide bearing the
C-terminal 15 amino acids of mouse PEA3.
A commercial rabbit polyclonal antibody reactive with the DNA
binding domain of GAL4 was used to quantify the abundance of GAL4-PEA3
on immunoblots (Upstate Biotechnology, Inc., Lake Placid, NY; catalog
no. 06-262). 125I-Labeled goat anti-mouse or goat
anti-rabbit IgG were used as secondary antibodies. Quantification of
expression levels of PEA3 and GAL4-PEA3 chimeras was performed by use
of a PhosphorImager (Molecular Dynamics) equipped with ImageQuant 3.3 software. In one series of experiments described here, the abundance of
several GAL4-PEA3 chimeras (GAL4-PEA3dlC100, GAL4-PEA3dlN42dlC100,
GAL4-PEA3dlC85 and GAL4-PEA3dlN42dlC85) was also measured using a
rabbit polyclonal primary antibody reactive with the DNA binding domain
of GAL4 (BAbCo; PRB-255C) and a secondary goat anti-rabbit antibody
coupled to peroxidase (KPL; 474-1506) using chemiluminescence
(PerkinElmer Life Sciences). Quantification of the amount of the
chimeric proteins using these reagents was accomplished with a Kodak
ImageStation and Kodak 1D image analysis software version 3.0. Unless
indicated otherwise in the figure legends, the specific transcriptional activity of the various PEA3 and GAL4-PEA3 proteins was determined in
duplicate within an experiment, and each experiment was repeated at
least twice using two different preparations of the various reporter
and effector DNAs.
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays--
Transfections were carried out as described above
except that the COS-1 cells were initially plated in 60-mm (diameter)
dishes at a density of ~3 × 105 cells/dish. The
cells were transfected with a total of 6 µg of DNA comprising 3 µg
of expression plasmid DNA and 3 µg of salmon sperm DNA as carrier.
The amount of the WT PEA3 expression vector used was varied (1, 2, or 3 µg) in each experiment to derive a normal curve, whereas the total
amount of the expression vector encoding the PEA3 deletion mutants was
set at 3 µg in each experiment.
Nuclear extracts were prepared 48 h after transfection as
described (32). EMSAs were carried out as described previously (8) with
the following modifications. The binding buffer used contained 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 0.4 mM spermidine, and 7% glycerol. 5 µg of nuclear extract
was used for each binding reaction. A PEA3-specific antibody was added
to these reactions where stated. A radiolabeled double-stranded
oligonucleotide (5'-AATTCGCGAGTGGCCGGAACCGGACG-3 and the complementary
sequence 5'-GATCCGTCCGGTTCCGGCCACTCGCG-3') bearing an optimal PEA3
binding site was used as a probe in these assays.2 Prior to
their use, the DNA strands were annealed and labeled by fill-in
reaction with the Klenow fragment of DNA polymerase I using
[ Figure Generation--
Original autoradiographs were scanned in
Adobe Photoshop 3.0, and the final figures were produced using Canvas
5. In several of the figures, lanes on the same autoradiogram were
juxtaposed to facilitate representation of the data. When lanes from
the same autoradiogram were spaced together, this was achieved in such
a way as to reveal a thin white line between adjacent lanes. Otherwise,
the final figures are faithful representations of the original autoradiograms.
Construction and Analysis of the Transcriptional Activity of PEA3
Deletion Mutants--
To define the functional domains in PEA3, we
constructed a set of unidirectional amino and C-terminal deletion
mutants (Fig. 1A). These were
expressed in COS cells from the pRc/RSV effector plasmid and their
capacity to stimulate luciferase expression from a PEA3-responsive
reporter plasmid was measured. The synthetic promoter of the reporter
plasmid comprises five repeats of an optimal PEA3 binding site
juxtaposed to a minimal TATA box. Commonly, WT PEA3 expressed from the
effector plasmid increased expression of luciferase from the reporter
plasmid to levels 10-20-fold higher than that achieved following
transfection of the reporter plasmid alone (data not shown). The mutant
proteins were rarely expressed at the same level as the WT protein.
Indeed, many of the mutants that we isolated did not encode stable
proteins and were not analyzed further. In consequence, it was
necessary to measure the transcriptional activity (represented by
luciferase activity) and the abundance of each mutant protein in the
same experiment to derive their specific activity. The specific
activity of each of the mutant proteins was calculated and normalized
to that of WT PEA3, which was set at 100%.
A schematic of the structure of the various amino and C-terminal
deletion mutants of PEA3 that were analyzed is illustrated in Fig.
1A. We report the average relative expression level and the
specific activity of each of the PEA3 mutants by comparison with that
of WT from several experiments in Fig. 1B. A representative immunoblot from a single experiment is shown in Fig. 1, C
and D, respectively.
Analyses of two amino-terminal truncation mutants revealed that
deletion of 29 or 45 amino acids from this terminus reproducibly increased the specific transcriptional activity of the resulting proteins 2-3-fold, implying that these sequences negatively regulate PEA3 activity (Fig. 1B). Mutants with amino-terminal end
points at residues 55, 65, 74, and 85 were progressively compromised in
their capacity to stimulate luciferase expression by comparison with
WT. Indeed, the specific activity of dlN85 was only 10% that of the WT
protein. These observations are consistent with the presence of
positively acting sequences, perhaps an activation domain, in this
amino-terminal region. Surprisingly, a mutant lacking the first 106 amino acids of PEA3 was 5-fold more active than dlN85 and only 2-fold
less active than WT PEA3. Hence, residues between amino acids 85 and
106 may comprise another negative regulatory region. The specific
activity of several other progressively larger amino-terminal deletion
mutants (dlN120, dlN130, and dlN145) was similar to that of dl106,
implying that residues between 106 and 145 play little role in the
activity of PEA3. However, amino-terminal deletions extending to
residues 226, 245, and 326 were increasingly reduced in their activity,
consistent with the occurrence of another activation domain in this
region. The ETS domain in PEA3 is located between residues 334 and 417;
deletions impinging on the ETS domain abolished transcriptional
activity probably by compromising DNA binding and consequently were not
characterized further (Ref. 41; data not shown).
Four C-terminal deletion mutants were isolated and characterized
(dlC450, dlC440, dlC430, and dlC420). Each of these was reproducibly more active (~2-fold) than the WT protein, implying that sequences between residues 480 (the C-terminal residue of PEA3) and 450 negatively regulate PEA3 activity. Taken together, the results of the
analyses of both the amino- and C-terminal deletion mutants suggested
the occurrence of both positively and negatively acting regions located
outside of the borders of the PEA3 ETS DNA binding domain.
Analyses of GAL4-PEA3 Chimeras--
The various deletion mutants
described previously may have lesions affecting either the
transactivation or the DNA binding function of PEA3. Deletions that
increased or decreased PEA3 activity may have compromised negatively or
positively acting domains affecting transactivation or negatively
acting regions regulating DNA binding. To distinguish the effect of the
mutations on each of these activities, we independently defined the
borders of the PEA3 activation domain and those of its DNA binding
domain. To map the location of the activation domain, we constructed
GAL4-PEA3 chimeras and measured their capacity to effect the
expression of luciferase from a reporter plasmid containing repeats of
a GAL4 DNA binding site located upstream of a minimal promoter. We
could not detect the expression of the GAL4 DNA binding domain encoded
by the expression plasmid pSG424 by immunoblotting. In consequence, we
always included a transcriptionally active chimera (GAL4-PEA3dlC85) as
a positive control in each experiment.
The GAL4-PEA3 chimera comprising full-length PEA3 was transcriptionally
inert (Fig. 2). As demonstrated
subsequently, this was true independent of its abundance in cells. The
inability of GAL4-PEA3 to stimulate luciferase expression was not
surprising, because it has been commonly observed that GAL4 chimeras
harboring one of several different DNA binding domains are poor
transcriptional activators. However, two different C-terminal deletion
mutants (dlC317 and dlC267) lacking the ETS DNA binding domain were
also impaired in their capacity to stimulate luciferase expression from
the reporter. Unlike GAL4-PEA3, which was inactive, these C-terminal
deletion mutants possessed measurable but low activity. Hence, the mere
presence of the ETS domain in the GAL4-PEA3 chimera was not solely
responsible for its lack of transcriptional activity.
Deletion of C-terminal residues distal to amino acid 267 increased the
specific activity of the resulting chimeras and commensurately decreased their expression levels (Fig. 2, A and
B). Indeed, a dramatic increase in transcriptional activity
of nearly 50-fold occurred when C-terminal sequences between residues
120 and 85 were deleted. One interpretation of these findings is that
there is a strong activation domain between residues 1 and 85 in PEA3, whose activity can be substantially inhibited by a regulatory region
located between amino acids 85 and 120. It is noteworthy that analyses
of amino-terminal deletion mutants of PEA3 also suggested the
occurrence of a negative regulatory region between residues 85 and 106 capable of suppressing the activity of an activation domain located
C-terminal of residue 106 (Fig. 1, A and B).
We noted that transcriptionally inactive GAL4-PEA3 was expressed at
20-fold higher levels than transcriptionally active
GAL4-PEA3dlC85 (Fig. 2B). This suggested the
potential that the apparent inactivity of GAL4-PEA3 may have been
due to transcriptional squelching when expressed in cells at high
levels. To test this possibility, we varied the amount of the
expression vector encoding these two GAL4-PEA3 fusion proteins in
transfection assays and compared their capacity to stimulate luciferase
expression when present in cells at equivalent levels (Fig.
3). This comparison showed that under
conditions where both proteins were expressed at similar levels,
GAL4-PEA3dlC85 was active, whereas GAL4-PEA3 was not. Indeed, GAL4-PEA3
was inactive when expressed over a 10-fold range. Hence, it would
appear that GAL4-PEA3 is inert and that its inability to transactivate
transcription does not result from squelching.
Two Regions Flanking the Amino-terminal Activation Domain
Independently Negatively Regulate Its Activity--
To further refine
the location of the PEA3 activation domain and putative negative
regulatory regions affecting transactivation, we isolated
amino-terminal deletion mutants of the GAL4-PEA3 chimera bearing the
first 85 amino acids of PEA3 (Fig.
4, A and
B). This chimera, GAL4-PEA3dlC85, possesses the putative
amino-terminal negative regulatory region but lacks the candidate
inhibitory sequences flanking the C-terminal border of the activation
domain. Deletion of the first 23 or 42 residues from GAL4-PEA3dlC85
progressively increased the activity of the resulting GAL4-PEA3
chimeras to levels 6- and 20-fold greater than that of GAL4-PEA3dlC85.
It is noteworthy that deletion of this region from PEA3 also increased the activity of the resulting amino-terminal deletion mutants (Fig. 1,
A and B; compare PEA3dlN29 and PEA3dlN45 with
PEA3). Another amino-terminal deletion mutant of GAL4-PEA3dlC85 lacking the first 55 residues of PEA3 (GAL4PEA3dlN55dlC85) was substantially reduced in its capacity to activate transcription, suggesting that this
deletion impinged on the activation domain (Fig.
4A). We interpret these results to indicate that
sequences between residues 1 and 42 independently inhibit the activity
of an activation domain whose amino-terminal border lies between
residues 42 and 55 and whose C-terminal border is located near amino
acid 85.
To learn whether the candidate negative regulatory region flanking the
C-terminal border of the activation domain could also function to
independently inhibit the activity of the activation domain and to
refine the location of these sequences, we isolated additional
C-terminal GAL4-PEA3 chimeras and measured their specific activity
(Fig. 4, C and D). By comparison with
GAL4-PEA3dlC85, GAL4-PEA3dlC100 was severely impaired in its capacity
to activate luciferase expression. Indeed, GAL4-PEA3dlC100 possessed
very low activity akin to that of GAL4-PEA3dlC120 (Fig. 2A).
This suggests that the C-terminal border of this negative regulatory
region is located between residues 100 and 85.
Deletion of the amino-terminal 42 residues of PEA3, corresponding to
the amino-terminal negative regulatory region, from GAL4-PEA3dlC100 stimulated the activation potential of the resulting chimera 15-fold (Fig. 4C). However, this chimera (GAL4-PEA3dlN42/dlC100) was
only 1% as active as GAL4-PEA3dlN42/dlC85, which lacks both negatively acting regulatory regions (Fig. 4, compare A and
C). Hence, the 15 residues between amino acids 85 and
100 potently and independently inhibit the activity of the activation
domain. Taken together, these findings suggest that two negatively
acting regions residing approximately between amino acids 1 and 42 and
between amino acids 85 and 100 act independently to suppress the
activity of a strong activation domain located between residues 42 and 85.
We noted that several of the aforementioned GAL4-PEA3 chimeras were
expressed at comparatively different levels in transfected COS cells;
this might have affected assessment of their specific transcriptional
activity. Hence, to verify the results reported above, we determined
the activity of these chimeras when expressed at approximately the same
concentration in COS cells. This condition was achieved by varying the
amount of transfected DNA encoding each GAL4-PEA3 chimera (Fig.
5). Transfection of differing amounts of
the expression vectors encoding the various GAL4-PEA3 chimeras (Fig.
5A) led to a linear dose-dependent increase in
the abundance of each chimera (Fig. 5B) and to a
dose-dependent increase in luciferase activity expressed
from the GAL4 reporter plasmid (Fig. 5C). Each chimera was
expressed at a level comparable with that of any other in cells
transfected with a specific dose of the GAL4-PEA3 expression vector DNA
(summarized in Fig. 5C). Comparison of the activity of the
chimeras revealed that GAL4-PEA3dlC100 was least active. This chimera
harbors a central activation domain flanked by both negative regulatory
elements. Deletion of either the amino-terminal or C-terminal negative
regulatory region increased the activity of the resulting chimera (Fig.
5C). This was true when these chimeras were expressed at
roughly the same level. For example, compare the activity of
GAL4PEA3dlC100 at a DNA dose of 250 ng with that of
GAL4-PEA3dlN42dlC100 at a DNA dose of 500 ng or with that of
GAL4-PEA3dlC85 at a dose of 250 ng. Deletion of both negative
regulatory regions from GAL4-PEA3dlC100 resulted in a chimera
(GAL4-PEA3dlN42dlC85) that was substantially more active than any of
the others. These observations illustrate that under conditions where
equivalent levels of each chimera are expressed in cells, they
nonetheless differ substantially in their capacity to activate
expression of the reporter plasmid. These findings substantiate our
contention that the amino-terminal 100 residues of PEA3 comprise two
negative regulatory elements that flank a central activation domain;
each negative regulatory region was independently capable of inhibiting
the activity of the activation domain.
Only the Amino-terminal Activation Domain Functions in the Context
of the GAL4 DNA Binding Domain to Activate Transcription--
The
analysis of the transcription activity of PEA3 deletion mutants
suggested the potential that PEA3 might possess at least two activation
domains, one near its amino terminus (residues 42-85) and another in
the central portion of the protein. The occurrence of the centrally
located activation domain was inferred from the observation that PEA3
deletion mutants lacking the amino-terminal activation domain (and both
flanking inhibitory regions) were only 2-fold reduced in activity
compared with WT PEA3 (Fig. 2). For example, PEA3dlN106, dlN120,
dlN130, and dlN145 were ~50% as active as WT PEA3. Furthermore,
progressive deletion of amino-terminal residues from amino acids
145-226 reduced the activity of the resulting deletion mutant 10-fold
to ~5% that of WT. Other amino-terminal deletion mutants with end
points at residues 245, 278, and 326 were similarly debilitated. These
findings are consistent with the occurrence of another activation
domain in PEA3 with an amino-terminal border between residues 145 and 226.
To learn whether this central portion of the protein harbored an
activation domain, we isolated a GAL4-PEA3 chimera devoid of the
amino-terminal activation domain and flanking negative regulatory
regions but including residues bearing this putative activation domain.
This chimera (GAL4-PEA3dlN147) contained the region between residues
147 and 480 in PEA3. We also isolated five carboxyl deletion mutants of
this chimera with end points at residues 420, 375, 326, 276, and 226. All of these chimeras were expressed, but none of them stimulated
luciferase expression from the GAL4-responsive reporter plasmid (data
not shown). Hence, this central portion of PEA3 apparently does not
possess an independent activation domain as assessed by this assay. It
is conceivable that this central region and others in the ETS domain or
in the C-terminal region of PEA3 are required together to constitute an
activation domain. However, because all the GAL4 chimeras bearing the
ETS domain that we have constructed are inactive, we have been unable
to verify this possibility.
The Latent DNA Binding Activity of PEA3 Is Unmasked by
PEA3-specific Antibodies--
We showed previously that the
PEA3 ETS domain, expressed as a GST fusion protein in E. coli, is necessary and sufficient for sequence-specific DNA
binding in vitro (8). To learn whether this was also true of
the native PEA3 protein isolated from mammalian cells, we measured the
capacity of the WT and various amino and C-terminal deletion mutants
expressed in COS cells to bind to an optimized PEA3 binding site by
EMSA. Initially, we compared the DNA binding activity present in
untransfected COS cells with that present in COS cells transfected with
the PEA3 expression vector. An activity capable of binding to an
optimized PEA3 binding was observed in nuclear lysates of untransfected
COS cells (Fig. 6; band
labeled B). However, COS cells do not express
PEA3 protein at levels detectable by immunoblot analysis (Fig. 1,
C and D), and hence this species is unlikely to
be due to endogenous simian PEA3. We suspect that it represents the
binding of some other Ets protein endogenous to COS cells.
Unexpectedly, no new protein-DNA complexes were detected when
lysates from COS cells transfected with the PEA3 effector plasmid were
used in these assays (Fig. 6, lane 5). Indeed,
the same endogenous protein-DNA species was detected at the same
relative abundance in nuclear lysates prepared from COS cells
transfected with the PEA3 expression vector (compare lanes
1 and 5). Our inability to detect a novel
PEA3-DNA complex using lysates from transfected COS cells in this assay
was not due to the failure of PEA3 to be expressed in these cells (Fig. 1C; data not shown). Hence, despite the presence of PEA3 in
the lysates, this protein was apparently incapable of binding to
DNA.
A number of different explanations could account for this observation.
For example, the PEA3-DNA complex and that formed between the
endogenous protein and DNA may comigrate in the gel system used here.
Alternatively, PEA3 may not have been expressed at high enough levels
to detect its binding to DNA using this assay. Or PEA3 ectopically
expressed in COS cells may not bind to DNA. In an initial attempt to
distinguish among these possibilities, we included a PEA3-specific
antibody in the DNA binding assays. Preincubation of nuclear lysates
from untransfected COS cells with a PEA3-specific monoclonal antibody
(MP16), a PEA3-specific polyclonal antibody (PC2) or a control
Myc-specific monoclonal antibody did not alter the electrophoretic
mobility of the protein-DNA complex due to the endogenous activity
(Fig. 6, compare lane 1 with lanes
2-4). This finding too suggests the absence of PEA3 in COS
cell lysates. By contrast, both PEA3-specific antibodies effected or
stabilized the binding of a nuclear protein to the optimized PEA3
binding site after incubation with nuclear extracts prepared from
transfected COS cells (compare lane 5 with
lanes 6 and 7). This species was not
detected when a Myc-specific antibody was preincubated with lysates
containing PEA3 (lane 8). Therefore, the novel
protein-DNA species detected in lysates from COS cells transfected with
the PEA3 effector plasmid probably represents a PEA3-antibody complex
bound to DNA. We show subsequently that the electrophoretic mobility of
this complex varies with the size of PEA3 encoded by various deletion
mutants, providing additional evidence that it comprises PEA3, the
PEA3-specific antibody, and DNA.
The requirement for PEA3-specific antibodies to detect DNA binding by
PEA3 in COS cell nuclear lysates may have been due to the abundance of
PEA3 in these extracts. The antibody may stabilize the binding of low
quantities of PEA3 in the lysates to DNA. To investigate this
potential, we compared the binding of equivalent amounts of PEA3
expressed in COS cells with that of GST-dlN145PEA3 isolated from
E. coli (Fig. 7). We used an
amino-terminal truncated version of GST-PEA3 lacking the first 144 residues of PEA3 in these analyses primarily because it is more stable
during isolation from E. coli than is a GST fusion protein
comprising full-length PEA3, thereby facilitating calculation of its
specific DNA binding activity.
GST-dlN145PEA3 bound to the optimized PEA3 binding site in a
dose-dependent fashion without a requirement for
preincubation with a PEA3-specific antibody (Fig. 7A).
Measurement of the specific DNA binding activity of this GST-PEA3
fusion protein in the absence and presence of this antibody revealed
that preincubation of the fusion protein with the PEA3-specific
antibody increased DNA binding between 2- and 3-fold (Fig. 7,
A-C). By comparison, the same amount of PEA3 in
nuclear lysates from COS cells was incapable of binding to DNA unless
it was preincubated with a PEA3-specific antibody. These observations
suggest that the DNA binding activity of PEA3 in COS cell nuclear
lysates is inhibited and that preincubation of the lysate with
PEA3-specific antibodies unmasks its DNA binding activity.
Two Modules Flanking the PEA3 ETS Domain Independently Negatively
Regulate DNA Binding--
Quantification of the data presented in Fig.
7 revealed that expression of PEA3 in COS cells over a 10-fold range
resulted in a linear increase in DNA binding activity that was
proportional to the amount of PEA3 protein in the nuclear lysates (Fig.
7C). None of the PEA3 amino- and carboxyl-truncated deletion
mutants analyzed previously (Fig. 1) were expressed at less than 10%
the level of the WT protein, suggesting that we could quantitatively compare the DNA binding activity of PEA3 with that of its mutant forms.
In consequence, we transfected expression vectors encoding PEA3 and its
deleted derivatives into COS cells and tested their DNA binding
capacity by EMSA in the presence of a PEA3-specific antibody. A
polyclonal antibody recognizing the C-terminal residues of PEA3 was
used to analyze the amino-terminal deletion mutants, whereas a
monoclonal antibody, which recognizes a centrally located epitope, was
used to characterize the C-terminal deletion mutants. The results of
three separate experiments are illustrated in Fig. 8, A-C. The top
portion of each panel illustrates an EMSA,
whereas the bottom portion of each
panel represents a corresponding immunoblot of PEA3 levels
in the lysates from the same experiment. Duplicate samples bearing
equal amounts of total nuclear protein from independently transfected
cultures were assayed. The structure of the PEA3 deletion mutants is
illustrated in D, and a summary of the results of several independent repetitions of each experiment is shown in
E.
All but one of seven amino-terminal deletion mutants was capable of
binding to DNA as well as the WT protein. Only PEA3dlN85, which was
expressed at ~10% the level of WT PEA3, was reduced in its DNA
binding activity to levels 50% that of PEA3. Deletion of
amino-terminal residues up to amino acid 226 did not affect DNA
binding, suggesting that these sequences play little role in this
process. Interestingly, three mutants, PEA3dlN245, PEA3dlN278, and
PEA3dlN326, reproducibly bound DNA 2-4-fold better than did the WT
protein, suggesting the occurrence of sequences that negatively regulate DNA binding between residue 245 and the amino-terminal border
of the ETS domain. It is also noteworthy that whereas these four
amino-terminal deletion mutants (PEA3dlN226, PEA3dlN245, PEA3dlN278,
and PEA3dlN326) bound to DNA as well as or better than WT PEA3, each of
these was nonetheless severely compromised in its capacity to activate
transcription (compare Fig. 1, A and B, with Fig.
7, D and E). This suggests that residues between 226 and 480, comprising the C-terminal half of PEA3, do not harbor an
independent activation domain.
We also analyzed the DNA binding activity of four C-terminal deletion
mutants (Fig. 9). Deletion of the
C-terminal 30 amino acids of PEA3 increased DNA binding ~2-fold.
Three other C-terminal deletion mutants displayed similarly increased
DNA binding capacity that varied between 2- and 3-fold that of WT PEA3.
It is noteworthy that each of these mutants also possessed
commensurately increased transcriptional activity (2-3-fold) by
comparison with PEA3 (Fig. 1). Hence, residues C-terminal of the ETS
domain of PEA3 also appeared to negatively regulate DNA binding and
apparently play no role in transcription activation.
We inferred the occurrence of two regions in the PEA3 protein flanking
the ETS domain that inhibit DNA binding. The amino-terminal border of
one of these is located between residues 245 and 278, whereas the
carboxyl terminus of the other is located between residues 480 and 450. In independent experiments, we found that the DNA binding activity of
deletion mutants bearing either of these negative regulatory regions
was manifest only in the presence of PEA3 specific antibody (data not
shown). Hence, deletion of only one of these two regions did not
relieve the requirement for a PEA3-specific antibody to uncover PEA3
DNA binding activity. To learn whether a PEA3 mutant lacking both
inhibitory sequences could bind to DNA in the absence of a
PEA3-specific antibody, we isolated such a mutant, PEA3dlN315dlC417,
and measured the capacity of the encoded protein to bind to DNA by EMSA
following its expression in COS cells.
As shown previously, WT PEA3 in COS cell nuclear lysates was incapable
of binding to DNA unless preincubated with a PEA3-specific antibody
(Fig. 10, compare lanes
1 and 2 with lanes 3 and
4). By contrast, PEA3dlN315dlC417, comprising essentially
only the PEA3 ETS domain, was capable of binding to DNA without prior
incubation with a PEA3-specific antibody (lanes 5 and 6). Hence, our analyses of the DNA binding activity of
PEA3 deletion mutants suggest the occurrence of two DNA binding
inhibitory regions flanking the ETS domain that function independently
to block DNA binding. Deletion of both regions is required to unmask
PEA3 DNA binding activity.
PEA3 Comprises an Amino-terminal Acidic Activation Domain, Which Is
Flanked by Modules That Independently Negatively Regulate Its
Activity--
A summary of our analyses of both the specific
transcriptional and DNA binding activity of PEA3 deletion mutants is
shown in Fig. 11. Deletion of the first
45 amino acids of PEA3 increased its transcriptional activity but did
not affect its DNA binding activity. This finding is consistent with
the occurrence of sequences that negatively regulate transcription
activation in this region (residues 1-45). By contrast, amino-terminal
truncations with end points at residues 55, 65, 74, and 85 were
progressively and markedly reduced in their capacity to activate
transcription but were essentially unaffected in their capacity to bind
to DNA. Hence, the region between residues 45 and 85 probably comprises an activation domain. Like several other activation domains, this region is rich in acidic amino acids. Surprisingly, extension of the
amino-terminal deletions to residues 106, 120, 130, and 145 progressively increased the transcription activity of PEA3 by
comparison with PEA3dlN85, which was essentially devoid of activity.
Indeed, the transcriptional activity of PEA3dlN106, PEA3dlN120,
PEA3dlN130, and PEA3dlN145 varied between 40 and 60% that of the WT
protein. None of these mutants were compromised in their capacity to
bind to DNA. These observations are consistent with yet another
negative regulatory region C-terminal of residue 85 affecting
transcription activation but not DNA binding. This finding also
suggested the occurrence of another activation domain C-terminal of
residue 145. This second activation domain must include sequences
between residues 145 and 226, because PEA3dlN226, unlike
PEA3dlN145, was significantly reduced in its capacity to activate
transcription but possessed DNA binding activity equivalent to that of
WT PEA3. Collectively, our observations are consistent with the
occurrence of two negative regulatory regions and two activation
domains within the first 226 amino acids of PEA3. These sequences
appear to play no role in DNA binding, because their deletion did not
affect this process.
This interpretation is largely supported and extended by our analyses
of GAL4-PEA3 chimeras. Analyses of such chimeras confirmed the
occurrence of two negative regulatory regions affecting transcription activation flanking a strong activation domain in PEA3. The negative regulatory regions mapped between residues 1 and 42 and between residues 85 and 100, respectively, whereas the activation domain was
located between residues 42 and 85. Chimeras harboring this acidic
activation domain and either one or the other negative regulatory
module (GAL4-PEA3dlC85 or GAL4-PEA3dlN42dlC100) were much less active
than the chimera bearing only the acidic activation domain
(GAL4-PEA3dlN42dlC85) (Figs. 4 and 5). Therefore, the function of the
activation domain was potently and independently inhibited by each of
the negative regulatory regions. It will be interesting to learn
whether these regulatory regions function through an intramolecular or
intermolecular mechanism and whether they demonstrate specificity for
particular activation domains.
Analyses of amino-terminal deletion mutants of PEA3 suggested the
occurrence of a second activation domain located broadly between
residues 106 and 226. However, we were unable to verify the occurrence
of an activation domain in this region by use of GAL4-PEA3 chimeras.
Whereas these negative findings may have numerous explanations, one
possibility is that the central portion of PEA3 serves a function
unrelated to either transactivation or DNA binding per se.
We are currently investigating this potential. A schematic illustrating
the location of these various functional motifs is shown in Fig.
12.
The activation domains of the two PEA3 subfamily members, human ERM and
mouse ER81, have also been broadly localized. Analyses of GAL4-ERM
chimeras in human HeLa cells and a rabbit kidney cell line (RK13)
revealed the occurrence of an activation domain in the first 198 residues of the 510-amino acid ERM protein and another in the
C-terminal 61 residues (33). The amino-terminal 72 amino acids, but not
the C-terminal 61 residues, function to stimulate transcription in the
budding yeast, S. cerevisiae, when fused to the GAL4 DNA
binding domain (34). Similar analyses of GAL4-ER81 chimeras in rabbit
kidney cells also revealed the occurrence of an activation domain
within the amino-terminal 182 residues of mouse ER81 (35). Hence, it
would appear that all three PEA3 subfamily members possess an
amino-terminal activation domain.
The amino-terminal activation domain and the acidic region are
coincident in PEA3. The acidic region is highly similar in sequence
(~85% sequence identity) among PEA3 subfamily members and may
constitute the activation domain of all three proteins (17). The acidic
region of each PEA3 subfamily member is predicted to comprise an
There is little data to suggest the occurrence of negative regulatory
domains affecting the function of the amino-terminal activation domains
of ERM and ER81. On the contrary, there is evidence that ERM does not
possess a negative regulatory module at its amino terminus (33).
Deletion of the amino-terminal 42 residues of ERM from a GAL4-ERM
chimera bearing residues 1-72 reduced the activity of the resulting
chimera up to 10-fold.
Our analyses of deletion mutants of PEA3 and of GAL4-PEA3 chimeras
provided little support for the occurrence of a C-terminal activation
domain in PEA3 similar to that in ERM (33). Deletion of this region
(residues C-terminal of the ETS domain) from PEA3 stimulated the
capacity of the resulting mutants to activate transcription directly
commensurate with their increased DNA binding activity (Fig. 11).
Moreover, deletion mutants of PEA3 harboring these sequences but
lacking the amino-terminal activation domain (i.e.
PEA3dlN245 and PEA3dlN326) did not activate transcription, yet these
mutants bound DNA as well as the WT protein (Fig. 11). Hence, analyses of PEA3 deletion mutants provided no evidence for an activation domain
in the C-terminal region of PEA3. Indeed, direct test of the potential
of a GAL4-PEA3 chimera bearing only the C-terminal residues of PEA3
(residues 419-480) to activate transcription in COS cells revealed
that it did so at levels only 0.2% that of the chimera bearing the
amino-terminal acidic activation domain (residues 42-85) (data not
shown). Hence, it seems unlikely that this weak C-terminal activation
domain plays a significant role in transactivation by PEA3.
Two Regions Flanking the PEA3 ETS Domain Negatively Regulate DNA
Binding--
The DNA binding activity of PEA3 expressed in COS cells
could not be detected unless nuclear lysates bearing the protein were preincubated with antibodies directed against PEA3. Two different PEA3-specific antibodies reactive with epitopes located in either the
center or the carboxyl terminus of the protein were independently capable of unmasking its DNA binding activity. The latent DNA binding
activity of COS cell-expressed PEA3 could also be uncovered by deleting
two inhibitory regions flanking the ETS DNA binding domain. The
amino-terminal border of one such negative regulatory module was
located between residues 245 and 278, and the C-terminal border of the
other was located between residues 480 and 450 (Fig. 12). Deletion of
either negative regulatory region separately did not overcome the
requirement for a PEA3-specific antibody to uncover DNA binding
activity, implying that each region acts independently to repress
binding of the ETS domain to DNA. Interestingly, a GST-PEA3 fusion
protein isolated from E. coli and bearing both negative
regulatory modules and the ETS domain bound to DNA in the absence of a
PEA3-specific antibody. Incubation of these proteins with a
PEA3-specific antibody modestly (2-3-fold) increased DNA binding.
Hence, fusion of GST to PEA3 simultaneously abrogates the function of
both negative regulatory modules.
Taken together, these observations suggest that native PEA3 in
mammalian cells exists in a dynamic equilibrium between two states, one
capable and another incapable of binding to DNA. We suspect that the
transition between these two states is normally regulated and is
accompanied by structural changes in the protein. This equilibrium may
be perturbed artificially by interaction with an antibody, linkage to
GST, or mutation. In vivo, the transition between the
inactive and active states for DNA binding is likely to be governed by
post-translational modification (i.e. phosphorylation, acetylation, proteolysis, etc.) and/or interaction with one or more
partner proteins.
We reported previously that mouse PEA3 synthesized in vitro
is incapable of binding to DNA (41). This finding has been confirmed and extended using zebrafish PEA3 synthesized in vitro in
reticulocyte lysates (10). Deletion of either one of two regions
flanking its ETS domain improves DNA binding, but this effect is
greatest when both regions are deleted. These negatively acting regions bordering the zebrafish ETS domain comprise about 90 amino acids (amino-terminal region) and 60 amino acids (C-terminal region) and
approximate the locations of those reported herein for mouse PEA3.
Expectedly, these negative regulatory regions in mouse and zebrafish
PEA3 share significant amino acid identity, 49% identity between the
two amino-terminal negative regulatory regions and 59% between the two
C-terminal negative regulatory regions. By comparison, the overall
sequence identity between mouse and zebrafish PEA3 is 54%, and that
between their ETS domains is 96%.
The sequences affecting DNA binding by the PEA3-related Ets protein ERM
have also been located (33). Full-length ERM synthesized in
vitro in reticulocyte lysates binds to DNA, but to a lesser extent
than do deleted versions of the protein lacking sequences flanking the
ETS domain. One of the negative regulatory regions at the carboxyl
terminus of human ERM occurs at the same position and shares sequence
similarity to the corresponding negative regulatory region of mouse and
zebrafish PEA3. However, the negative regulatory region amino-terminal
of the ERM ETS domain does not correspond in either location or
sequence to that of the mouse and zebrafish proteins. The reason for
this difference is not clear.
Several other Ets proteins including Ets-1 (5, 38), Elk-1 (39), and
PU.1 (40) also possess regulatory modules flanking their ETS domain
that negatively affect DNA binding. The location and structure of these
regions have been extensively characterized for Ets-1, which has served
as a model for studies of auto inhibition of DNA binding by
sequence-specific transcription factors (41). The regulatory modules in
Ets-1 comprise PEA3 Transcription Activity Is Regulated at Multiple
Levels--
Our deletion analyses reveal that the activity of PEA3 is
negatively regulated at the level of both transactivation and DNA binding. This suggests that its activity is tightly regulated by
mechanisms that are currently poorly understood. We imagine that the
interaction of PEA3 with other proteins and or its post-translational modification may affect both of these functions of the protein. In this
regard, it is noteworthy that PEA3 is phosphorylated at serine residues
by mitogen-activated protein kinases and that some of these sites occur
within the negative regulatory regions identified
here.6 Moreover, we have
recently identified several proteins that physically interact with
PEA3, although the functional consequences of these interactions have
yet to be uncovered.7
Clearly, it will be of interest to unravel the mechanisms accounting for the regulation of PEA3 activity and to learn more of its
developmental and physiological roles.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP. The double-stranded oligonucleotide was
purified by polyacrylamide gel electrophoresis, and 0.3 ng of this DNA
was used in the binding assays. DNA binding activity was quantified by
using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) as
described above. The specific DNA binding activity of the various
PEA3-related proteins was determined by normalizing DNA binding
activity to the expression level of each PEA3 deletion mutant. The
specific DNA binding activity of WT PEA3 was set as 100%. Unless
stated otherwise in the figure legends, all DNA binding experiments
were carried out a minimum of three times with at least two independent preparations of the effector DNAs encoding PEA3 or its deleted derivatives.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of regions in PEA3 affecting
its transcriptional activity in COS cells. A,
schematic illustrating the structure of WT PEA3 and deletion
derivatives thereof. B, comparative expression level and
specific activity of the deletion mutants. The transcriptional activity
and expression level of WT PEA3 was set at 100%. The data were derived
from four independent experiments. C, representative
immunoblot illustrating the abundance of PEA3 and amino-terminal
deletion mutants thereof in equal amounts (20 µg) of total cell
protein from transfected COS cells. Lane 1,
lysate from COS cells transfected with 0.8 µg of the empty expression
vector, pRC/RSV. Lanes 2-5, COS cell lysate from
cells transfected with 0.2, 0.4, 0.6, or 0.8 µg of pRC/RSV-PEA3
encoding WT PEA3. Lanes 6-17, lysate from COS
cells transfected with 0.8 µg of pRC/RSV-PEA3 encoding the
amino-terminal deletion mutants, N45,
N55,
N65,
N74,
N85,
N106,
N120,
N130,
N145,
N226,
N245, and
N326, respectively. The polyclonal antibody, PC2, was used to detect
PEA3 and its derivatives in these samples. Note the occurrence of a
cellular cross-reacting protein in all the lanes that migrates between
the 48- and 34-kDa markers. D, representative immunoblot
showing the abundance of PEA3 and PEA3 deletion mutants in equal
amounts (20 µg) of total cell protein from transfected COS cells.
Lane 18, lysate from COS cells transfected with
0.8 µg of the empty expression vector, pRC/RSV. Lanes
19-21, COS cell lysate from cells transfected with 0.2, 0.4, or 0.8 µg of pRC/RSV-PEA3 encoding WT PEA3, respectively.
Lanes 22-26, lysate from COS cells transfected
with 0.8 µg of pRC/RSV-PEA3 encoding
N29,
C420,
C430,
C440, and
C450, respectively. The monoclonal antibody, MP16, was
used to detect PEA3 and its deleted derivatives in these samples.
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Fig. 2.
Transcriptional activity of GAL4-PEA3
chimeras. A, schematic illustrating the
structure of the GAL4-PEA3 chimeras. Each chimera bears the GAL4 DNA
binding domain and either all of PEA3 or various amino-terminal
portions of PEA3. The abundance (relative expression) of the various
GAL4-PEA3 chimeras relative to that of GAL4-PEA3dlC85 ( C85), which
was set as 1.0, is shown. Similarly, the specific activity of the
GAL4-PEA3 chimeras by comparison with that of GAL4-PEA3dlC85 is also
shown. The data from four independent experiments were averaged.
B, representative immunoblot of the abundance of the
GAL4-PEA3 chimeras in COS cell lysates resulting from a single
experiment. Equal amounts of total cellular protein (30 µg) was
loaded onto the gel prior to immunoblotting. Protein samples were
prepared from COS cells transfected with 0.5 µg of the pSG424-PEA3
expression vectors encoding
C317 (lane 1),
C267 (lane 2),
C177 (lane
3),
C128 (lane 4),
C120
(lane 5),
C85 (lane 6),
C55 (lane 7), and GAL4-PEA3 (lane
8). The GAL4-PEA3 chimeras were detected with an antibody
that recognizes determinants in the GAL4 DNA binding domain.
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Fig. 3.
GAL4-PEA3 is transcriptionally inactive.
A, table illustrating the activity and abundance
of GAL4-PEA3 chimeras. COS cells were transfected in duplicate with 500 ng of pSGS424, which encodes the GAL4 DNA binding domain (Gal4 DBD),
with 20, 50, or 100 ng of the vector encoding GAL4-PEA3 (PEA3) or with
100, 250, or 500 ng of that encoding GAL4PEA3dlC85 (PEA3 C85). Cell
lysates from the transfected cells was used to measure luciferase
activity, which was recorded as relative light units. The abundance of
the GAL4 chimeras in 30 µg of cell protein from cells transfected in
the same experiment was expressed in arbitrary units relative to that
of GAL4-PEA3dlC85 (set to 1) following PhosphorImager analysis.
B, representative immunoblot of a single experiment carried
out in duplicate. COS cells were transfected with various amounts of
the expression vector encoding either GAL4-PEA3 or GAL4-PEA3dlC85 as
described above. The chimeras were detected with an antibody that
recognizes determinants in the GAL4 DNA binding domain.
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Fig. 4.
Mapping the borders of two negative
regulatory regions flanking the amino-terminal activation domain of
PEA3. A and C, schematic
representations of GAL4-PEA3 chimeras. The abundance and
specific activity of these chimeras were determined as described in the
legend to Fig. 3. The data from four independent experiments were
averaged. B and D, representative immunoblots
illustrating the abundance of the various GAL4-PEA3 chimeras in COS
cells. Note that the photographs were derived by splicing groups of
lanes from the same individual autoradiograms. A thin
white area was left between the
lanes to clearly illustrate this fact. The GAL4-PEA3
chimeras were detected as described in the legend to Fig. 3.
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Fig. 5.
The PEA3 amino-terminal activation domain is
flanked by negative regulatory modules. A,
schematic illustration of the GAL4-PEA3 chimeras.
B, representative immunoblot of a single experiment
illustrating the dose-dependent increase in the expression
of GAL4-PEA3 chimeras following transfection of cells with differing
amounts of the various expression vectors. COS cells were transfected
with 100, 250, or 500 ng of the indicated expression vector. The
abundance of the GAL4-PEA3 chimeras in 50 µg of cell protein was
assessed following transfection by use of a chemiluminescence protocol
as described under "Experimental Procedures." The abundance of the
chimeras was expressed in arbitrary units. C,
table illustrating the activity and abundance of the various
GAL4-PEA3 proteins following transfection of COS cells. An
approximately equal amount (5 µg) of cell protein from transfected
cells was used to measure luciferase activity, which was recorded as
relative light units expressed per µg of cell protein. The abundance
of the GAL4 chimeras in 50 µg of cell protein from cells transfected
in the same experiment was expressed in arbitrary units.
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Fig. 6.
The cryptic DNA binding function of PEA3 is
unveiled by incubation with PEA3-specific antibodies. DNA binding
reactions were carried out as described under "Experimental
Procedures" using 5 µg of nuclear extracts isolated from COS-1
cells transfected with the empty expression vector ( PEA3)
or that encoding PEA3 (+PEA3). The arrows
indicate the position of protein-DNA complexes. A,
antibody-PEA3-DNA complexes; B, protein-DNA complexes formed between a
nuclear protein endogenous to COS cells and the PEA3 binding site;
C, the free labeled DNA bearing an optimal PEA3 binding
site. MP16 and PC2 refer to a monoclonal and polyclonal antibodies
(Ab), respectively, that were preincubated with nuclear
lysates before the DNA binding assays were carried out. Each of these
antibodies binds to different antigenic determinants in PEA3. Myc
refers to an antibody reactive with the c-Myc protein and was used as a
negative control in these experiments.
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Fig. 7.
Comparison of the specific DNA binding
activity of recombinant GST-PEA3 and PEA3 isolated from mammalian
cells. A, EMSA illustrating the binding of varying
amounts of purified GST-PEA3dlN145 or PEA3 in COS cell nuclear extracts
to the PEA3 binding site. Increasing quantities of GST-PEA3dlN145 (1, 2.5, 5, or 10 ng) and 5 µg of nuclear extract isolated from COS-1
cells transfected with either 1, 2, or 3 µg of the PEA3 expression
plasmid were assayed in duplicate as described under "Experimental
Procedures." The MP16 antibody (Ab) was preincubated with
GST-PEA3dlN145 or with the nuclear extracts where indicated.
B, immunoblot illustrating the amount of GST-PEA3dlN145 or
PEA3 in the various samples used in A. The abundance of PEA3
in the samples was measured using the polyclonal antibody PC2, which
recognizes antigen determinants at the carboxyl terminus of PEA3.
C, quantitation of the specific DNA binding activity of
GST-PEA3dlN145 and PEA3 in COS cell nuclear extracts. Calculation of
the abundance of GST-PEA3dlN145 and PEA3 and their specific DNA binding
activity was performed as described under "Experimental Procedures"
using the data in A and B.
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Fig. 8.
Analyses of the DNA binding activity of
amino-terminal deletion mutants. A-C, EMSA
(top panel) and accompanying immunoblot
(corresponding bottom panel) illustrating the DNA
binding activity and abundance of the amino-terminal PEA3 deletion
mutants. The data illustrated in each panel were derived
from a separate experiment. 5 µg of nuclear extract from transfected
COS cells was used in binding reactions in the presence of the PC2
polyclonal antibody. 8 µg of nuclear extracts was used with the PC2
polyclonal antibody to detect PEA3 in the nuclear lysates by
immunoblotting. D, schematic
representation of PEA3 deletion derivatives that were used
in these analyses. E, illustration of the DNA binding
activity, expression levels and calculated specific DNA binding
activity of PEA3 and its amino-terminal deletion derivatives. The
specific DNA binding activity of PEA3 or its derivatives was calculated
from the total DNA binding activity present in 5.0 µg of nuclear
extract and dividing by the relative amount of PEA3 as determined by
immunoblotting in these extracts as described under "Experimental
Procedures." The values shown represent the average of four
independent experiments.
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Fig. 9.
Analysis of the DNA binding activity of
C-terminal deletion mutants of PEA3. A, EMSA assay of
the indicated mutants. 8 µg of nuclear extracts from transfected COS
cells was used with the MP16 monoclonal antibody. B,
immunoblot illustrating the abundance of PEA3 and the PEA3 deletion
mutants in nuclear lysates from transfected COS cells. The abundance of
PEA3 was assessed with the PN1 polyclonal antibody. C,
schematic representation of the PEA3 deletion
mutants. D, DNA binding activity, abundance, and specific
DNA binding activity of the PEA3 C-terminal deletion mutants. The
results represent the average of four independent experiments.
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Fig. 10.
Deletion of both inhibitory regions flanking
the PEA3 ETS domain relieves the requirement for a cross-reacting
antibody for DNA binding. Duplicate samples of nuclear extract (5 µg) from independently transfected COS cells were analyzed. The PC2
polyclonal antibody was added where indicated. The arrows
indicate specific PEA3-DNA complexes (A and C),
the protein-DNA complex due to the binding of an endogenous protein in
COS cell nuclear extracts (B), and the free PEA3
double-stranded oligonucleotide used in the assay (D). The
schematic depicts the structure of PEA3 and the
PEA3 N315
C417 deletion mutant, which harbors only the ETS
domain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 11.
Summary of the transcriptional and DNA
binding activities of the amino and C-terminal deletion mutants of
PEA3. A, schematic representation
of the structure of the PEA3 deletion mutants. B,
bar diagram illustrating the specific
transcriptional and DNA binding activity of the PEA3 deletion mutants.
The data represented in this figure was taken from Figs. 1,
8, and 9.
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Fig. 12.
Schematic illustrating the functional
domains, negative regulatory regions, and sequence motifs in mouse
PEA3. AD, acidic domain; ETS, ETS domain.
-helix (36, 37). Indeed, a structural analysis (circular dichroism)
of a peptide bearing this region of human ER81 demonstrates that it can
assume an
-helix, albeit in a nonphysiological hydrophobic solvent
(34). However, NMR analyses of a 128-residue amino-terminal
fragment of PEA3 comprising the acidic activation domain and both
negative regulatory modules suggest that this region is unstructured in
physiological solutions.5
Hence, if this region assumes an
-helical structure in
vivo, then this may be induced by its interaction with a partner protein.
-helical regions that are thought to allosterically
inhibit the function of another
-helical motif, termed helix 1, in
the ETS domain that is required for DNA binding. There are no obvious
sequence similarities among the inhibitory regions bordering the ETS
domains of these various Ets proteins, but the potential that they
comprise similar structural modules seems a likely possibility.
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ACKNOWLEDGEMENTS |
---|
We thank Laura Hastings for technical assistance and Brian Allore and Dinsdale Gooden of the Mobix Central Facility for providing oligonucleotides and automated DNA sequencing services. We also thank Silvia Arber and Thomas Jessell for providing PEA3-specific polyclonal antibodies. Finally, thanks are due to Michael Rudnicki for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by the Canadian Institutes of Health Research, the National Cancer Institute of Canada, and the Canadian Breast Cancer Research Initiative.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institute for
Molecular Biology and Biotechnology, 1280 Main St. W., Hamilton, Ontario L8S 4K1, Canada. Tel.: 905-525-9140 (ext. 27217); Fax: 905-521-2955; E-mail: hassell@mcmail.cis.mcmaster.ca.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M005509200
2 S. Bowman and J. A. Hassell, manuscript in preparation.
3 L. Hastings and J. A. Hassell, unpublished results.
4 S. Arber and T. Jessell, personal communication.
5 J. Barrett, L. MacIntosh, A. Edwards, and J. A. Hassell, unpublished data.
6 S. Perron, R. Tozer, U. Ashraf, S. Walbi, and J. A. Hassell, manuscript in preparation.
7 J-H. Xin and J. A. Hassell, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PEA3, polyoma enhancer activator 3; ETV1, -4, and -5, Ets translocation variant 1, 4, and 5, respectively; EMSA, electrophoretic mobility shift assay; WT, wild type; GST, glutathione S-transferase.
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