From the Department of Pathology, Harvard Medical School, Boston, Massachussetts 02115
Received for publication, January 4, 2001, and in revised form, March 2, 2001
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ABSTRACT |
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Genetic studies of the Drosophila erect
wing (ewg) gene have revealed that ewg
has an essential function in the embryonic nervous system and is
required for the specification of certain muscle cells. We have found
that EWG is a site-specific transcriptional activator, and we report
here that evolutionarily conserved regions of EWG contribute both
positively and negatively to transcriptional activity. Using gel
mobility shift assays, we have shown that an EWG dimer binds
specifically to DNA. In transfection assays, EWG activated expression
of a reporter gene bearing specific binding sites. Analysis of deletion
mutants and fusions of EWG to the Gal4 DNA binding domain has
identified a transcriptional activation domain in the C terminus of
EWG. Deletion analysis also revealed a novel inhibitory region in the N
terminus of EWG. Strikingly, both the activation domain and the
inhibitory region are conserved in EWG homologs including human nuclear
respiratory factor 1 (NRF-1) and the sea urchin P3A2 protein. The
strong conservation of elements that determine transcriptional activity
suggests that the EWG, NRF-1, and P3A2 family of proteins shares common
mechanisms of action and has maintained common functions across evolution.
The erect wing gene is essential for
Drosophila embryogenesis, and analysis of mutants has
revealed that ewg1
function is required for proper development of the embryonic nervous
system as well as certain muscles (1). Viable alleles of erect
wing give rise to flies with defects in indirect flight muscle
development, leading to the erect wing phenotype. Immunostaining has
shown that EWG is a nuclear protein expressed in neurons during the
embryonic and larval stages (2). During pupal development, EWG is
expressed in the myoblasts that give rise to the indirect flight
muscles (3). The expression of EWG is regulated posttranscriptionally; the regulated efficiency of the splicing of the ewg
transcript leads to an enrichment of the mRNA encoding the 116-kDa,
733-amino acid-long form of the EWG protein in the nervous
system (4). The expression of a cDNA encoding the 116-kDa
form of EWG in neurons rescues the lethality caused by loss-of-function
mutations in ewg, and broad expression of this cDNA also
restores muscle development, indicating that the 116-kDa form of the
protein can provide all known ewg activities (2, 3).
EWG is a member of a family of related proteins that includes the human
NRF-1 (also called In this study we show that EWG functions as a transcriptional
activator and that evolutionarily conserved regions of EWG contribute both positively and negatively to transcriptional regulation. In gel
mobility shift assays, an EWG dimer binds specifically to a consensus
NRF-1 binding site. In Drosophila tissue culture cells, EWG
activates transcription from a promoter bearing binding sites for EWG.
Deletion analysis of EWG reveals a C-terminal activation domain and an
N-terminal domain that inhibits transactivation. The core of the
activation domain is highly conserved, and the mutation of
evolutionarily conserved residues in the activation domain reduces
activity. Our studies suggest that EWG participates in neurogenesis and
indirect flight muscle development by directly binding to and
regulating the expression of target genes important for differentiation
or maintenance of these tissues. The finding that evolutionarily
conserved regions of EWG contribute to transcriptional regulation
suggests that EWG and its homologs in other species have maintained
common mechanisms of action and regulation across evolution.
Plasmids and Cloning--
EWG deletion mutants were generated by
polymerase chain reaction and cloned into pPac for expression from the
Drosophila actin promoter or pT
For expression of Gal4 + EWG fusions from the Drosophila
actin promoter, EWG fragments generated by polymerase chain reaction were cloned in frame C-terminal to Gal4-(1-147) in pPacG4. The EWG
residues present in the Gal4 fusions are fAD amino acids 351-733, N1
amino acids 351-654, N2 amino acids 351-631, N3 amino acids 351-564,
N4 amino acids 351-542, N5 amino acids 351-480, C3 amino acids
564-733, C4 amino acids 631-733, C5 amino acids 654-733, M1 amino
acids 542-654, M2 amino acids 542-631, M3 amino acids 564-654, M4
amino acids 564-631, M5 amino acids 542-564, and M6 amino acids
631-654. The EWG inserts were confirmed by sequencing. Site-directed
mutagenesis was used to substitute the following pairs of amino acids
with alanine: Thr-591 and Thr-594; Ile-592 and Val-595; Ser-642 and
Tyr-644; Val-639 and Val-641. The mutant EWG M3 fragments were
sequenced and introduced into pPacG4.
G5-E1B-luc has been described (16). EWGBS3-luc was generated by cloning
three copies of the wild-type EWG binding site upstream of the TATA box
in E1B-luc.
DNA Binding Studies--
Complementary oligonucleotides were
annealed and used in the binding reactions. The sequences of the top
strand oligonucleotides were: wild-type,
5'-GTAGACTGCGCATGCGCATCTGGA-3'; 5G, 5'-GTAGACTGCGGATGCGCATCTGGA-3'; and
5G8C, 5'-GTAGACTGCGGATCCGCATCTGGA-3'. Annealed oligonucleotides were
end-labeled and purified using a Qiagen nucleotide removal kit. Gel
mobility shift assay conditions were essentially as described (10).
Binding reactions were performed in 30 µl of TM buffer (25 mM Tris (pH 7.9), 6.25 mM
MgCl2, 0.5 mM EDTA, 0.5 mM
dithiothreitol, 50 mM KCl, and 10% (v/v) glycerol)
containing 2 µg of sonicated salmon sperm DNA, 1 µl of in
vitro translated protein (Promega), and 0.1 pmol of labeled
oligonucleotide. For competition experiments, unlabeled
oligonucleotides in 10, 50, and 250 M excess were added to
the binding reaction prior to adding labeled oligonucleotide. After
incubation at room temperature for 20 min, the samples were electrophoresed on a 4% polyacrylamide gel
(acrylamide:bisacrylamide, 19:1) in 0.5 × TBE with 1%
glycerol. Gels were then dried and autoradiographed.
Transfections--
1.5 ml Drosophila S2 cells at
1 × 106 cells/ml were plated in 35-mm dishes,
and 24 h later the cells were transfected by calcium phosphate
precipitation. For analysis of full-length EWG and deletion mutants,
100 ng of the indicated pPacEWG plasmid was co-transfected with 200 ng
of either EWGBS3-luc or E1B-luc, 20 ng of pRL-tk as an internal
control, and carrier DNA to bring the total to 3 µg. For analysis of
G4 + EWG fusion proteins, 2 ng of the pPacG4 + EWG plasmid was
co-transfected with 100 ng of G5E1B-luc, 20-50 ng of pRL-tk as an
internal control, and carrier DNA to 3 µg. After 40-48 h, cells were
harvested and luciferase assays were performed using the dual
luciferase kit from Promega. All transfections were done in triplicate
on at least three occasions.
Immunoblot Analysis--
The expression of EWG derivatives was
analyzed following transfection of 3 µg of the appropriate pPac-EWG
or pPacG4 + EWG construct into Drosophila S2 cells. Total
cell lysates were analyzed using affinity-purified rabbit sera directed
against EWG-(1-350), EWG Is a Sequence-specific DNA-binding Protein--
EWG is highly
related to human NRF-1, chicken IBR/F, and sea urchin P3A2 proteins in
regions that correspond to the nuclear localization, DNA binding, and
dimerization domains identified in these homologs (7, 9, 17, 18). In
addition, an alignment of EWG with NRF-1 reveals patches of high
homology in the C-terminal half of these proteins as well (Fig.
1). EWG residues 146-174 are 79%
identical to the nuclear localization signal identified in NRF-1 (18).
The predicted nuclear localization signal in EWG is likely to be
functional because immunostaining has shown EWG to be nuclear in the
neurons of Drosophila embryos (2). The high degree of
sequence similarity between EWG residues 185-343 and the DNA binding
and dimerization domain of NRF-1 predicts that EWG is also a
sequence-specific DNA binding-protein. Using a gel mobility shift
assay, we observed full-length EWG bound to a 24-base pair
oligonucleotide containing a palindromic NRF-1 consensus site (Fig.
2A) (8, 9). As expected, the
addition of an unlabeled wild-type oligonucleotide efficiently competed the observed gel mobility shift. Much higher concentrations of an
oligonucleotide with a single nucleotide substitution (5G) were needed
to compete for EWG binding, and a mutant oligonucleotide with
substitutions in both halves of the palindromic site (5G8C) did not
compete for binding by EWG. Consistent with these results, when labeled
5G and 5G8C oligonucleotides were used in gel mobility shift assays,
EWG was found to bind very poorly to the 5G oligonucleotide and not at
all to the 5G8C oligonucleotide (data not shown). Thus, EWG is a
sequence-specific, DNA-binding protein with a sequence specificity
similar to that of NRF-1.
We also used a gel mobility shift assay to investigate if EWG binds to
this palindromic site as a dimer (19). A truncated form of EWG
(residues 1-350) translated in vitro was found to bind to
the NRF-1 consensus site, but similar to full-length EWG the truncated
form bound very poorly to the 5G and not at all to the 5G8C mutant
oligonucleotides (Fig. 2B and data not shown). When
full-length EWG and EWG350 were cotranslated and used in the gel
mobility shift assay, a single additional band of intermediate mobility
was observed, indicating that EWG binds DNA as a dimer (Fig.
2B). When full-length EWG and EWG350 were translated
individually and then mixed no intermediate band was observed,
suggesting that EWG forms stable dimers in solution. Thus, EWG amino
acids 1-350 are sufficient to bind specifically to DNA as a dimer,
indicating that these evolutionarily conserved sequences share a common function.
EWG Is a Transcriptional Activator--
The observation that EWG
is a sequence-specific, DNA-binding protein raised the possibility that
EWG might regulate transcription through its cognate binding sites
either as an activator like NRF-1 or as a repressor like P3A2. We
therefore introduced three EWG binding sites upstream of a consensus
TATA box driving luciferase expression to generate an EWG-responsive
reporter gene, EWGBS3-luc. Cotransfection of a plasmid expressing
full-length EWG stimulated luciferase expression from this reporter
gene ~30-fold but did not stimulate a reporter gene lacking EWG
binding sites (Fig. 3A).
Consistent with our findings, EWG has recently been shown to activate
expression of an NRF-1-responsive cytochrome c promoter in
transfection assays (20). Unlike full-length EWG, the EWG350 deletion
mutant did not stimulate expression from the EWGBS3-luc reporter (Fig.
3B). Immunoblot analysis indicated that EWG350 was expressed
in the transfected cells at levels equal to or greater than full-length
EWG (data not shown). Because EWG350 is able to bind DNA and form
heterodimers with full-length EWG (Fig. 2B), we tested to
see if this mutant could act as a dominant negative. Cotransfection of
a plasmid expressing full-length EWG with increasing amounts of a
plasmid expressing EWG350 resulted in a dose-dependent inhibition of luciferase expression (Fig. 3B). Thus, EWG is
a transcriptional activator, and sequences in the C terminus are necessary for the activation function.
The EWG Activation Domain Is Highly Conserved--
Having found
that the C terminus of EWG was necessary for transcriptional
activation, we tested to see whether the C terminus was in fact
sufficient to function as an activation domain. EWG amino acids
351-733 were fused to the DNA binding domain of Gal4 (to generate fAD)
and cotransfected into Drosophila tissue culture cells along
with a reporter gene bearing five Gal4 binding sites upstream of a
consensus TATA box driving luciferase expression (G5-luc). In this
assay the C terminus of EWG was a very potent activation domain because
the Gal4 + EWG fAD fusion stimulated transcription 1,200-fold more than
Gal4-(1-147) alone (data not shown). We then generated and tested N-
and C-terminal deletions to further define the regions of EWG that
contribute to activation. The expression of all Gal4 + EWG fusions was
confirmed by immunoblot analysis with anti-Gal4 antisera (data not
shown). As shown in Fig. 4, the minimal
EWG activation domain is contained in deletion M3, residues 564-654,
that activates transcription to 25-30% of the level of the entire
C-terminal fragment. With the exception of the N2 deletion, which has
only about 10% of the activity of the full-length Gal4 + EWG fusion,
all of the active Gal4 + EWG fusions, N1, C3, M1, and M3 retain
residues 564-654. The EWG minimal activation domain contains a highly
conserved core, residues 631-654. However, both the highly conserved
region and the adjacent region contribute to activation because neither
alone is sufficient for activity (see M4 and M6 in Fig. 4B).
Although not sufficient, the highly conserved region (residues
631-654) is important for activation because deletion of this region
significantly reduces the activation function (compare N1
versus N2, M1 versus M2, and M3 versus
M4).
To confirm the importance of conserved residues in the activation
domain, double alanine substitution mutants were analyzed in the
context of the minimal activation domain, M3, amino acids 564-654.
Alanine substitution of either Ser-642 and Tyr-644 or Val-639 and
Val-641 in the highly conserved region significantly reduced activation
(Fig. 5). Substitution of bulky
hydrophobic residues in the adjacent region with alanine (Ile-592 and
Val-595) also reduced activity, whereas the substitution of two
hydrophilic residues in this region (Thr-591 and Thr-594) had no
effect. Thus, particular conserved residues throughout the minimal
activation domain contribute to activity.
Deletions of EWG Confirm the Importance of the Conserved Activation
Domain and Reveal an N-terminal Inhibitory Domain--
To confirm that
the activation domain identified in Gal4 fusions contributes to
activation by EWG, we tested additional deletion mutants in EWG for
their ability to activate expression from the EWGBS3-luc reporter. The
expression of all EWG deletions was confirmed by immunoblot analysis
(data not shown). As shown in Fig.
6A, EWG654, which lacks the
sequences C-terminal to residue 654, was able to activate expression of
the reporter gene, although less well than the full-length EWG. In
contrast, EWG631, which lacks the evolutionarily conserved core of the
activation domain identified in the Gal4 fusions, was essentially
inactive. The deletion end point in EWG631 corresponds to the Gal4 + EWG N2 fusion indicating that, although this C-terminal deletion
retains about 10% activity as a Gal4 fusion, it does not have
significant activity in the context of EWG. A deletion mutant lacking
the minimal activation domain identified in the Gal4 fusions, EWG564,
was as inactive as EWG350, which lacks the entire C terminus.
Thus, these data confirm that sequences in the C terminus of EWG are
required for activation and support a critical role for the conserved
core of the activation domain, residues 631-654.
We have found that evolutionarily conserved sequences in the N terminus
of EWG down-regulate transcriptional activation. Although deletion of
nonconserved residues prior to the first methionine at position 87 (EWG
initiates with CTG) (2) did not reduce the activity of EWG, deletion of
the first 144 residues was unexpectedly found to increase activation
35-fold (Fig. 6A). This was surprising because previous
studies of NRF-1 have shown that deletion of the residues from the N
terminus to the nuclear localization signal reduced DNA binding (21),
and EWGN We report here that evolutionarily conserved regions of the
Drosophila erect wing gene product, EWG, function to
regulate transcription. Using in vitro DNA binding assays
and transfection assays in Drosophila tissue culture cells,
we have shown that EWG is a sequence-specific, DNA-binding protein that
activates transcription from its cognate binding sites. Our analysis of EWG deletion derivatives has revealed functional roles in
transcriptional regulation for each of the regions of EWG that are
conserved in the human NRF-1 and sea urchin P3A2 homologs with the
exception of residues 543-564. In contrast, our studies failed to
reveal a critical role for the nonconserved regions of EWG. Because of this strong correlation of sequence conservation with function, it
seems likely that conserved residues 543-564 contribute to EWG
activity in certain physiological contexts not reproduced in our
assays. Consistent with the proposal that the highly conserved region
of EWG from residues 146-343 participates in nuclear localization, DNA
binding, and dimerization, we have found that deletion of the sequences
C-terminal to amino acid 350 does not impair DNA binding or
dimerization, and deletion of the sequences N-terminal to position 144 does not reduce, but rather stimulates, site-specific activation. We
have mapped the minimal activation domain to residues 564-654, which
includes a highly conserved core, residues 631-654, necessary for
activation. Unexpectedly, a conserved region in the N terminus was
found to inhibit activation by EWG. The identification of
evolutionarily conserved regions that both positively and negatively influence transcriptional activation by EWG raises the possibility that
the activity of EWG and its homologs may be regulated by common
mechanisms dependent upon cell type or promoter context.
A wide variety of unrelated amino acid sequences has been shown to
function as activation domains (22); thus it is particularly striking
that the EWG activation domain is so highly conserved. An analysis of
NRF-1 deletions has revealed a major role for NRF-1 residues 449-477
in activation that corresponds well to the highly conserved core that
we have shown to be critical for the function of the EWG activation
domain (18). In the case of NRF-1, the mutation of hydrophobic residues
in the activation domain also significantly reduced activation,
although mutation of multiple glutamines in the activation domain had
no effect (18). NRF-1 has been shown to bind the coactivator PGC-1
through its DNA binding domain, but no targets of the NRF-1 activation
domain have been identified (23). The high sequence conservation of the
activation domain across evolution strongly suggests that EWG and NRF-1
activate transcription by a common mechanism. As shown in Fig. 5, the
core activation domain is well conserved in the sea urchin P3A2
homolog, which has been shown to repress transcription (13, 14). It is
interesting to note that in P3A2 there is alanine at the position equivalent to Val-641 in EWG because we have found that a double mutant
in which alanine replaced both Val-639 and Val-641 was severely
impaired for activation. Thus, it is possible that P3A2 does not
activate transcription because of changes in the region homologous to
the activation domain or, alternatively, that this function may be
conserved and P3A2 may function both as an activator and as a repressor.
Analysis of EWG deletion derivatives revealed the presence of a novel
conserved inhibitory domain in the amino terminus. Deletion of residues
87-144 resulted in a 55-fold increase in activation (Fig.
6B). Modulation of transcriptional activation by EWG may be
critical because overexpression of EWG in Drosophila,
particularly outside the nervous system, is lethal (4). Previous
studies with NRF-1 found that deletion of the N terminus ( Expression of the 116-kDa form of EWG is enriched in the
Drosophila nervous system because of differential efficiency
of splicing (4). Several other splice variants of ewg
mRNA have been described with the potential of encoding
additional EWG isoforms. Our identification of
functional domains in EWG supports predictions of the functions of other forms of EWG protein that may be expressed. All of the observed splice variants encode the N terminus, DNA binding, and dimerization domains. Alternative splicing of exon D (amino acids 386-540), which is not conserved between EWG and NRF-1, has been observed in both neuron-rich heads and neuron-poor bodies. In our
analysis of Gal4 + EWG fusions, residues encoded by exon D were not
required for activity; therefore our data suggest that EWG forms both
containing and lacking exon D should function as transcriptional
activators. Interestingly, the highly conserved region that is critical
for function of the EWG activation domain is present in a single small
exon, exon H, which encodes amino acids 627-668. Thus, we predict that
EWG proteins derived from the observed splice variants lacking exon H
would fail to activate transcription. Although transgenic studies have
shown that the 116-kDa form of EWG rescues development of the indirect
flight muscle, the form of EWG expressed in muscle has not been
identified. It is of obvious interest to determine whether EWG activity
in the nervous system and in myoblasts requires the same functional domains.
We have identified evolutionarily conserved regions of EWG that
contribute to DNA binding and dimerization, transcriptional activation,
and regulation of the activation function. Our studies suggest that the
essential function of EWG in the developing Drosophila nervous system is to regulate expression of specific target genes. Two
ewg mutants have been molecularly characterized; both
introduce early stop codons before the DNA binding/dimerization
domain3 (4). Characterization
of additional ewg mutant alleles should help confirm that
the functional domains we have identified contribute to EWG function
in vivo. NRF-1 has been shown to regulate expression of
nuclear encoded genes important for mitochondrial function, and NRF-1
binding sites have been implicated in the regulation of many genes
involved in growth-responsive metabolic pathways as well as
neuron-specific genes (8, 10, 11, 24, 25). Identification of target
genes regulated by EWG will provide new insights into the role of this
transcription factor in development of the nervous system and
specification of the indirect flight muscles.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Pal), chicken IBR/F, zebrafish Nrf, and
sea urchin P3A2 proteins (2). Although these proteins share regions of
significant homology, diverse activities have been reported for EWG
homologs in different species. Similar to ewg, zebrafish
nrf is an essential gene expressed in the developing nervous
system (5). NRF-1, IBR/F, and P3A2 were identified as sequence-specific
DNA-binding proteins, although the DNA binding domain in these proteins
does not appear to correspond to a known structural motif (6-9). Human
NRF-1 has been shown to activate the expression of several nuclear
genes involved in mitochondrial function, including cytochrome
c and mitochondrial transcription factor A, supporting the
hypothesis that NRF-1 acts to coordinate nuclear and mitochondrial
functions (8, 10-12). In sea urchins, P3A2 contributes to early
pattern formation in the embryo by repressing expression of the CyIIIA
cytoskeletal actin gene in oral ectoderm (6, 13, 14). In contrast to
IBR/F, which has been found to repress transcription of the histone
h5 gene by binding to a site overlapping the start site of
transcription (9, 15), P3A2 acts at a distance to repress
transcription. The high degree of sequence similarity between EWG,
NRF-1, and P3A2 raises the possibility that EWG may also function to
regulate transcription either as an activator or a repressor.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
stop for use in in
vitro transcription/translation reactions. EWG350 encodes amino
acids 1-350, EWG564 encodes amino acids 1-564, EWG631 encodes amino
acids 1-631, and EWG654 encodes amino acids 1-654. For analysis of
N-terminal deletions, a C-terminal flag epitope was added by polymerase
chain reaction. Flag-tagged derivatives include full-length EWG,
EWGN
86 (amino acids 87-733), EWGN
144 (amino acids 145-733),
EWG
87-130, EWG
87-144, and EWG
130-144.
-Flag (Sigma), or
-Gal4-(1-147) (Upstate
Biotechnology, Lake Placid, NY).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The Drosophila EWG protein
is highly related to human NRF-1. A schematic of the EWG and NRF-1
proteins is shown with the extent of sequence similarity indicated. The
NRF-1 nuclear localization sequence (NLS), DNA binding, and
dimerization domains are all highly conserved in EWG (>70% homology).
In addition, two small stretches in the C terminus, EWG residues
543-564 and 631-654, also share >70% homology with NRF-1. A region
in the N terminus and one in the C terminus share >50%
similarity.
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Fig. 2.
EWG binds specifically to an NRF-1 consensus
site as a dimer. A, EWG is a sequence-specific
DNA-binding protein. EWG was translated in vitro and used in
a gel mobility shift assay with a radiolabeled oligonucleotide
containing the consensus binding site for NRF-1. In lane 1 an unprogrammed lysate was used, indicated as "no EWG." Increasing
amounts (10-, 50-, and 250-fold excess) of unlabeled wild type
(WT), 5G mutant, or 5G8C double mutant competitor
oligonucleotide were included in the binding reaction as indicated. The
sequence of the wild type NRF-1 consensus binding site is indicated as
WT, and the substitutions in the 5G and 5G8C mutant sites
are indicated. B, EWG binds DNA as a dimer. Full-length
EWG or a C-terminal truncation of EWG (EWG350) was translated
in vitro and analyzed in a gel mobility shift assay using
the wild type binding site. Full-length EWG and EWG350 were in
vitro translated individually, co-translated, or translated
separately and mixed as indicated. The unbound oligonucleotide is not
shown here.
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Fig. 3.
The C terminus of EWG is necessary for
transcriptional activation. A, EWG activates
transcription from a reporter gene with EWG binding sites. A plasmid
expressing EWG or empty vector was cotransfected into
Drosophila S2 cells along with a luciferase reporter gene
bearing three EWG binding sites upstream of a consensus TATA box
(EWGBS3-luc) or the control lacking EWG sites (E1B-luc). The luciferase
activity obtained with empty expression vector and EWGBS3-luc was set
at 1. The experiment was performed in triplicate; error
bars indicate the standard deviation. B, EWG350
functions as a dominant negative. Plasmids expressing full-length EWG
or the C-terminal deletion EWG350 were cotransfected with the
EWGBS3-luc reporter as indicated. When increasing amounts of the EWG350
expressing plasmid (50, 100, and 200 ng) were cotransfected with 100 ng
of a plasmid expressing full-length EWG, the resulting luciferase
activity was reduced in a dose-dependent manner. The
expression of full-length EWG and EWG350 in transfected cells was
confirmed by immunoblot (data not shown). The luciferase activity
obtained with empty expression vector and EWGBS3-luc was set at 1. The
experiment was performed in triplicate; error
bars indicate the standard deviation.
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Fig. 4.
A highly conserved region comprises the
core of the EWG activation domain. A, fusions of
C-terminal fragments of EWG to the DNA binding domain of Gal4 (residues
1-147) were cotransfected into Drosophila S2 cells with a
reporter bearing five Gal4 sites upstream of a luciferase reporter gene
(G5-luc). The regions of EWG contained in the Gal4 fusions are
indicated schematically as described in Fig. 1. The activity of the
Gal4 + fAD fusion containing EWG residues 351-733, which activated
expression 1,200-fold more than Gal4(1-147) alone (not shown), was
set at 100%. The relative activity of N- and C-terminal deletions
of the EWG activation domain is indicated. The experiment was performed
in triplicate; error bars indicate the standard
deviation. B, EWG amino acids 564-654 constitute the
minimal activation domain. The activity of the indicated Gal4 + EWG
fusions was determined as described in A.
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Fig. 5.
Mutations in the conserved core of the EWG
activation domain reduce activity. A, schematic of the
minimal activation domain of EWG (residues 564-654) with the positions
of double amino acid substitutions indicated. An alignment of the
evolutionarily conserved core of the activating region is shown.
Identical residues are shaded and boxed; similar
residues are boxed. Shown are EWG residues 631-654, NRF-1
residues 446-469, and P3A2 residues 388-410. Asterisks
indicate the positions of alanine substitutions in EWG. B,
Mutations in conserved residues reduce activity of the
minimal activation domain. Double alanine substitutions at positions
Thr-591 and Thr-594, Ile-592 and Val-595, Val-639 and Val-641, or
Ser-642 and Tyr-644 were analyzed in the context of Gal4 + EWG M3.
Plasmids expressing wild-type or the indicated mutant derivatives of
Gal4 + EWG M3 were cotransfected with the G5luc reporter. The activity
of wild type Gal4 + EWG M3 was set at 100%. The experiment was
performed in triplicate; error bars indicate the
standard deviation.
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Fig. 6.
Evolutionarily conserved sequences in both
the N- and C-terminal regions of EWG affect the activation
function. A, the core activation domain is necessary
for activation by EWG. Plasmids expressing wild-type or the indicated
C-terminal deletion mutants of EWG were cotransfected with the
EWGBS3-luc reporter. The regions of EWG contained in the derivatives
are indicated schematically as described in Fig. 1. The luciferase
activity stimulated by full-length EWG was set at 100%. Expression of
all constructs was verified by immunoblotting (not shown). The
experiment was performed in triplicate; error
bars indicate the standard deviation. B, the
conserved N terminus of EWG inhibits activation. Plasmids
expressing wild-type EWG or the indicated N-terminal deletions with a
C-terminal Flag tag were cotransfected with the EWGBS3-luc reporter.
The activity of full-length EWG was set at 100%. The activity of the
N-terminal deletions was determined as described in A. Note
that the scale is different in panels A and
B.
144 was also found to bind DNA with reduced affinity in gel
mobility shift assays.2 The
deletion of EWG residues 87-144 resulted in a dramatic 55-fold increase in activity. Smaller internal deletions of residues 87-130 led to a 35-fold increase in activity, whereas deletion of residues 130-144 had only a modest effect. The fact that similar levels of activation were not obtained by increasing the amount of wild-type EWG plasmid used in the transfection together with the results of
immunoblot analysis suggest that increased protein levels are not
sufficient to explain the increased activity (data not shown). It is
interesting to note that the inhibitory region is absent in the Gal4 + fAD fusion, which activated transcription about 40-fold better than
full-length EWG in our assays. A fusion of EWG-(1-144) to the Gal4 DNA
binding domain did not activate or repress transcription.2
Thus, these studies confirm that the C-terminal activation domain that
we have identified is the only region of EWG that is both necessary and
sufficient for activation. Interestingly, the inhibitory domain in the
N terminus of EWG is evolutionarily well conserved, suggesting that the
transcriptional activity of EWG and its homologs, NRF-1 and P3A2, may
be modulated by a common mechanism.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
77)
resulted in decreased DNA binding, which in the case of NRF-1 was shown to be due to a defect in dimerization (21). It was therefore surprising
that deletion of the N terminus of EWG resulted in increased
activation, particularly because deletion of EWG residues 1-144 was
also found to reduce DNA binding activity in
vitro.2 Our results suggest that the N terminus of
NRF-1 may also function to inhibit activation. Interestingly,
phosphorylation of the N terminus of NRF-1 in response to extracellular
signals has been shown to increase transcriptional activation by NRF-1
because of an increase in DNA binding (although not dimerization) (12, 21). The N terminus of NRF-1 has also been reported to interact with
dynein light chains, although the functional significance of this is
unknown (20). The conserved inhibitory region is rich in acidic
residues, but the sequence provides no clues as to whether inhibition
involves interactions in cis with other regions of EWG or in
trans with corepressor proteins. If the latter mechanism
applies, it is possible that this amino-terminal domain contributes to
transcriptional repression by P3A2. It will be interesting to determine
whether the inhibitory activity of the EWG N terminus is modulated in
response to extracellular signaling pathways or through interactions
with other transcription factors at specific promoters.
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ACKNOWLEDGEMENTS |
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We thank Catherine Huang for initiating studies of EWG and Kalpana White and Matthias Soller, Brandeis University, for the ewg cDNA and helpful discussions. We also thank T. Keith Blackwell for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by March of Dimes Birth Defects Foundation Basil O'Connor Starter Scholar Research Award Grant 5-FY98-0555 (to G. G.).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.
Present address: Cell and Molecular Biology Program, Duke
University, Durham, NC 27710.
§ To whom correspondence should be addressed: Dept. of Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0985; Fax: 617-432-1313; E-mail: grace_gill@hms.harvard.edu.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M100080200
2 I. K. Fazio and G. Gill, unpublished data.
3 T. A. Bolger and G. Gill, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ewg, erect wing; NRF-1, nuclear respirator factor 1; luc, luciferase.
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