From the Department of Molecular and Cellular
Physiology, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267-0576 and the § Department of
Molecular and Cellular Biology, Harvard University,
Cambridge, Massachusetts 02138
Received for publication, December 19, 2002
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ABSTRACT |
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Interferon regulatory factor (IRF)-7 is activated
in response to virus infection and stimulates the transcription of a
set of cellular genes involved in host antiviral defense. The mechanism by which IRF-7 is activated and cooperates with other transcription factors is not fully elucidated. Activation of IRF-7 results from a
conformational change triggered by the virus-dependent
phosphorylation of its C terminus. This conformational change leads to
dimerization, nuclear accumulation, DNA-binding, and transcriptional
transactivation. Here we show that activation of IRF-7, like that of
IRF-3, is dependent on modifications of two distinct sets of Ser/Thr
residues. Moreover, we show that different virus-inducible cis-acting
elements display requirements for specific IRFs. In particular, the
virus-responsive element of the ISG15 gene promoter
can be activated by either IRF-3 or IRF-7 alone, whereas the P31
element of the interferon- Viral infection of vertebrate cells results in the early secretion
of a number of cytokines. These include interferons
(IFNs),1 tumor necrosis
factors, and several chemokines and interleukins that signal the
occurrence of the infection and orchestrate the innate immune response
directed against the invading virus (1-4). The production and action
of these cytokines depends in large part on specific modulations of
gene expression.
Expression of type I IFN genes is controlled at least in
part by IFN regulatory factors (IRFs), such as IRF-3, IRF-5, and IRF-7.
These IRFs differ in their pattern of expression: IRF-3 is a
constitutively and ubiquitously expressed protein, whereas IRF-5 and
IRF-7 are expressed at different levels in various tissues, and their
synthesis can be further stimulated following exposure to IFNs. In
cells where the constitutive levels of IRF-7 are low, this
IFN-dependent up-regulation of IRF-7 is crucial for
expression of its target genes (e.g. most IFN- Normally, IRF-3 and IRF-7 associate with each other, and upon virus
infection they further interact with the coactivators p300 and CBP
(CREB-binding protein) to form a stable complex termed VAF
(virus-activated factor). VAF alone is sufficient to activate the
transcription of IFN- and virus-inducible genes through their ISRE
(IFN-stimulated response element), where it binds with high affinity.
However, it is necessary but not sufficient to activate the
IFN- The activity of IRF-7 is modulated by virus-dependent
post-translational modifications, but the exact mechanism by which it activates transcription is not fully elucidated. In particular, it
would be of interest to identify the amino acids involved in IRF-7
activation, and to determine whether IRF-7 can activate transcription
independently from other transcription factors or from coactivators. In
this study, we examined IRF-7-dependent transcription in
mammalian cells and in insect cells (S2 cells, derived from
Drosophila melanogaster embryos). Insect cells have a number
of advantages that alleviate limitations associated with the use of
mammalian cells: 1) they do not have any apparent IRF ortholog (IRF
family members have overlapping binding specificity and can associate
with each other); 2) the role of mammalian coactivators can be assessed
(Drosophila CBP ortholog is functionally different enough
that it cannot coactivate transcription with IRF-3 (16)); 3) the
IFN-dependent feedback loop that leads to IRF-1 and IRF-7 up-regulation cannot operate in insect cells (no IFN or IRF orthologs); 4) there is no stimulation of virus-activated signal transduction pathways by DNA transfection alone.
Here, we show that, unlike IRF-3, IRF-7 exhibits intrinsic
transcriptional activity (16). Nevertheless, binding of the mammalian p300/CBP coactivators further stimulates the ability of IRF-7 to
activate transcription. Whereas IRF-3 and IRF-7 can independently activate transcription from the ISRE, efficient activation of the P31
sequence from the IFN- Plasmid Constructs and Sequence Analysis--
Effector
constructs for transient transfections of mammalian and insect cells
were cloned into pcD Cell Culture and Transfections--
HEC-1B (HTB-113, ATCC) cells
were derived from a human endometrial carcinoma and are resistant to
IFN; SAN cells were derived from a human glioblastoma and are lacking
type I IFN genes; 293T cells are a SV40 large T antigen
expressing highly transfectable derivative of 293 cells, which were
derived from human embryonic kidney cells transformed with human
adenovirus type 5. These cell lines were grown at 37 °C, 5%
CO2, in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. Sendai virus was obtained from SPAFAS and used at
200 hemagglutinating units/ml. S2 cells were grown at
26 °C, in Schneider's Drosophila medium containing 12%
fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin.
Transfections using the calcium phosphate coprecipitation technique
were as described (17). Mammalian cells in 100-mm dishes were
transfected with 1 ml of a precipitate containing 10 µg of reporter,
5 µg of effector plasmid (except for Gal4, 1 µg), 5 µg of
pCMV-lacZ and pSP72 to a total of 25 µg (5-9 µg) for 18 h,
trypsinized, aliquoted for further treatments, and harvested 3 days
after transfection.
S2 cells were seeded in 6-well plates (3 million cells in 3 ml),
transfected the next day with 0.3 ml of a precipitate containing 250 ng
of hsp82lacZ, 500 ng of reporter plasmid, and effector plasmid mixtures
as indicated in the figure legends (with pPac added to a total of 5.75 µg), and harvested 2 days after transfection. CAT activity and
In Vitro Translation, Cell Extract, EMSA, and
Western--
In vitro translation in rabbit reticulocyte
lysates were performed exactly according to the manufacturer's
recommendations using the TNT kit (Promega), linearized
pcD Pull-down Experiments--
GST-CBP-N, -M, -C, -p300-N, -M, and C
were described previously (20), GST-CBP-N1, -N2, -N3, -C1, -C2, -C3,
and GST-p300-C1, -C2, -C3 were generated by subcloning PCR products and
verified by sequencing. GST and GST fusions were expressed in
Escherichia coli BL21 and purified as recommended by the
manufacturer (Amersham Biosciences), and dialyzed against
phosphate-buffered saline, 10% glycerol.
35S-Labeled in vitro translated proteins were
incubated with GST fusion proteins immobilized on glutathione-Sepharose
beads in 150 mM KCl, 20 mM Tris, pH 8.0, 0.5 mM dithiothreitol, 50 µg/ml ethidium bromide, 0.2%
Nonidet P-40, and 0.2% bovine serum albumin (binding buffer) for
1 h at 4 °C, followed by two washes with binding buffer and two
washes with binding buffer without bovine serum albumin. Extracts from
HEC-1B cells labeled in vivo with [32P]orthophosphate were prepared and treated with
deoxycholate and Nonidet P-40 exactly as described (13). After 50-fold
dilution with binding buffer and preclearing on glutathione-Sepharose
beads, the 32P-labeled proteins were incubated with
immobilized GST fusions for 2 h at 4 °C, followed by three
washes with binding buffer. Proteins bound to the beads were eluted
with RIPA buffer and immunoprecipitated with SL-12 (anti-IRF-3) or
affinity-purified anti-IRF-7 (HU36) as described (13). Pulled down and
immunoprecipitated proteins were then analyzed by SDS-PAGE and autoradiography.
Transcriptional Activity of IRF-7 Mutants in Insect Cells--
In
mammalian cells, IRF-7 is constitutively associated with IRF-3, which
is present ubiquitously, and it is thus unclear whether IRF-7 can
activate transcription on its own. The ability of IRF-7 to activate
transcription independently from other transcription factors or from
coactivators was tested in S2 cells. Because S2 cells lack the IFN
system, IRF-7 cannot be activated through the virus-dependent pathway. However, IRF-7 can be made
constitutively active in mammalian cells either by deletion of an
internal inhibitory domain or by glutamic acid (Glu) substitutions of
several critical Ser residues in the C terminus (5, 21). These residues
have been proposed to be the targets of virus-dependent
phosphorylation, and they fall into two sets based on sequence
comparison (Fig. 1A). We used
constructs expressing human IRF-7B wild-type (WT), IRF-7E2, IRF-7E6,
and IRF-7E8 (mutants of one or both sets of target residues), and
IRF-7
Expression of IRF-7B WT, IRF-7E6, or IRF-7E8 alone resulted in minimal
activity of the ISREx3CAT reporter, whether the p300/CBP coactivators
were coexpressed or not. By contrast, expression of IRF-7
The unexpected lack of activity of IRF-7E6 in insect cells might
reflect poor DNA binding in the absence of IRF-3 with which it is
normally associated. We tested this possibility using Gal4-IRF-7 fusions and a reporter, G5E1bCAT, driven by five copies of a
Gal4-binding site. Gal4-IRF-7WT, E2, E6, and E8 had very low level
activity in S2 cells, which was minimally stimulated by p300/CBP
coexpression (Fig. 1C, middle panel). The
activity of E6 was slightly stronger than that of Gal4-IRF-7 WT, which
could be accounted for by the difference in their expression levels
(Fig. 1C, right panel). By contrast,
Gal4-IRF-7
Thus, IRF-7 Transcriptional Activity of IRF-7 Mutants in Human Cells--
We
first tested our IRF-7E6 mutant in 293T cells and confirmed that, as
described for IRF-7(D475-487) (21), it was constitutively active and
virus infection did not appreciably stimulate its activity (data not
shown). However, the use of 293T cells for such experiments might be
problematic (limitations 1-4, see Introduction). Therefore, we
examined the phenotypes of a panel of hIRF-7B mutants by doing cotransfections in SAN cells. These cells lack type I
IFN genes, avoiding the complication of a feedback
loop, and transfection per se does not activate
virus-dependent pathways as effectively as in 293T cells
(limitations 3 and 4). We used the P31x2CAT reporter, which is only
weakly inducible by virus alone but strongly stimulated by WT IRF-7,
thus allowing the phenotypes of mutations to be clearly assessed
(13).
The sequence and phenotype of each IRF-7B point mutants are shown in
Fig. 2A. If a residue is
normally phosphorylated in response to viral infection, mutations to
Ala are expected to decrease virus-dependent activity. By
contrast, mutations to Glu could lead to constitutive activity or to a
decrease in virus-dependent activity depending on how
effectively Glu functions as a phosphoserine mimetic. Consistent with
previous observations, expression of full-length IRF-7B strongly
stimulated the activity of P31x2CAT, in both uninfected and infected
cells (13). Mutation of either set of Ser residues to Ala (IRF-7A2,
-A6, and -A8) drastically reduced the ability of IRF-7 to stimulate
P31x2CAT activity in virus-infected cells, suggesting that both sets of
residues are involved in virus activation. Mutation of the first set to
Glu (IRF-7E2) also strongly suppressed IRF-7 activity in virus-infected cells. By contrast, mutation of part or all of the second set of
residues to Glu (IRF-7Em3 and E6) had no effect on virus-activated levels, but substantially increased IRF-7 constitutive activity. Combining the IRF-7E6 mutation with either Ala or Glu substitution in
the first set (IRF-7A2E6 and -E8), however, led to a strong decrease in
the ability of IRF-7 to stimulate P31x2CAT activity both in uninfected
and virus-infected cells, suggesting that Glu residues do not serve
as good phosphomimetics in the first set.
In addition, we also assayed each of these mutants as fusion proteins
with the Gal4 DNA-binding domain by doing cotransfections with
G5E1bCAT. This approach minimized the possibility of endogenous IRF
proteins being associated with the ectopically expressed IRF mutants
(limitation 1). Most mutants displayed no transcriptional activity
(Fig. 2A, right panel), suggesting that the
activity detected with the P31x2CAT reporter was the result of their
association with endogenous IRF-3 or IRF-7 molecules. Gal4-IRF-7B WT
activity was still inducible but the basal activity was significantly
higher than for the native protein. Intriguingly, Gal4-IRF-7E6 showed very strong basal activity and virus infection enhanced it to levels
even higher than those achieved with Gal4-IRF-7
The fact that the activity of IRF-7E6 or Gal4-IRF-7E6 could be further
stimulated upon virus infection in SAN cells unless the first set of
residues were mutated to Ala or Glu strongly suggests that residues
within both sets were phosphorylated in response to viral infections.
Furthermore, the constitutive activity displayed by IRF-7E6 or
Gal4-IRF-7E6 in SAN or 293T cells is best accounted for by
phosphorylation of the first set of residues upon transfection because
this constitutive activity was absent when these residues were mutated
or when IRF-7E6 or Gal4-IRF-7E6 were expressed in insect cells.
We also took advantage of the activity of IRF-7
The phenotypes of IRF-7B deletion mutants are shown in Fig.
3C. The shortest truncation (aa 449) led to a complete lost
of virus responsiveness, and further deletions removed inhibitory sequences and uncovered a weak constitutive activation domain (aa 388, 308, and 266). By contrast, expression of an internal deletion mutant,
IRF-7 IRF-7 Interacts with Multiple Domains of
p300/CBP--
The functional interaction between IRF-7 and
p300/CBP as seen in the transfection experiments suggest that these
proteins may also interact physically. We first examined the
interaction between IRF-7 and the coactivators using metabolically
32P-labeled protein and three non-overlapping fragments of
CBP fused to GST (see "Experimental Procedures"). As shown in Fig.
4B, IRF-7 associates
specifically with the C-terminal 550 aa of murine CBP in a
virus-dependent manner, although the interaction is much weaker than what was observed with IRF-3 in a similar experiment (16).
Thus, endogenous virus-activated IRF-7 can interact with the C-terminal
domain of CBP.
We next used in vitro translated IRF proteins in pull-down
experiments to further map this interaction (a representative
experiment is shown in Fig. 4C, and binding values referred
to in the text below correspond to the average of at least three
independent experiments). Both IRF-7BWT and IRF-7
For p300, IRF-7WT and IRF-7 Mapping IRF-7 Domains--
Latent IRF-7 does not bind DNA but
virus activation or truncation of the C-terminal domain results in DNA
binding activity (13). We conducted EMSA experiments using the ISRE of
the ISG15 gene as a probe, WT or mutant IRF-7B proteins
expressed in vitro, and GST-CBP fusion proteins. We detected
very little ISRE binding for IRF-7B or IRF-7
In the presence of GST-CBP-N, a slow migrating complex was detected
with IRF-7B, IRF-7
These interactions were also assayed independently from DNA binding by
pull-down assays (Fig. 5B). Interestingly, IRF-7
When tested in S2 cells, IRF-7B1-388 displayed no
transcriptional activity, whereas further deletion to 308 showed some activity (Fig. 5C), as was the case in mammalian cells (Fig.
2B). Taken together, these results indicated that there is
no straightforward correlation between the ability of IRF-7B to bind
DNA, interact with the p300/CBP coactivators, and activate
transcription in either mammalian or insect cells. Indeed,
IRF-7B1-388 bound DNA most efficiently, interacted
robustly with p300/CBP, and yet completely failed to activate
transcription. Rather, these results suggest that IRF-7 functional
domains are arranged in a complex pattern, where the domains involved
in DNA binding, transcriptional activity, and interactions with
coactivators appear to be distinct and partially overlapping (Fig.
5D).
IRF-3, IRF-7, p300, and CBP Synergistically Activate
Transcription--
We have shown that constitutively active forms of
either IRF-3 or IRF-7 can each activate transcription in insect cells,
i.e. independently from the presence of any endogenous IRF
(Fig. 1 and Ref. 16). In mammalian cells, however, IRF-7 is found to be
constitutively associated with IRF-3, and ectopic expression of both
proteins leads to maximal transcriptional activation (13), suggesting
these proteins have evolved to regulate the expression of
virus-inducible genes in a cooperative manner. We therefore examined
the effect of cotransfecting IRF-3 and IRF-7 on two reporters, one
driven by the ISRE of the ISG15 gene, and the other driven by the IRF-binding P31 element of the IFN-
In these experiments, we used a constitutively active form of IRF-3,
IRF-3E7, where both sets of Ser/Thr residues have been changed to Glu
residues (16). When equal amounts of IRF-7
We also examined the effect of increasing amounts of IRF-3E7,
IRF-7
Thus, we conclude that IRF-3 and IRF-7 can strongly synergize in the
presence of p300/CBP, as the combination of these transcription factors
was 60 times more potent than what would be expected if their effects
were purely additive. This synergy and the dramatic increase in
transcription over a small increment in amounts of transfected plasmids
further suggest that the assembly of IRF-3, IRF-7, p300, and CBP into
an activating complex on the P31 sequence is highly cooperative.
IRF-7, c-Jun, and CBP Synergistically Activate
Transcription--
A reporter driven by a single copy of the P31
element fails to respond to virus infection in mammalian cells.
However, P431, which includes the AP-1-binding site of the IFN-
These results suggest that the ability of IRF-7 and c-Jun to interact
with CBP might be more important for their ability to synergize with
other transcription factors than to activate transcription by
themselves. Interfering with the interaction between these transcription factors and CBP by coexpressing a N- or C-terminal GST-CBP fusion protein inhibited the synergistic activation of P431x3CAT by c-Jun/IRF-7 IRF-7 is a transcription factor found associated with the promoter
of virus-inducible genes only in virus-infected cells and IRF-7 is
critical for the full activation of type I IFN genes (5,
13). Because of a number of limitations in the experimental systems
used so far, several aspects of the mechanism by which IRF-7 activates
transcription remain unresolved (see Introduction). Complementing our
studies with experiments in insect cells that bypass these limitations,
we were able to address some of these issues.
IRF-7 Activated Transcription Directly--
First, it was unclear
whether IRF-7 could activate transcription independently from other
transcription factors, because it is known to be constitutively
associated with the IRF-3 protein, which is ubiquitous in mammalian
cells, and it has the potential to interact with other IRFs. Here we
showed that a constitutively active form of IRF-7, IRF-7
It was also unclear whether IRF-7 activity is dependent on
coactivators, as no physical interactions had been detected between these proteins (21), and elimination of the coactivators by gene
targeting results in embryonic lethality. We found that IRF-7 IRF-7 Activation Depends on Modifications within Two Distinct Sets
of Residues--
We have shown that in the case of IRF-3, two sets of
residues cooperate in achieving maximal transcriptional activation
(16). However, the identity of phosphorylated residues remained unclear for IRF-7. Levy and colleagues (5) suggested that murine (m)IRF-7 activation depends on phosphorylation of the first set of residues (Ser425-426, which correspond to Ser471-472
in hIRF-7A, Ser443-444 in hIRF-7B, and
Ser385-386 in hIRF-3) (5). By contrast, Hiscott and
colleagues (21) proposed that phosphorylation of the second set of Ser
residues between aa 475 and 487 of hIRF-7A is responsible for
activation. In particular, these investigators found that
IRF-7(D475-487) is a strong constitutive activator in 293T cells.
We undertook a systematic analysis of the role played by both sets of
residues (Fig. 2). The observation that most mutants with only one set
of Ser residues mutated to unphosphorylatable amino acids are still
robustly virus-inducible, while mutants with both sets mutated are not,
strongly suggests that residues within both sets of Ser were
phosphorylated following viral infection. The constitutive activity of
IRF-7E6 is relatively weak in SAN cells, very strong in 293T cells, and
completely absent in insect cells. These results suggest that IRF-7E6
must be further modified upon transfection of mammalian cells. These
differences in activity could best be accounted for by phosphorylation
of the first set of residues by a mammalian-specific kinase, possibly
activated by transfection (limitation 4), as suggested for IRF-3
(16).
Thus, Glu residues only partially mimicked the effect of Ser
phosphorylation, which could be expected because of the intrinsic differences between their side chains. In the case of IRF-3, the Glu
substitution was least efficient for the first set, but IRF-3E7 (where
both sets were substituted with Glu residues) was nevertheless active
(16). In the case of IRF-7, phosphorylation of the first set of
residues was essential for activity as substitution to non-phosphorylatable residues blocked activity. Unlike what was observed for IRF-3, substitution of both sets with Glu residues in
IRF-7 failed to activate transcription (Figs. 1 and 2). This failure to
activate was not because of lack of DNA binding, as Gal4 fusions of the
same mutants displayed as little activity as Gal4-IRF-7WT (Fig. 1).
Rather, phosphorylation of the first set seems to be essential for the
conformational change of IRF-7 that leads to dimerization, DNA-binding,
and transcriptional activation. Nevertheless, in the context of
IRF-7 IRF-3, IRF-7, p300, and CBP Activate Transcription
Synergistically--
The fact that IRF-3 and IRF-7 can act
independently does not mean that they do so in physiological
conditions. In fact, IRF-3 and IRF-7 can associate with each other, and
this association is constitutive, unlike their
virus-dependent association with p300/CBP (13, 21).
When the ISREx3CAT reporter was used, IRF-3 was a more potent activator
than IRF-7 (Fig. 6), in agreement with the observation that IRF-3 binds
more efficiently to the ISRE than IRF-7 (whereas the reverse is true
for P31) (13). The combination of IRF-3 and IRF-7 was more potent than
each activator alone, and these results are consistent with the
analysis of ISG15 gene expression in embryonic fibroblasts
derived from IRF-3 and IRF-9 double knock-out mice (23).
Activation from the P31x4CAT reporter was very low with IRF-3 or IRF-7
alone, but the combination of these two factors strongly synergized
(60-fold, Fig. 6), as they do in mammalian cells (13). Synergy between
IRF-3 and IRF-7, like IRF-3 transcriptional activity, was entirely
dependent on the presence of the coexpressed mammalian coactivator.
Synergy is the functional counterpart of cooperativity in physical
association, which is itself dependent on multiple contacts between
interaction partners. Multiple protein-protein interactions are indeed
involved in the formation of VAF, a complex containing IRF-3, IRF-7,
p300, and CBP: IRF-3 and IRF-7 each interact with p300/CBP through two
and four distinct domains, respectively (Ref. 16 and this study); both
IRF-3 and IRF-7 homodimerize; IRF-3 further interacts with IRF-7
through two distinct domains (24); and p300/CBP also interact with each
other through multiple domains.2 These multiple
interactions may account for the unusual stability of VAF as compared
with most other transcription factor-coactivator complexes, which
typically cannot be detected in mobility shift assays.
Cooperativity in VAF formation is further suggested by the
observation that a small increment in amounts of plasmids expressing
IRF-3, IRF-7, p300, and CBP resulted in a dramatic increase in
transcription of the P31-driven reporter in insect cells.
Synergy between IRF-7 and c-Jun Is
CBP-dependent--
The presence of mammalian coactivators
is essential for IRF-3 activity. Therefore, the possible mechanisms by
which these coactivators promote synergy between IRF-3 and IRF-7, such
as scaffolding a nucleoprotein complex or recruitment of the
transcriptional machinery, could not be independently ascertained. By
contrast, both IRF-7 Our data indicate that IRF-7 could activate transcription
independently from other transcription factors and coactivators, and
are most consistent with virus infection leading to the phosphorylation of IRF-7B within two sets of Ser residues at its C terminus. We show
that IRF-7 interacted with four distinct regions of the p300/CBP coactivators and that these interactions were playing an essential role
in the ability of IRF-7 to strongly synergize with other transcription
factors. Such synergy presumably reflected the cooperative assembly of
nucleoprotein complexes where coactivators could be playing a
scaffolding role. VAF is uniquely activated by virus infection or
double-stranded RNA treatment and its cooperative assembly would ensure
that p300/CBP molecules, which are thought to be present in the cell at
limiting concentrations, would be redirected from other signaling
pathways to participate in the antiviral transcriptional response
required for the organism to survive a viral infection.
gene is robustly activated
only when IRF-3, IRF-7, and the p300/CBP coactivators are all present.
Furthermore, we find that IRF-7 interacts with four distinct regions of
p300/CBP. These interactions not only stimulate the intrinsic
transcriptional activity of IRF-7, but they are also indispensable for
its ability to strongly synergize with other transcription factors,
including c-Jun and IRF-3.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
genes) (5). A similar situation may exist for IRF-5 (6). The activation
of these IRFs involves the phosphorylation of a stretch of serine (Ser) and threonine (Thr) residues at their C-terminal ends. This
phosphorylation results in a conformational change that allows nuclear
accumulation, DNA-binding, and transcriptional activation of target
genes (5-16).
gene promoter, where it binds with much less
affinity. In this case, two other virus-inducible activator proteins
ATF-2/c-Jun and NF-
B are required, and together with VAF they
assemble into a unique complex at the IFN-
gene promoter
(13).
gene promoter requires both IRF-3 and IRF-7, as well as the p300/CBP coactivators. Multiple interactions, between IRF-3 and IRF-7, and between each transcription factor and
p300/CBP, account for the synergistic activation of the P31 cis-acting
element and the unusual stability of the VAF complex. In addition,
IRF-7 also synergized with c-Jun, and this synergy was facilitated by
recruitment of the p300/CBP coactivators.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
A and pPac vectors, respectively, using standard
methods (17). In these constructs, the coding sequence of human IRF-7B
is preceded by a histidine tag (H6IRF-7) or a sequence
encoding three flag tags, MDYKDHDGDYKDHDIDYKDHDE (F3IRF-7).
Alternatively, the coding sequence of IRF-7 was fused to the Gal4
DNA-binding domain (aa 1-147). Mutants of IRF-7 were generated by PCR
and were all verified by sequencing. Reporter constructs have been
described (13, 18), and contain the CAT gene driven by a
minimal TATA box, that of the adenovirus E1b gene, and
multiple copies of cis-acting elements as indicated.
-galactosidase activity were measured in extracts of transfected
cells (17), and CAT activity was expressed in arbitrary units after
normalization to
-galactosidase activity to control for transfection efficiency.
A effector plasmids, and T7 RNA polymerase. Whole cell extract
preparation, binding, and PAGE conditions for EMSA were as described
(13), except that 0.5 µg of poly(dI-dC) were added for EMSA involving
in vitro translated IRF-7. Immunoblotting, after SDS-PAGE
was performed as described (19), using M2 (anti-FLAG, Sigma) as primary
antibody, and anti-mouse horseradish peroxidase conjugates as secondary antibody. The chemiluminescence detection system was from PerkinElmer Life Sciences.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
i (lacking an internal inhibitory domain) with N-terminal FLAG
tags. These plasmids were cotransfected with a reporter driven by three
copies of the ISRE of the ISG15 gene (i.e. a high
affinity binding site for IRFs), in the presence or absence of
mammalian p300/CBP (Fig. 1B).
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Fig. 1.
The transcriptional activity of IRF-7 mutants
in insect cells. A, alignment of the C-terminal cluster
of Ser/Thr residues that are potentially phosphorylated in
virus-infected cells for human IRF-7B, murine IRF-7, human IRF-3,
murine IRF-3, and chicken IRF-3. B, transcriptional activity
in S2 cells of transfected (2 µg) IRF-7B constructs in the presence
or absence of cotransfected p300/CBP (1.5 µg of a 1:1 mixture) on the
ISREx3CAT reporter (left panel), and expressed proteins were
detected by immunoblotting (anti-FLAG antibody, right panel;
n-sp stands for nonspecific, FL for full-length).
C, transcriptional activity in S2 cells of transfected (1 µg) Gal4-IRF-7B and Gal4-IRF-7 i (left panel) and
Gal4-IRF-7B point mutants (middle panel, note the difference
in scale) in the presence or absence of cotransfected p300/CBP (1 µg
of a 1:1 mixture) on the G5E1bCAT reporter, and expressed proteins were
detected by Western blotting (right panel, using anti-Gal4
antibody). In IRF-7E2, the first set of Ser residues was substituted
with Glu residues; in IRF-7E6, the second set was replaced by Glu
residues; in IRF-7E8, both sets of residues were substituted (see Fig.
2 for exact sequence); in IRF-7
i, aa 267-438 were spliced out of
IRF-7B.
i
significantly stimulated ISREx3CAT activity in the absence of
transfected p300/CBP (Fig. 1B). Coexpression of p300/CBP further stimulated IRF-7
i activity by about 40%. This stimulation was even more significant when the concentration of IRF-7
i was low
(see below).
i was a very strong activator, which was further
stimulated by p300/CBP coexpression (3-4-fold). The fact that
Gal4-IRF-7
i was much more potent than IRF-7
i in S2 cells (compare
Fig. 1, B with C, left panel, and note
the different scales), suggests that IRF-7
i bound DNA relatively weakly.
i activated transcription in insect cells, demonstrating
that IRF-7 can activate transcription independently from other IRFs.
However, IRF-7E6 failed to do so, despite the ability of a very similar
construct, IRF-7(D475-487), to function as a constitutive activator in
293T cells. IRF-7(D475-487) is a mutant form of hIRF-7A, where the Ser
residues modified in IRF-7E6 have been mutated to aspartic acid (Asp)
instead of Glu (21); IRF-7A differs from IRF-7B used in this study by
an extra 29 aa after position 226. To understand the basis for this
unexpected difference, we examined the activity of a variety of IRF-7B
mutants in human cells.
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Fig. 2.
Mapping of residues in IRF-7 required for
virus-dependent transcriptional activity.
Transcriptional activity in SAN cells of His-tagged IRF-7B point
mutants on the P31x2CAT reporter (left graph) or as Gal4
fusions on the G5E1bCAT reporter (right graph); the sequence
of WT and each point mutant is indicated on the left of the
graphs. C, control, uninfected cells; V, Sendai
Virus-infected cells.
i (see Fig.
3C).
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Fig. 3.
Effect of point mutations in the context of
IRF-7 i and mapping of IRF-7 activation
domains. In
iE2, the first set of Ser residues
(Ser443-444, see Fig. 1A) is mutated to Glu; in
iE3 the first three Ser residues of the second set
(Ser447-449) are mutated to Glu;
iE5 combines both
iE2 and
iE3 mutations. A, transcriptional activity in
S2 cells of transfected IRF-7
i point mutants (0.5 µg) in the
presence or absence of cotransfected p300/CBP (1.5 µg of a 1:1
mixture) on the ISREx3CAT reporter (top panel), and
expressed proteins were detected by Western blotting (bottom
panel, using anti-FLAG antibody). B, transcriptional
activity in HEC-1B cells of transfected IRF-7
i point mutants (5 µg) on the P31x2CAT reporter in the presence of absence of Sendai
virus infection as indicated (top panel), and expressed
proteins were detected by Western blotting (bottom panel,
using anti-FLAG antibody). C, transcriptional activity of
IRF-7B full-length and deletion mutants on the P31x2CAT reporter
(left panel) or as Gal4 fusions on the G5E1bCAT reporter
(right panel) in SAN cells; 7C corresponds to the
splice variant IRF-7C. C, control, uninfected cells;
V, Sendai Virus-infected cells.
i in insect cells to
test the effect of Ser residue substitutions in the context of this
construct. Interestingly, all the substitutions tested were more active
than IRF-7
iWT in S2 cells (Fig. 3A). When the IRF-7
i
mutants were tested in mammalian cells, we observed the same pattern of
activity as in insect cells (Fig. 3B for HEC-1B cells, we
obtained very similar data for SAN cells, not shown). Thus, whereas our
data indicate that Glu substitutions in IRF-7 cannot sufficiently mimic
the virus-activated form of IRF-7 in insect cells, these mutations can
still increase the transcriptional activity of IRF-7 in the context of
the internal deletion mutant, underscoring the importance of both sets
of residues in IRF-7 activation.
i, where aa 267-438 were removed, led to a strong activation
of P31x2CAT in uninfected cells that was further increased upon virus
infection (about 2-fold). It should be noted that the level of
activation by IRF-7
i is comparable with that achieved with
virus-activated IRF-7B in this assay, whereas similar internal deletion
constructs described by other investigators are much more active than
WT IRF-7 (21, 22). As observed in insect cells, Gal4-IRF-7
i had very
strong transcriptional activity, about 20 times stronger than that of
IRF-7
i, consistent with relatively weak IRF-7
i DNA binding.
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Fig. 4.
IRF-7 interacts with multiple domains of
p300/CBP. A, primary structure of mCBP: the position of
functional domains is indicated, and regions fused to GST protein and
used in this study are mapped below. B, extracts from
control (C) or virus-infected (V) HEC-1B cells
labeled in vivo with [32P]orthophosphate were
treated with deoxycholate/Nonidet P-40 to dissociate IRF proteins from
p300/CBP, the detergent concentration was decreased by dilution, and
the diluted proteins were incubated with the indicated GST fusions
immobilized on glutathione-Sepharose. Proteins retained on the GST
fusions were eluted and immunoprecipitated with anti-IRF-7 antibodies.
Immunoprecipitated proteins were analyzed by SDS-PAGE and
autoradiography. C, 35S-labeled IRF-7B WT and
i were produced by in vitro transcription/translation
using rabbit reticulocyte lysates and incubated with the indicated GST
fusions of murine CBP and human p300 immobilized on
glutathione-Sepharose for pull-down experiments. Proteins retained on
the GST fusions were analyzed by SDS-PAGE and autoradiography. 25% of
IRF-7 proteins input is shown on the right.
i proteins bound
preferentially to the N-terminal region of CBP (~18 and ~57% of
input, respectively) as compared with the C-terminal region (~0.3 and
~1% of input, respectively), whereas IRF-3 displayed a reversed
preference (16). As was the case for IRF-3, however, binding to the N
and C regions of CBP was further mapped to the N2 and C2 domains, and
interestingly binding to C2 was substantially stronger than binding to
C (~0.9 and ~6% for IRF-7WT and IRF-7
i, respectively).
i also bound to the N (~12 and ~30%
of input, respectively) and C (~3 and ~12% of input, respectively) regions, but binding to the C region was further mapped to two independent domains, C1 and C2. For both p300 and CBP, weak (~2% of
input) but reproducible binding to IRF-7 proteins was also detected
with the middle part of each coactivator, M. Importantly, binding of
IRF-7
i to either the N or C region, but not the M region of p300/CBP
was consistently stronger than that of IRF-7BWT (about 3 times). Thus,
internal deletion of aa 267-438 in IRF-7B favors a conformation that
cannot only bind DNA and activate transcription on its own, but also
interact more strongly with p300 and CBP. Moreover, IRF-7 proteins made
more distinct contacts with the coactivators (3 for CBP and 4 for p300)
than IRF-3 proteins (2 distinct contacts, (16)). Furthermore, the fact
that the IRF-coactivator interactions were detected using protein
produced in vitro (in rabbit reticulocyte lysates, Fig.
4C) and GST fusions produced in bacteria suggests that these
interactions were direct and in particular did not involve IRF-3. It is
noteworthy that the pull-downs using recombinant proteins were more
sensitive than those using native proteins. The more stringent
pull-down immunoprecipitation experiments shown in Fig. 4B
failed to detect the interaction with CBP-N, presumably because this
interaction is affected by the presence of detergent.
i in the presence of GST
alone (Fig. 5A, lanes 4 and 7), and the same held true for IRF-7E6, E8,
iE2,
iE3, and
iE5 (data not shown). By contrast, truncation to
aa 388 led to detectable ISRE binding (Fig. 5A, lane
10), despite a significantly lower expression level in rabbit
reticulocyte lysate (Fig. 5A, right panel). No
supershift was detected in the presence of GST-CBP-C2, despite the
ability of this fusion protein to interact with IRF-7 (Figs.
4C and 5B).
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Fig. 5.
Mapping of the functional domains of
IRF-7. A, proteins were produced by in vitro
transcription/translation using rabbit reticulocyte lysates and
cDNAs encoding WT and the indicated IRF-7B mutants; control protein
(ctrl) is luciferase; translated proteins were analyzed in
the presence of GST, GST-CBP-N, and GST-CBP-C2 by EMSA using the
ISG15 ISRE as a probe (left panel); translated
proteins were detected by Western blotting (right panel).
B, 35S-labeled IRF-7BWT, i, 1-388, 1-308,
and 1-266 were produced by in vitro
transcription/translation and incubated with the indicated GST fusions
immobilized on glutathione-Sepharose for pull-down experiments.
Proteins retained on the GST fusions were analyzed by SDS-PAGE and
autoradiography. 20% of IRF-7B protein input is shown on the
right. C, transcriptional activity in S2 cells of
transfected IRF-7B deletion mutants (2 µg) in the presence or absence
of cotransfected p300/CBP (1.5 µg of a 1:1 mixture) on the ISREx3CAT
reporter. D, schematic of IRF-7 deletions and mapping of
domains involved in dimerization, intrinsic transcriptional activity,
and coactivator interactions.
i, and IRF-7B1-388. The
CBP-N-supershifted IRF-7
i complex showed an intensity similar to
that of the supershifted IRF-7 WT complex, despite its lower expression
level (right panel), indicating that this construct indeed
interacts more strongly with this region of CBP (compare Fig. 5,
A and B). Truncation to aa 388 lead to a small
increase in binding to those regions of p300/CBP as compared with WT,
whereas further truncation to aa 308 or 266 resulted in weaker binding
to these coactivators.
i, which
corresponds to the last 37 aa of IRF-7B spliced immediately after aa
266, bound much more strongly to p300/CBP in this assay than either IRF-7B WT or 1-266, whereas the removal of these last 37 aa (in IRF-7B1-388) had relatively little impact on the ability of IRF-7 to interact with the coactivators (Fig.
5B). As was the case for the EMSA assay, the properties of
IRF-7E6 and -E8, and of IRF-7
iE2, -
iE3, and -
iE5 in the
pull-down assay were indistinguishable from that of IRF-7B WT and
IRF-7
i WT, respectively (data not shown).
gene promoter
(Fig. 6).
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Fig. 6.
Synergistic activation by IRF-7 with IRF-3 or
c-Jun in insect cells. A, transcriptional activity in
S2 cells on the ISREx3CAT reporter of transfected IRF-7 i and IRF-3E7
(0.5 µg each) in the presence or absence of cotransfected p300/CBP
(1.5 µg of a 1:1 mixture). B, transcriptional activity in
S2 cells on the P31x4CAT reporter of transfected IRF-7
i (2 µg) and
IRF-3E7 (1.5 µg) in the presence or absence of cotransfected p300/CBP
(1.5 µg of a 1:1 mixture). C, transcriptional activity on
the P31x4CAT reporter in S2 cells: transfection of increasing amounts
of IRF-3E7, IRF-7
i, and p300/CBP (1:1 mixture), each 0.1, 0.2, 0.4, 0.8, and 1.6 µg of expression plasmids (gray bars);
activation achieved with the smallest amount of expression plasmids
(100 ng each) extrapolated linearly is indicated by the lower
line (triangles); activation achieved with the highest
amount of expression plasmids (1.6 µg each) interpolated linearly is
indicated by the upper line (squares).
D, transcriptional activity in S2 cells on the P431x3CAT
reporter of transfected IRF-7
i (0.5 µg) and c-Jun (0.5 µg) in
the presence or absence of cotransfected CBP (1 µg). E,
transcriptional activity in S2 cells on the P431x3CAT reporter of
transfected IRF-7
i (0.5 µg) and c-Jun (0.5 µg) in the presence
or absence of cotransfected CBP (1 µg) and GST-CBP fusions (1 and 3 µg in the presence of CBP, 1 µg in its absence) as indicated.
F, schematic of the virus-responsive elements in the human
IFN-
gene promoter, located from
99 to
55 relative to the
transcription initiation site: positive regulatory domain
(PRD) IV, from
99 to
91, which binds AP-1 family
members; PRD III and PRD I, from
90 to
65, which bind IRF family
members and together form a virus-inducible enhanson known as P31; PRD
II, from
64 to
55, which binds NF-
B/rel family members.
Reporters used in this study are driven by multimers of the P31 (
90
to
65) and P431 (
99 to
65) sequences.
i or IRF-3E7 expression
plasmids were transfected into S2 cells, IRF-3 proved a stronger
activator than IRF-7 on the ISREx3CAT reporter, consistent with the
higher affinity of IRF-3 for this sequence (13). In the absence of
p300/CBP, cotransfection of IRF-3 and IRF-7 did not lead to any synergy
in activation (Fig. 6A). In the presence of p300/CBP,
however, the levels of activation achieved by the combination of IRF-3
and IRF-7 was much higher than by either activator alone. The extent of
the synergy was even more dramatic for the P31x4CAT reporter: each
constitutive activator, IRF-7
i or IRF-3E7, activated transcription
about 2.5-fold over background, whereas together they activate
transcription about 300-fold above background.
i, p300, and CBP on their ability to synergistically activate transcription from the P31x4CAT reporter (Fig. 6C). The
amounts of transfected plasmids were increased by a factor of two from 100 ng to 1.6 µg for each of IRF-3E7, IRF-7
i, and p300/CBP. The upper and lower curves on the graph were computed from the highest or
lowest levels of activation, respectively, assuming a linear relationship between amount of transfected plasmid and transcriptional activation, i.e. as if there was no cooperative association
of the factors involved in activation of the reporter. Remarkably, there was a dramatic threshold effect, a sharp increase in
transcriptional activity over the last 2-fold increase in amounts of
expression plasmids transfected (Fig. 6C).
gene
promoter, PRDIV, next to P31 (Fig. 6F), can confer virus
inducibility to a reporter as a single copy (18). We tested the ability
of IRF-7 and c-Jun to synergize in activating the P431x3CAT reporter in S2 cells (Fig. 6D). Transfection of c-Jun alone activates a
PRDIVx6CAT reporter, and the transcriptional activity of c-Jun is only
modestly stimulated by cotransfection of CBP in this assay (data not
shown). On the P431x3CAT reporter, the activity of c-Jun or IRF-7
i
was further increased by CBP coexpression. IRF-7
i synergized with c-Jun in the presence or absence of CBP. The mammalian coactivator had
relatively modest effects on either IRF-7 or c-Jun alone, but
stimulated the combination of these transcription factors by about
10-fold.
i/CBP. GST- CBP1-1100 was a
more potent inhibitor than GST-CBP1892-2441, consistent
with the observation that both IRF-7 and c-Jun interact more strongly with CBP N terminus (Fig.
4).2 Either fusion protein
alone, or in combination, had little effect on the level of activation
achieved by IRF-7
i/c-Jun, indicating that an intact CBP is required
for maximal transcriptional synergy.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
i, could
activate transcription in cells lacking any IRF family member (Fig. 1).
The contribution of Drosophila transcription factors to
IRF-7 activity is unlikely as we used reporters containing as
regulatory elements only a minimal TATA box and IRF-binding sites.
i activity in insect cells was further stimulated by coexpression of the
mammalian p300/CBP coactivators, up to 3-4-fold (Figs. 1 and 3). This
is in contrast to IRF-3E7, whose activity in insect cells is entirely
dependent on the expression of the mammalian coactivator (16) (Fig. 6).
These results suggest that IRF-7 had intrinsic transcriptional activity
or that Drosophila CBP was able to partially
coactivate with IRF-7. We favor the former possibility because mapping
experiments showed no direct correlation between interactions with
coactivators and transcriptional activity (Fig. 5D).
i, Glu substitution of residues within both sets increased
transcriptional activity (Fig. 3), further suggesting that residues
within both sets are modified upon virus infection. Whereas our data
identify both sets of residues as functionally important for
virus-dependent activation of IRF-7, further studies will
be required to determine exactly which Ser residues within each set are
phosphorylated upon virus infection.
i and c-Jun have intrinsic transcriptional
activities that were only moderately stimulated by coexpression of the
mammalian coactivators. IRF-7
i or c-Jun had little activity alone,
or in the presence of CBP, but coexpression of IRF-7
i, c-Jun, and
CBP resulted in very strong synergy on the P431x3CAT reporter.
Interfering with the interaction between these transcription factors
and CBP by coexpressing fragments of CBP inhibited this synergy. These results are not due to squelching, i.e. interference with
CBP transcriptional activity, as we previously demonstrated (16). Rather, these results suggest that the ability of IRF-7
i and c-Jun
to interact with CBP is more important to their ability to synergize
with other transcription factors than to activate transcription by
themselves. Taken together, these data suggest that in the activation
of the P431x3CAT reporter, CBP does not only serve as an adaptor
between the general transcription machinery and the activators, but it
can stabilize the formation of an IRF-7·c-Jun·P431 nucleoprotein
complex through simultaneous interactions with both IRF-7 and c-Jun.
The flexible nature of CBP may be crucial for accommodating the
specific arrangement of activator proteins on the IFN-
promoter as well as on other complex gene regulatory elements (25).
CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank T. Collins, R. Goodman, and D. Livingston for kindly providing reagents.
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FOOTNOTES |
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* This work was supported by a Dean Research Award (to M. G. W.) and National Institutes of Health Grant AI20642 (to T. Maniatis, Harvard University) during its initial phase.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: Dept. of Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115.
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Physiology, University of Cincinnati College of Medicine,
231 Albert Sabin Way, Cincinnati, OH 45267-0576. Tel.: 513-558-4515;
Fax: 513-558-5738; E-mail: marc.wathelet@uc.edu.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M212940200
2 H. Yang, C. H. Lin, G. Ma, and M. G. Wathelet, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; CAT, chloramphenicol acetyltransferase; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; ISGF-3, IFN-stimulated gene factor 3; ISRE, IFN stimulated response element; VAF, virus-activated factor; WT, wild type; aa, amino acid(s).
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REFERENCES |
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