Báo cáo khoa học: Mechanism for transcriptional synergy between interferon regulatory factor (IRF)-3 and IRF-7 in activation of the interferon-b gene promoter docx

Mechanism for transcriptional synergy between interferon regulatory
factor (IRF)-3 and IRF-7 in activation of the
interferon-
b gene promoter
Hongmei Yang
1
, Gang Ma
1
, Charles H. Lin
2,
*, Melissa Orr
1
and Marc G. Wathelet
1
1
Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA;
2
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA
The interferon-b promoter has been studied extensively a s a
model system for combinatorial transcriptional regulation.
In virus-infected cells the transcription factors ATF-2, c-Jun,
interferon regulatory factor (IRF)-3, IRF-7 a nd NF-jB, and
the coactivators p300/CBP play critical roles in the activa-
tion of this and other promoters. It remains unclear, how-
ever, why most other combinations of AP-1, IRF and Rel
proteins fail to activate the interferon-b gene. Here we have
explored how different IRFs may cooperate with other fac-
tors to activate transcription. First we showed in undiffer-
entiated embryonic carcinoma cells that ectopic expression
of either IRF-3 or IRF-7, but not IRF-1, was sufficient
to allow virus-dependent activation of the interferon-b

promoter. Moreover, the activity of IRF-3 and I RF-7 was
strongly affected by promoter context, with IRF-7 prefer-
entially being recruited to the natural interferon-b promoter.
We fully reconstituted activation of this promoter in insect
cells. Maximal synergy required IRF-3 and IRF-7 but not
IRF-1, and w as strongly dependent on the presence of p 300/
CBP, even when these coactivators only modestly affected
the activity of each factor by itself. These results suggest that
specificity in activation of the interferon-b gene depends on a
unique promoter context and on the role played by coacti-
vators as architectural factors.
Keywords: coactivator; interferon; IRF; synergy; virus.
Specificity in transcriptional regulation is thought to derive
in part from the combinatorial assembly of unique
complexes of transcription factors a t target p romoters.
Studies of the virus-inducible interferon (IFN)-b gene
promoter support this paradigm but the molecular basis
for its tissue- and stimulus-specific expression remains
incompletely understood (re viewed i n [1]).
Cells from vertebrate organisms respond to viral infection
by activating antiviral enzymes a nd by modulating t he
expression levels of a set of cellular gen es, some of w hich
encode cytokines such as IFNs (reviewed in [2,3]). These
cytokines s ignal t he occurrence of a n infection to other cells,
allowing coordination of the a daptive response a t the
organismal level. The IFN-b gene plays a crucial role in
initiating and s ustaining t his r esponse t hrough i ts early
direct transcriptional a ctivation in infected cells and through
its ability to e nhance the induction by virus o f the family of
IFN-a genes (reviewed in [4]).

The molecular basis for the regulation of IFN-b tran-
scription h as been partially elucidated (Fig. 1A). A compact
virus-inducible enhancer controls this intronless gene
(reviewed in [1]), and flanking scaffold/matrix-attachment
regions (S/MARs) insulate the t ranscription unit from the
influence of o ther regulatory elements (reviewed in [5]).
In vivo, IFN-b is essentially silent in uninfected cells, with
less than one copy of mRNA detected per 100 000 c ells [6].
The uninduced state i s maintained, at least in part, through
the i nhibitory effects of a n NF-jB r egulating f actor ( NRF;
[7,8]), YinYang 1 [9] and nucleosomes. Nucleosomes are
ordered immediately upstream from the gene [10,11] and
their histone tails are hypoacetylated [12]. The treatment
of cells with histone deacetylase inhibitors also leads to
significant transcription from the IFN-b gene promoter in
the absence of virus infection [13,14].
The IFN-b promoter contains binding sites for members
of the AP-1, IRF and Rel families. These ci s-acting e lements
are called positive r egulatory domains (PRDs) and are
located between )99 and )55 relative to the transcription
initiation site . Virus infection r esults in the coordinate
activation o f ATF-2/c-Jun, virus-activated factor (VAF)
and NF-jB [15]. ATF-2/c-Jun binds to PRD IV ()99 to
)91); VAF contains IRF-3/IRF-7 and binds to PRD III-
PRD I (known as P 31, )90 to )64) while the p 50/p65
NF-jB dimer binds to PRD II ()66 to )55) (Fig. 1A). VA F
also contains the coactivators p300 and CREB binding
protein (CBP), which are thought to play a critical role in
activation of the IFN-b promoter because interactions
between p300/CBP and both ATF-2/c-Jun and NF-jB

could compensate for the l ow intrinsic affinity of IRF-3/7
Correspondence to M. G. Wathelet, D epartment of Molecular and
Cellular Physiology, University of Cincinnati College of Medicine,
231 Albert Sabin Way, Cincinnati, O H 45267-0576, USA.
Fax: +1 513 558 5738, Te l.: +1 513 558 4515,
E-mail:
Abbreviations: CAT, c hloramphenicol acetyl transfe rase; CREB,
cAMP response element binding protein; CBP, CREB-binding pro-
tein; DOC, deoxycholate; GST, glutathione S-transferase; IFN,
interferon; IRF, IFN regulatory factor; ISRE, IFN stimulated
response element; NRF, NF-jB r egulatory fa ctor; PRD, positive
regulatory domain; VAF, virus activated factor; WT, wild type.
*Present a ddress: Department of Cellular and Molecular Medicine,
UCSD School of Medicine, La Jolla, CA 92093, USA.
(Received 26 April 2004, revised 20 July 2004, accepted 28 July 2004)
Eur. J. Biochem. 271, 3693–3703 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04310.x
for this promo ter (revie wed in [1]). ATF-2, c-Jun, IRF-3,
IRF-7, p50 and p65 are found associated with the IFN-b
promoter in vivo in virus-infected cells [15]. Their binding
to the IFN-b promoter is accompanied by the localized
acetylation of histone tails in neighboring nucleosomes [12],
remodeling of these nucleosomes, recruitment of the tran-
scriptional machinery and transcriptional activation [11].
Besides virus infection, many stressing or inflammatory
stimuli can coordinately activate members of the AP-1, IRF
and Rel families of transcription factors. However, it
remains unclear why only the set of f actors activated upon
virus infection (or upon lipopolysaccharide treatment in
some cells [16]) is able to turn the IFN-b gene on.
Comparison of the sets of factors activated by different

stimuli suggests that a key determinant in specificity is the
nature of the IRF molecules involved. Specifically, most
stimuli that activate AP-1 and NF-jB also induce IRF-1
(e.g. interleukin-1, tumor necrosis factor [17]) but fail to
activate the IFN-b gene substantially. By contrast, virus
infection [ 15] o r lipopolysaccharide treatment additionally
activate IRF-3 and/or IRF-7, and consequently the IFN-b
gene. M oreover, it is not understood why type I IFN genes
can be activated by virus i nfection in most a dult cells but not
in pluripotent cells, such as embryonic stem cells or
undifferentiated embryonic carcinoma cells [18,19].
Here we explore the mechanism by which different IRFs
functionally interact with AT F-2/c-Jun and NF-jBto
activate IF N-b. Ectopic expression of eith er IRF-3 or IRF-
7, but not IRF-1, was sufficient to allow virus-dependent
activation o f the IFN- b promoter in undifferentiated
embryonic carcinoma cells. These cells, as well as insect
cells,wereusedtodefinetheroleplayedbyeachtranscrip-
tion factor and c oactivator in activation of the IFN-b
promoter. We s how that activation of the IFN-b promoter
was critically dependent on the nature of the IRF involved.
Moreover, we show that synergy between different tran-
A
B
Arbitrary Units
P31x2CAT in P19 cells IFNβCAT in P19 cells
0
2
4
6

8
10
12
14
16
18
values
0.02 0.02 0.41 0.38 0.02 1.52 0.05 0.22 0.22 12.0
Co SV Co SV Co SV Co SV Co SV
pcDßA IRF-1 IRF-3 IRF-7 IRF-3/7
0
2
4
6
8
10
12
14
values
0.06 0.11 0.44 0.42 0.06 0.98 0.37 3.21 0.50 9.81
Co SV Co SV Co SV Co SV Co SV
pcDßA IRF-1 IRF-3 IRF-7 IRF-3/7
Fig. 1. The IFN-b gene locus and pro moter context dependence. (A) Schematic representation of the human IFN-b gene locus, including flanking S/
MARs; the virus-respon sive element (VRE ) and the fact ors binding to it in the uninduced and v irus-induce d states are i ndic ated. (B) Ac tivity o f
P31·2CAT and )110IFNbCAT in P19 cells. P19 cells in 6-well p lates were cotransfected with 1 lg cytomegalovirus (CMV)-lac Z, 2 lgreporter
plasmid P31·2CAT (leftpanel)or)110IFNbCAT (right panel) a nd CMV-driven vectors directing the expression of the indicated transcription
factors (HuIRF-1, 3 lg; H
6
HuIRF-3, 1 lg; F
3

HuIRF-7B, 2 lg). CAT activity and b-galactosidase activity were m easured in extracts of transfected
cells infected with Sendai v irus (SV) or m ock-infected as c ontrol (Co ). CAT activit y was no rmalized to b-galactosid ase activity to con trol for
transfection efficiency and i s expressed i n arbitra ry units, r ather t han fold i nduction , so t hat t he re lative stre ngth of d ifferent rep orters c an b e
compared. Fold indu ction can be comp uted from the values listed u nder the g raph.
3694 H. Yang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
scription factors was strongly dependent on the presence
of p300 or CBP, even when these coactivators had only
a modest effect on the trans criptional activity o f each
transcription factor a lone.
Experimental procedures
Plasmid constructs and sequence analysis
Effector constructs for transient transfections of mamma-
lian and insect cells are in the pcDbA and pPac vectors,
respectively. Reporter constructs consist of one or more
copies of a cis-acting element driving e xpression of the
chloramphenicol acetyl transferase (CAT) gene t hrough the
E1b TATA box, except the IFN-b promoter ( )110 to +20)
construct, which is d riven by its own T ATA box [15,20–23].
Cell culture and transfections
P19 cells were grown at 37 °C, 5% (v/ v) CO
2
, in Dulbecco’s
modified Eagle’s medium containing 10% (v/v) fetal bovine
serum, 50 UÆmL
)1
penicillin and 50 lgÆmL
)1
streptomycin.
S2 cells were grown at 26 °C, in Schneider’s Drosophila
medium containing 12% (v/v) fetal bovine serum,

50 UÆmL
)1
penicillin and 50 lgÆmL
)1
streptomycin.
Transfections using the calcium phosphate c oprecipitat-
ion technique were as described previously [24]. P19 cells
were seeded in 6-well plates (300 000 cells in 3 mL),
transfected the n ext day with 0.3 mL of a precipitate
containing 2 lg reporter, 1 lg pCMV-lacZ and 1–3 lgof
effector plasmid (with pcDbAaddedtoatotalof6lg) for
18 h. Cells were then washed three times with NaCl/P
i
and
further incubated with medium until harvested 2 days after
transfection. Sendai virus was added for the last 1 8 h of
transfection. Sendai v irus was obtained f rom SPAFAS
(North Franklin, CT, USA) and used at 200 hemagglutinin
unitsÆmL
)1
.
S2 cells were seeded in 6-well plates (3 million cells in
3 mL), transfected the next d ay with 0.3 mL of a precipitate
containing 250 ng hsp82lacZ, 500 ng reporter plasmid and
effector plasmid mixes as indicated in the figure legends
(withpPacaddedtoatotalof5.75lg), and harvested
2 days after transfection.
CAT and b-galactosidase activities were measured in
extracts of transfected cells [24], and CAT activity
was expressed in arbitrary units after normalization to

b-galactosidase activity to control for trans fection efficiency.
Variation in transfection efficiency between samples was
normal. Arbitrary units rather than fold activation was used
in most Figures h erein so that the relative strength of
reporters can be compared. Basal activity of a reporter
displayed the most variation from experiment to experi-
ment, presumably because t he effect of small fluctuations is
most visible on low values of C AT activity. As a re sult, the
net fold activation for the IFN-b promoter is different in
different experiments.
Pull-down experiments
The GST–p300/CBP f usions were described [25], a s were the
GST–ATF-2 and GST–c-Jun fusions [22]. GST–IRF-3/7
were generated b y subcloning previously described cDNA
inserts [20,21] a nd verified by sequencing. GST fusions were
expressed in Escherichia coli BL21 and purified as recom-
mended (Pharmacia), and dialyzed against phosphate-
buffered saline/10% (v/v) glycerol.
In vitro translation in rabbit reticulocyte lysates wa s
performed as recommended using the T nT kit (Promega),
appropriately linearized pcDbA effector plasmids and T7
RNA polymerase.
35
S-Labeled in vitro translated proteins were incubated
with GST fusion proteins i mmobilized on glutathione–
sepharose beads in 150 m
M
KCl, 20 m
M
Tris, pH 8.0,

0.5 m
M
dithiothreitol, 50 lgÆmL
)1
ethidium bromide, 0.2%
(v/v) NP-40 and 0.2% (w/v) BSA (binding buffer) for 1 h at
4 °C, followed b y two washes with binding buffer and two
washes with binding buffer without BSA. Proteins bound to
the beads were eluted with SDS loading buffer and analyzed
by SDS/PAGE, visualized by autoradiography and quan-
tified with a phosphoimager.
Results
Transcriptional activity of IRF-3 and IRF-7 is dependent
on promoter context
The activity of a transcription factor depends on the
promoter context, which refers both to the specific arrange-
ment of the cis-acting elements in the promoter and to the
nature of the factors they bind. To investigate the effect of
promoter context on the transcription al activity of IRFs, we
used undifferentiated P19 cells and two reporter plasmids,
P31·2CAT and )110IFNbCAT. P31 is the binding site for
IRFs in the IFN- b gene promoter (Fig. 1A) and P31·2CAT
contains two copies of P31 d riving the expression of the
CAT reporter through the E1b TATA box. This a rtificial
context isolates the contribution of P31 from that of
PRD IV and PRD II. In the )110IFNbCAT reporter, in
contrast, P31 is in its natural context. P19 cells were chosen
for these experiments because in the absence of c otrans-
fected IRF both r eporters had very little i ntrinsic activity
and this activity was not significantly stimulated upon virus

infection (Fig . 1B). Cotransfection of IRF-1, a constitutive
activator, stimulated each r eporter to a similar extent and
had no effect o n their virus-inducibility. Cotransfection of
either IRF-3 or IRF-7 made both reporters virus-inducible,
while cotra nsfection of both IRF-3 and IRF-7 h ad a
synergistic effect, making both reporters strongly virus-
inducible (Fig. 1B). Intriguingly, the effects of IRF-3 and
IRF-7 were d ramatically affected by context. IRF-3 stimu-
lated P31·2CAT activity in P19 cells infected by Sendai
virus  75-fold, as compared to  11-fold for IRF-7 (and
 600-fold for IRF-3 + IRF-7). By contrast, IRF-3 stimu-
lated virus-induced )110IFNbCAT activity only  nine-
fold, while IRF-7 stimulated it  29-fold (and  90-fold for
IRF-3 + IRF-7). Thus, the ability of IRF-3 to stimulate
P31 was about eight times stronger in an isolated context
than within its natural conte xt, while the a bility of IRF-7 to
stimulate P31 was about 2.6 times stronger in its natural
context than in isolation. We conclude that both proteins
are required for maximal a ctivation of the IFN-b gene and
that there are interactions unique to the arrangement of
regulatory elements in the promoter that favor the involv-
ment of IRF-7 in its activation.
Ó FEBS 2004 Synergy in activation of the IFN-b gene (Eur. J. Biochem. 271) 3695
Benefits of using insect cells to reconstitute the
activation of the
IFN-
b gene
Some of the genes encoding factors involved in IFN-b
expression have been inactivated by gene targeting
(reviewed in [26]). However, functional redundancy in

transcription fac tor families and the lack of viability
resulting from gene targeting of either p300 or CBP places
restrictions on the use of mammalian cells to dissect the
activation mechanism of the IFN-b gene. The IFN system
is restricted to vertebrates and insect ce lls do not contain
IRFs orthologs. Moreover, insect cells contain a p300/CBP
ortholog that is sufficiently distinct from the mammalian
proteins that it cannot substitute for them to enable
IRF-3-dependent transcription [21], making insect cells an
ideal system to dissect the roles of individual factors in
activation of the IFN-b gene. Furthermore, mammalian
ATF-2/c-Jun, IRF-1 and NF-jBhavebeenshowntobe
transcriptionally active in the Schneider S2 cell line [23]. In
contrast to IRF-1, both IRF-3 and IRF-7 require virus-
dependent pho sphorylation of specific r esidues i n their
C-termini to display transcriptional activity. These modi-
fications cannot take place in S2 cells, as they lack the
relevant kinase(s), but we have shown t hat mutant forms of
these proteins, IRF-3E7 and IRF-7Di, which are active in
mammalian cells, are also transcriptionally active in S2 cells
[20,21] (Fig. 2).
Transcription factor activities are selectively affected
by coactivators
Interactions between individual transcription factors or
between transcription f actors and coactivators that are
specific to the IFN-b promoter must account at least in p art
for the results obtained in P19 cells.Therefore,weinves-
tigated these interactions both at t he physical a nd functional
levels. First, we tested the ability of mammalian p300 and
CBP, alone or in combination, to affec t the activity of

mammalian transcription factors in S2 cells (Fig. 2). As
described previously, IRF-3E7 did n ot activate transcription
from an IFN stimulated r esponse element (ISRE)-driven
reporter in the absence of murine ( m)CBP in S2 cells [21].
Coexpression of either mCBP or human (h)p300 allowed
IRF-3-dependent transcription (Fig. 2A). By c ontrast, IRF-
7Di displayed intrinsic transcriptional activity [20], which
was f urther stimulated by either hp300 (approximately
twofold) or mCBP (approximately fourfold). Coexpression
of p300/CBP had little e ffect, if any, on I RF-1 transcrip-
tional activity in S2 cells. Thus, mCBP proved twice as
active as hp300 for both I RF-7Di a nd IRF-3E7, consistent
with the observation that both IRFs interact m ore strongly
with mCBP than with hp300 [20,21] (Fig. 3D). Interestingly,
the combination of p300/CBP was more effective than
either coactivator alone in the case of IRF-3E7, while a
similar s ynergy was not o bserved with IRF-7Di.
0
5
10
15
20
25
30
35
40
Vec t o r IRF-3E7 IRF-7∆I F3E7/F7∆I
Vector hp300 mCBP hp300/mCBP
0
5

10
15
20
25
30
35
40
0.0
0.5
1.0
1.5
2.0
2.5
3.0
D
0
10
20
30
40
50
60
70
Vector IRF-1 IRF-7
∆
I IRF-3 E7
Vector hp300 mCBP hp300/mCBP
Arbitrary Units
Arbitrary Units
ISREx3CAT in S2 cells P31x4CAT in S2 cells

PRDIV
x6CAT in S2 cells PRDIIx3CAT in S2 cells
Arbitrary Units
Vector hp300 mCBP hp300/mCBP
-
+
-
+
-
+
-
+
ATF-2/c-Jun NF-κB
+
-
+++
Vector hp300 mCBP
hp300/mCBP
Arbitrary Units
C
AB
Fig. 2. Effects of hp300 and mCBP expression o n the a ctivity of transcription factors in S2 cells. (A) Transcriptional activity of IRF-1, IRF-7Diand
IRF-3E7 (0.5 lg) in the presence or absence of c otransfected hp300, mCBP and hp300/m CBP (1.5 lg) on the ISRE·3CAT reporter. The value for
vector alo ne was 0.24 and the value for IRF-3E7 without c oactivator was 0.16. (B) Transcriptional activity of IR F-3E7 (1.5 lg) a nd IRF-7Di(2 lg),
alone or in c ombination a nd in the presence or ab sence of c otransfected hp300, mCBP and hp300/mCBP (1.5 lg) on the P31·4CAT reporter. The
value for vector alone was 0.22. (C) Transcriptional activity of ATF-2 (0.15 lg) and c-Jun (1.5 lg) in the presence o r a bsence of cotransfected
hp300, mCBP and hp300/mCBP (1.5 lg) on the P RDIV·6CAT reporter. (D) Tran scriptional activity of p50 (0.1 lg) and p65 (0.15 lg) in the
presence or absence of cotransfected hp30 0, mCBP and hp300/mCBP (0.5 lg) on the P RDII·3CAT reporter. The value for vector a lone was 0.06.
3696 H. Yang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
IRF-3 and IRF-7 each bind with much higher a ffinity to

the ISRE o f IFN- a nd virus-inducible genes than to the P31
sequence within the IFN-b promoter [15]. Accordingly,
IRF-3 a nd IRF-7 can individually activate an ISRE-driven
reporter, but significant activation of a P31-driven reporter
requires cooperation between IRF-3 and IRF-7 in S2 cells
[20]. As shown in Fig. 2B, hp300 was relatively ineffective
in promoting synergy between IRF-3 and IRF-7 on the
P31·4CAT reporter as c ompared to mCBP, and the p300/
CBP combination stimulated activity to an intermediary
level.
Expression of ATF-2/c-Jun i n S2 cells re sulted in
increased activity of a PRDIV-driven reporter, and coex-
pression o f p300 and/o r CBP further stimulated it up to
twofold (Fig. 2C). By contrast, expression of the N F-jB
dimer p50/p65 (known as nfkb1/RelA) led t o a strong
activation of a PRD II-driven reporter that, if anything, w as
slightly inhibited by coexpression of the p300/CBP coacti-
vators. Thus all the transcription factors known to bind the
IFN-b gene promoter in virus-infected c ells can be e xpressed
and activate transcription in insect cells, and the p300 and
CBP coactivators have distinct and specific effects on their
transcriptional activity.
Transcription factors interact with multiple domains
of coactivators
We have previously mapped the domains responsible for
interactions between IRF-3 or IRF-7 a nd p300/CBP [20,21]
(summarized in Fig. 3). S imilarly, others have mapped
interactions of hATF-2, c-Jun, p65 or IRF-1 with p300/
CBP. However, not all domains of p300 and CBP were
tested in these experiments and the results were somewhat

conflicting [ 25,27–30]. Theref ore, we conducted a systematic
analysis of the domains within p300 and CBP that interact
with mATF-2
195
(the shorter murine activating form of
ATF-2), c -Jun, p 50, p65 and IRF-1, and the result of these
experiments are summarized in Fig. 3. Binding of mATF-
2
195
to GST-p300/CBP was undetectable in our standard
assay conditions, but lowering the salt concentration from
150 t o 75 m
M
salt allowed detection of relatively weak
(binding £ 4% input) interactions with CBP-N, C BP-C2,
p300-N, p300-M and p300-C2. Binding of c-Jun was
stronger (up to 40% input) but mapped to the same
domains. T hus both ATF-2 and c-Jun c an bind to p300 and
CBP through multiple domains, with a preference of c-Jun
for the N- and C-terminal regions and of ATF-2 for the
central region of the coactivators.
Binding of p50 (amino acids 1–503 of p105) to GST–
p300/CBP was v ery weak o verall. Bindin g to CBP–N
averaged to 1.5% of input and binding to other GST–p300/
CBP fusions did not exceed 0.5% of input. By contrast, p65
bound strongly to the N-, C1- and C2-regions of p300, and
to the N- and C2-regions of CBP. Thus, the bulk of the
interaction between NF-jB and p300/CBP is mediated by
the p65 subunit through the N- and C-terminal regions of
the coactivators.

The pattern of IRF-1 b inding to p300 and CBP domains
closely resembled that observed for IRF-7 [20], with r elatively
strong binding to CBP–N (  21%), –N2 ( 6%) and –C2
( 6%) ( but weak binding to CBP-C,  1%), and to p300–N
( 11%), –C ( 14%), –C1 ( 7%) and –C2 ( 27%).
Synergy between ATF-2/c-Jun and IRF-3/IRF-7
The affinity of ATF-2/c-Jun and of IRF-3/IRF-7 for their
target sites within the IFN-b promoter are significantly
lower than that for optimal binding sites. For example, a
reporter driven by a single P31 is not virus-inducible, while a
reporter driven by a single ISRE, which binds IRF-3/IRF-7
with higher affinity, is v irus-inducible; three copies of P31
are required t o make a reporter strongly virus-inducible [15].
nfkb1 (p50)
c-Jun
ATF-2
195
IRF-7∆i
RelA (p65)
771 1069 1459 2441
267
462 661
1
223120361892
hp300
1855
2370
596 744 1571
1
22102010

IRF-1
mCBP
IRF-3E7
Fig. 3. Mapping of domains of hp 300/mCBP interacting with t ranscription factors. Summ ary o f in teraction stud ies b etwe en do mains o f t he p300 and
CBP coactivators and the transcription factors that can bind to the IFN-b gene promoter (primary data not shown and [20,21]). The fo llowing
domains were used in pull-down experiments (with the amino acid coordi nates indicated in parentheses): CBP–N1(1–267); CBP–N2(267–462);
CBP–N3(462–661); CBP–N(1–771); CBP–M(1069–1459, or 1069–1892 for testing ATF-2); CBP–C1(1892–2036); CBP–C2(2036–2231); CBP–
C3(2231–2441); CBP–C(1892–2441); p300–N(1–596); p300–M(744–1571); p300–C1(1855–2010); p300–C2(2010–2210); p300–C3(2210–2414); and
p300–C(1571–2370). The intensity of binding, e xpressed as percentage of input bound, is indicated by different shades of gray and only interactions
resulting in binding to more th an 1.5% o f input are sh own.
Ó FEBS 2004 Synergy in activation of the IFN-b gene (Eur. J. Biochem. 271) 3697
However, a single copy of the sequence encompassing the
PRD IV and P31 sites, termed P431, confers significant
virus-inducibility t o a reporter gene in mammalian cells [22],
suggesting that ATF-2/c-Jun and IRF-3/IRF-7 cooperate
to synergistically activate this reporter. We explored the
mechanism underlying this synergy by coexpressing these
transcription f actors and t he p300/CBP c oactivators in
S2 cells (Table 1). ATF-2/c-Jun, IRF-3 and IRF-7 each
stimulated the P431·3CAT reporter less than threefold in
the absence of mammalian p300/CBP, and less than 10-fold
in their p resence. However, the combination of transcrip-
tion factors and coactivators resulted in very strong
activation of this reporter (> 1000-fold), indicating that
these proteins bound cooperatively to the P431 element and
synergistically activated transcription. S ynergy was compu-
ted by dividing the fold induction obtained experimentally
for a given combination of proteins by the value obtained
when the fold induction for e ach of the proteins present in
the combination were added (Table 1). There was little

synergy i n the absence of cotransfected p300/CBP, and this
synergy involved only ATF-2/c-Jun and IRF-7, suggesting
these factors physically interact on the P431 site. In the
presence of mammalian p 300/CBP, however, very s trong
synergy was observed when all the transcription factors
were combined (> 200-fold), and removing a single f actor
ledtomuchlowerlevelsofsynergy.
Proteins binding to the
IFN-
b promoter interact weakly
with each other
Three forms of IRF-3 were p roduced by in vitro translation
and tested for their ability to interact with ATF-2, c-Jun,
IRF-3 and IRF-7 immobilized on beads as GST fusion
proteins. W ild type IRF-3 (IRF-3wt) interacted poorly, if a t
all, with the other proteins (Fig. 4). By contrast, IRF-3E7,
which partially mimics virus-activated IRF-3, interacted
weakly with all proteins tested. Virus i nfection leads to a
conformational change and dimerization of IRF-3, but
IRF-3E7 is mostly a monomer [21]. Therefore we also tested
a truncation of IRF-3 that dimerizes more efficiently and we
found that i ndeed IRF-3
1–328
bound much more strongly to
the GST fusion proteins than either IRF-3wt or IRF-3E7.
Similarly, we tested three forms of IRF-7, namely IRF-7wt,
IRF-7DiandIRF-7
1–388
, and found that IRF-7
1–388

bound
more efficiently to ATF-2, c-Jun and IRF-3 than either
IRF-7wt or IRF-7Di.
ATF-2 and c-Jun strongly interacted with each other as
expected for these heterodimerization partners, while bind-
ingtoIRF-3andIRF-7wasmuchweaker(Fig.4).
Similarly, interactions with p50 o r p65 were weak but
detectable with all GST fusions tested. The strength of the
interactions among transcription factors was, with the
exception of that between ATF-2 a nd c-Jun, much weake r
than the ir inte ractions w ith the p300/CBP coa ctivators.
However, even weak interactions could play a determining
role in the context of a given promoter if the arrangement
of cis-acting elements allows them to occur.
Synergistic activation of the
IFN-
b gene promoter
in insect cells
The ability o f the IFN-b promoter to be activated in S2 cells
in response to various combinations of factors was inves-
tigated (Figs 5 and 6). We first tested the effects of ATF-2/
c-Jun, IRF-1, IRF-3/IRF-7 and NF-jB (p50/p65), in the
presence or absence o f m ammalian p 300/CBP, on the
Table 1 . Synergistic activation by ATF-2/c-Jun, IRF-3 and IRF-7.
Transcriptional activity of the in dicated combination of t he tran-
scription factors (0.5 lg each of ATF-2, c-Jun, IRF-3E7 and IRF-7Di)
in the presence or absen ce of co transfected p 300/CB P (1.5 lg) o n the
P431·3CAT reporter. Syn ergy was c omputed b y divid ing t he fold
induction obtained experimentally for a given combination of proteins
by the value obtained when the fold induction for each of the proteins

present in the comb ination we re added.
Vector p300/CBP
Vector ATF-2/c-Jun Vector ATF-2/c-Jun
Fold induction
P431·3CAT
Vector 1.0 2.9 0.9 3.4
IRF-3E7 0.9 2.7 1.0 6.9
IRF-7DI 1.8 28.8 8.2 116.8
IRF-3E7/
IRF-7DI
2.6 34.6 56.3 1131.3
Synergy vs. additive
P431·3CAT
Vector 1.0 1.0 1.0 1.0
IRF-3E7 1.0 1.0 1.3 2.1
IRF-7DI 1.0 7.8 4.7 27.7
IRF-3E7/IRF-7DI 1.0 7.8 22.7 227.4
p65
GST pull-downs
mATF-2
p50
c-Jun
IRF-3WT
IRF-3E7
IRF-3
328
IRF-7WT
IRF-7∆i
IRF-7
388

IRF-1WT
IVT proteins
GST
INPUT
IRF-7WT
ATF-2
c-Jun
IRF-3WT
IRF-3E7
IRF-7∆i
GST fusions
Fig. 4. Physica l inte ractio ns a mong transcription factors.
35
S-labeled
IRF-3WT, IRF-3E7, IRF-3(1–328), IRF-7WT, IRF-7Di, IRF-7(1–
388), mATF-2(195), c-Jun, nfkb1(p50) and RelA (p6 5) were incubated
with the indicated GST fusions of ATF-2, c-Jun, IRF-3 and IRF-7
immobilized on glut athione sepharose. Proteins retained on the GST
fusions an d 20% of the protein input were analyzed by SDS/PAGE
and autoradiography; a representative experiment is shown.
3698 H. Yang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
transcription o f t he )110IFNbCAT r eporter ( Fig. 5A).
Each transcription factor pair or IRF-1 could activate this
reporter on their own. To investigate synergy, their amount
was t itrated so that they would each minimally activate the
reporter (< 1 .5-fold f or all apart from IRF-1, which w as
 2.7-fold). Pairwise combinations of ATF-2/c-Jun, IRF-1
or NF-jB did not stimulate transcription more than the
sum of their individual e ffects, whether p300/CBP were
present or n ot. By c ontrast, IRF-3/IRF-7 with ATF-2/c-Jun

or with NF-jB showed syn ergy that w as entirely dependent
on the presence of mammalian c oactivators ( 5.3-fold and
 3.3-fold, respectively). The ATF-2/c-Jun, IRF-1 and NF-
jB combination displayed very little synergy (£ 1.4-fold),
which was not augmented in the presence of p300/CBP. In
marked contrast, the ATF-2/c-Jun, IRF-3/IRF-7 and NF-
jB combination strongly synergized ( 27.6-fold) but only
in the presence o f p300/CBP. Thus, maximal activation was
dependent on the s imultaneous presence of the mammalian
coactivators and on the set of factors activated upon virus
infection.
Threshold effect in synergistic activation
We next investigated the mechanism of this synergy.
Synergy is the functional equ ivalent of physical cooperativ-
A
B
0
30
60
90
120
150
All - IRFs All + IRF3/7 All + IRF1
IFNβCAT in S2 cells
Fold Activation
0
10
20
30
40

50
60
Vector
0.70 1.05 1.87 0.81 0.77 2.26 1.54 1.09 2.12 1.17 3.21 2.05
p
300/CBP
0.91 1.67 1.67 1.61 1.05 2.59 7.29 1.78 1.81 7.08 3.27 39.8
Arbitrary Units
IFNβCAT in S2 cells
ATF-2/c-Jun
IRF-1
IRF-3/IRF-7
NF-κB
- + - - - + + + - - + +
- - + - - + - - + - + -
- - - + - - + - - + - +
- - - - + - - + + + + +
Fig. 5. IRF-3/IRF-7 but not IRF-1 synergize with ATF-2/c-Jun, p50/
p65 and p300/CBP i n activation of the IFN-b promoter. (A) Activity o f
the indicated combinations of transcription factors pairs (0.5 lgfor
ATF-2, c-Jun, IRF-3E7 an d IRF-7Di; 1 lg for IRF-1; 12 ng for p50
and 18 ng for p65) in the presence or absence o f cotransfected p300/
CBP ( 1.5 lgofa1:1mix)onthe)110IFNbCAT reporter. (B )
Threshold effect in activation of the )110IFNbCAT reporter by ATF-
2/c-Jun, p50/p65 and p300/CBP (All – IRFs, circles), in the presence of
IRF-1 (All + IRF-1, triangles) or IRF -3E 7/IR F-7Di(All+IRF3/7,
squares); 8X corresponds to the amo unt of transcriptio n factors used
in (A), with or without 1 lg of IRF-1 or of an IRF3/7 m ix and 0.75 lg
of CBP; 4X, 2X and 1X correspond to d ecre ase of the amount used in
8X by factors o f 2, 4 a nd 8, r espective ly.

0
10
20
30
40
50
60
TFs
++
-
+++++
A
Arbitrary Units
IFNβCAT in S2 cells
p300 CBP p300/CBP
0
10
20
30
40
50
60
70
Vector
All
- ATF-
2
-
c-Jun
- AJ

- IRF-3
- I
RF-7
- F3/
7
- p50

-

p65
- NFkB
- CBP
- p300
- p/C
B
IFNβCAT in S2 cells
Arbitrary Units
C
Arbitrary Units
0
10
20
30
40
50
Ve cto r +IRF7 0.1 ug +IRF7 0.25 ug +IRF7 0.5 ug
Vector All - IRF3 All + IRF3 0.25 ug All + IRF3 0.5 ug
IFNβCAT in S2 cells
Fig. 6. Mechanism of synergistic activation of the IFN-b pr omoter in S2
cells. (A) Activation of t he )110IFNbCAT reporter by the transcrip-

tion factors (T Fs; 500 ng each of ATF -2, c -Jun, IRF-3E7 and IRF-
7Di, 1 00 ng p50 and 150 ng p65) in the presence or a bsence of 0.75 or
1.5 lg of the p300, CBP or p 300/CBP coactivators. The value in the
absence of TFs was 0.09. (B) Activation of the )110IFNbCAT
reporter by cotransfection with all t he transcription factors [All; ATF-
2/c-Jun (1 lg), IRF-3E7 /IRF-7Di(1lg), p50 (100 ng), p65 (150 ng)
and p300/CBP (1.5 lg)] or All minus the indicated factors. The value
with vector alone was 0.16. (C) Activation of the )110IFNbCAT
reporter by the factors ATF-2/c-Jun (1 lg), p50 (100 ng), p65 ( 150 ng)
and p300/CBP (1.5 lg), in the p resence or absence of 0.25 o r 0.5 lgof
IRF-3 E7 and in the presence or a bsence of 0.1, 0.25 or 0.5 lgofIRF-
7Di, as indicated. The value with vector alone was 0.17.
Ó FEBS 2004 Synergy in activation of the IFN-b gene (Eur. J. Biochem. 271) 3699
ity in the assembly of the components required for the
function. Cooperative assembly of transcription f actors is
expected to show a strong d ependence on small changes
in their concentrations near the threshold at which the
complex can form. The amount of transfected plasmids w as
serially increased by a factor of two over an eightfold range,
and the experiment was performed in the presence o r
absence of IRF proteins (Fig. 5B). Transfection o f i ncreas-
ing amounts of ATF-2/c-Jun, NF-jBandCBPledtoa
linear increase in r eporter activity (lower curve, c ircles).
Remarkably, adding IRF-3/IRF-7 to t his mix resulted in an
exponential increase in reporter activity (upper curve,
squares). At the two lowest amounts of t ransfected
plasmids, the addition of IRF-3/IRF-7 had little effect on
transcription. Past that threshold, however, there was a
sharp increase i n transcriptional activity over the last two
twofold i ncreases in amounts of expression plasmids

transfected, resulting in an approximately 1 15-fold increase
in reporter a ctivity over an eightfold increase in the amounts
of transfected plasmids. By contrast, when IRF-1 i nstead of
IRF-3/IRF-7 was used (middle c urve, triangles), reporter
activity rose  5.6-fold over an eightfold i ncrease in
amounts of transfected plasmids, and  2.9-fold over the
last twofold increase. Thus, these results indicate that
transfection of S2 cells reproduced the essential features of
the specific transcriptional activation of the IFN- b promoter
in response to distinct stimuli.
Contribution of each individual factor to synergy
Either p300 or CBP was able to promote the synergistic
activation of the IFNbCAT reporter in S2 cells and
displayed dose-dependent effects (Fig. 6A). CBP proved
more efficien t than p300, and the combination of p300/CBP
displayed an intermediary efficiency, as was the case for
IRF-3/IRF-7 on t he P31 ·4CAT reporter ( Fig. 2B). In
Fig. 6B, we tested the effect of removing individual factors.
Removal of either ATF-2 or p50 led t o an increase in
activity from the IFNbCAT reporter, suggesting c-Jun and
p65 homodimers are stronger activators than the A TF-2/
c-Jun and p 50/p65 heterodimers in this context. By contrast,
removal of either IRF-3 or IRF-7 l ed to a decrease in
activity from the IFNbCAT reporter, and the decrease was
more significant when IRF-7 was absent.
Both IRF-3 and IRF-7 are required for full activation
of the
IFN-
b promoter
Whether the two virus-activable IRFs are both required for

transcription from the IFN-b promoter is unclear. We
examined the dose–response to both IRF-3 and IRF-7 in
the context of the IFN- b promoter (Fig. 6C). Transfection
of S2 cells with ATF-2/c-Jun, NF-jB and p300/CBP led to a
level o f r eporter activation that was only modestly stimu-
lated by the addition of IRF-3. However, in the presence of
even small amounts o f IRF-7, addition of IRF-3 resulted in
a s trong stimulation of the reporter activity. IRF-7, together
with ATF-2/c-Jun, NF-jB and p300/CBP, could lead to
substantial activation of the reporter even in the complete
absence of IRF-3. Nevertheless, maximal activation of the
IFN-b promoter depended on the p resence of both IRF-3
and IRF-7 (Fig. 6B,C), as observed in mammalian cells
(Fig. 1B; [15]).
We have previously shown that r eporters driven by
multiple copies of either PRD III or PRD I fail to respond
to virus infection. Two or more copies of P31 (i.e. PRD III-
PRD I as a single unit), however, confer virus-inducibility,
suggesting that interactions between factors bound to
PRD III and PRD I are required to activate transcription
in virus-infected cells at physiological levels of IRFs. As
shown in Fig. 7B, overexpression of IRF-3 but not IRF-7
led to virus-dependent activation of the PRDIIIx10CAT
reporter. By contrast, PRDI·7CAT was more strongly
activated by IRF-7 than by IRF-3 in virus-infected cells.
Importantly, i t was the c ombination of IRF-3 and IRF-7
that proved the most potent for both rep orters. Taken
together, our data strongly suggest that maximal activation
of the IF N-b promoter requires the cooperative assembly of
a nucleoprotein complex containing p300/CBP, ATF-2/

c-Jun, NF-jB and both IRF-3 and IRF-7.
Discussion
The current paradigm for specificity in transcriptional
activation holds that the physiological concentration o f
transcription factors typically is such that a s ingle f actor
does not activate transcription on its o wn. The need for
several factors to cooperate allows for a combinatorial
principle to operate, which could account for specificity.
B
A
PRDIII, PRDI and IRF binding sites
0
10
20
30
40
Co
0.06 0.06 0.02 0.06 0.06 0.09 0.78 1.16
SV
0.04 5.79 0.03 13.1 0.05 6.17 20.0 32.7
Vector IRF-3 IRF-7 IRF-3/7 Vector IRF-3 IRF-7 IRF-3/7
PRDIIIx10CAT PRDIx7CAT
Arbi
trary Units
PRDIIIx10- & PRDIx7-CAT in P19 cells
Fig. 7 . IRF-3/IRF-7 maximally activates both PRD III and PRD I. (A) Sequence of PRD III, PRD I and optimal binding sites for IRF-1, IRF-3
and IRF-7 [47,48]. ( B) Effect of IRF-3 (1 lg), IRF-7 (2 lg) or IRF-3 and IRF-7 o n t he ac tivity of PRDIII·10CAT or PRDI·7CAT in P19 cells
uninfected (Co) or infected with Sendai virus (SV).
3700 H. Yang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Model for synergistic activation of the

IFN-
b promoter
The IFN-b promoter is approximately six times more potent
when PRD IV is converted to a higher affinity site [22].
Likewise, IRF-3 and IRF-7 bind to P31 with much less
affinity than to the ISRE present in some IFN-inducible
genes [15] and the IFN- b promoter is  28 times m ore
potent when P31 is converted t o such an ISRE. By contrast,
PRD II binds NF-jB with high affinity [31,32]. Thus, in
order to a ctivate the IFN- b promoter, a physiological
stimulus must not only activate t ranscription factors of the
AP-1, IRF and Rel families, but also a specific combination
that can bind the promoter cooperatively to overcome the
low intrinsic affinities of PRD IV and P31 for their cognate
factors.
IRF-1-dependent activation of the
IFN-
b promoter
ATF-2/c-Jun, IRF-1 and NF-jB could each activate the
IFN-b promoter in insect cells. However, the combination
of these f actors a ctivated the IFN-b promoter to a level that
did not exceed the sum of individual contributions, under
conditions where each factor is limiting (Fig. 5). This result
is consistent with the observations that (a) stimuli that
activate this combination of transc ription factors in mam-
malian cells do not activate the IFN-b gene, and that (b)
these factors bind the IFN-b promoter anticooperatively
in vitro, due to steric hindrance between IRF-1 and NF-jB.
In the latter experiments, the high mobility group (HMG)-I/
Y protein is able to neutralize this anticooperativity but

binding re mains noncooperative [23]. We t ested t he effect of
expressing HMG-I in S2 cells on activation of the IFN-b
promoter by ATF-2/c-Jun, IRFs, NF-jB and coactivators.
We found no statistically significant effect, one way or the
other, over a wide range of HMG-I concentrations, w hether
IRF-1 or IRF-3/7 were used (H.Yang and M. G. Wathelet,
unpublished data). H owever, we note t hat D1, a Dro sophila
ortholog of HMG-I [33], i s present in S2 cells in large
amounts [E. Kas ( CNRS, UMR5099, Toulou se, France),
personal communication] and thus could mask any effect
of transfected HMG-I.
IRF-3/IRF-7-dependent activation of the
IFN-
b promoter
Unlike IRF-1, IRF-3 E7/IRF-7Di strongly synergized with
ATF-2/c-Jun, NF-jB and p300/CBP to a ctivate the IFN-b
promoter (Fig. 5A). Presumably, the difference between the
level of a ctivation achieved w ith the IRF-3/7-containing set
of factors vs. that ach ieved with the IRF-1-containing set
would be much greater if the virus-activated IRF-3/7
proteins could be used instead of the mutant forms, not only
because of the difference in affinity for DNA, but also for
the coactivators. Nevertheless, the observation of a strong
thresholdeffect,evenwiththeIRF-3E7/IRF-7Di-containing
set (Fig. 5B), further suggests t hat the bindin g of the set of
virus-activated transcription factors to the IFN-b promoter
is highly cooperative.
Some synergy was evident in the absence of either ATF-2/
c-Jun or NF-jB. This is consistent with the observation that
it is possible to bypass the requirement for both factors

provided that the concentration of IRF-3 is above physio-
logical levels [34]. Interestingly, no synergy was observed in
the absence of either p300/CBP o r IRF-3/IRF-7, suggesting
that VAF s erves as a keystone in the assembly of a functional
activator/coactivator complex at the IFN- b promoter. VAF
formation depends on multiple protein–protein interactions:
(a) virus-dependent homodimerization of IRF-3 [35] and o f
IRF-7 [36]; (b) constitutive interactions between IRF-3 and
IRF-7 [15,37,38]; and (c) virus-dependent modifications of
these factors that result in their association with several
domains of the coactivators p300 and CBP [20,21].
The role of promoter context in ensuring specificity
We show that IRFs could interact with ATF-2, c-Jun, p50
and p65 (Fig. 4). These interactions were rather weak but
could be important in the context of t he IFN-b promoter if
the arrangement of the PRDs allows them to occur. The
importance of these interactions was tested f unctionally and
our results indicate that the balance of positive and negative
interactions between IRF-1 and ATF-2/c-Jun or NF-jB
prevented cooperative binding in the context of the IFN-b
promoter. By contrast, such a balance favored cooperative
binding when IRF-3/IRF-7 was used inst ead of I RF-1
(Fig. 5). IRF-7 but not IRF-3 drives synergy with ATF-2/
c-Jun when binding to P431 (Table 1), which suggests that
IRF)7 has unique interactions with ATF-2/c–Jun Such
interactions might acco unt, at l east in part, for the
observation that IRF-7 is a stronger activator when binding
to P31 in its natural context than in isolation (while the
reverse was true of IRF-3, Figs 1B and 6).
The role of coactivators in promoting synergy and

specificity
Synergistic activation of the IFN-b promoter was entirely
dependent on thepresence of m ammalian coactivator (Figs 5
and 6 ), consistent with the inhibitory effect of E1a on
induction of the IFN-b gene in response to dsRNA [39].
Importantly, ATF-2/c-Ju n, IRF-7 DiandNF-jB had intrin-
sic transcriptional activities that were only moderately
stimulated by coexpression of the mammalian coactivators
(Fig. 2). Nevertheless, this c ombination of f actors had little
activity in the absence of coactivators but strongly synergized
in their presence (Figs 6B.C). Thus, the ability of c -Jun, IRF-
7Di and RelA to interact with coactiva tors was more
important to their ability to synergize with other transcrip-
tion factors than to activate transcription by th emselves.
Taken t ogether, these data suggest that in the a ctivation of
the IF N- b promoter, coactivators not only s erve as an
adaptor between the general t ranscription m achinery and the
activators, but also act as a scaffold by stabilizing the
formation of a nucleoprotein complex through simultaneous
interactions with transcription f actors. The flexible nature of
p300 and CBP may b e crucial for accommodating the specific
arrangement o f activator proteins on the IFN-b promoter as
well as on other c omplex gene regulatory elements [40]. Such
a scaffolding role for these coactivators has been hypothes-
ized [1,41], but not demonstrated. Our data lend strong
support to this important paradigm.
If p300 or CBP bind simultaneously to two or more
transcription factors, it must do so through different
domains. It is t herefore somewhat puzzling that all the
factors tested interacted most strongly with the N2 and C2

Ó FEBS 2004 Synergy in activation of the IFN-b gene (Eur. J. Biochem. 271) 3701
fragments. Moreover, we have shown that in the case of
IRF-3, all of its interactions with the coactivators were
indispensable for transcriptional activity [21]. Similarly, we
have shown that the ability of IRF-7 to synergize with
either c-Jun or IRF-3 was depe ndent of its contacts with
the coactivators [ 20]. These observations could be reconciled
considering that t hese domains in p300 and CBP are
relatively large a nd it is thus possible for more than one
factor to bind at once. Furthermore, more than one
molecule of coactivator is likely r ecruited to t he IFN-b
promoter in virus-infected cells, because p300 and CBP can
interact with each other (H. Yang, C. H. Lin & M. G.
Wathelet, unpublished d ata) and VAF con tains at least two
molecules of p300/CBP [15]. Thus, we favor a model
(Fig. 1A) where at least two molecules of coactivator
contribute to the cooperative assembly of a nucleoprotein
complex at t he IFN-b promoter.
Tissue-specificity in activation of the
IFN-
b promoter
It has been documented that the IFN-b promoter is not
induced by virus in e mbryonic stem cells or undifferentiated
embryonic carcinoma cells [18,19]. The data presented in
Fig. 1B suggest that in P19 cells (a pluripotential teratocar-
cinoma line) the failure to activate the IFN-b promoter was
not due to the absence of the pathway leading to activation of
IRF-3 and IRF-7. The e ndogenous levels of IRF-3 and IRF-
7 in P19 cells were apparently to o l ow to support induction of
the transiently transfected )110IFNbCAT reporter by virus,

but ectopic expression of either factor was sufficient to confer
virus-inducibility to this reporter. Additional experiments
will be required to determine if this observation holds true
for the endogenous IFN-b gene. In early passage primary
embryonic fibroblasts, by contrast, IRF-3 is expressed
at normal levels while IRF-7 is expressed at low levels.
Elimination of IRF-3 by gene targeting does not block IFN-b
mRNA induction but results in lower levels, indicating that
these low IRF-7 levels are biologically significant. Inactiva-
tion of the IRF-9 gene in these IRF-3 null cells results in
undetectable levels of IRF-7 mRNA and a complete block in
IFN-b induction [42]. However, i n later passage embryonic
fibroblasts or in spleen cells of IRF-3 null mice, which express
higher levels of IRF-7, induction of the IFN-b mRNA is
similar to wild type [43]. T hese results are congruent with our
observations in P19 a nd S2 cells (Figs 1B and 6) that indicate
that (a) either IRF-3 or IRF-7 is sufficient to activate the
IFN-b promoter; (b) maximal activation is achieved in the
presence of both IRF-3 and IRF-7; and (c) IRF-7 preference
for t he context of the IFN-b promoter favors its recruitment
to the promoter even when e xpressed at low l evels. Because
viruses can interfere with antiviral defenses [44], i ncluding
production of IFN and targeting of IRF-3 [45] or IRF-7 [ 46],
the existence of some redundancy in th e function of IRF-3
and IRF-7 might help m inimize the influence of this later
class of virulence factors.
Acknowledgements
We would like to thank T. Collins, R. Goodm an and D. Livingston for
kindly providing r eagents and N. Horseman for critical reading of the
manuscript. This w ork was supported by a Dean Research A ward to

M.G.W.
References
1. Maniatis, T., Falvo, J.V., Kim, T.H., Kim, T.K., Lin, C.H.,
Parekh, B.S. & Wathelet, M.G. (1998) Structure and Function of
the Interferon-b Enhanceosome. Cold Sprin g H arbor Laboratory
Press, Cold Spring Harbor, NY.
2. Sen, G.C. (2001) Viruses and interferons. Annu. Rev. Microbiol.
55, 255–281.
3. Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H. &
Schreiber, R.D. (1998) How cells respond to interferons. Annu.
Rev. Biochem. 67, 227–264.
4. Levy, D.E., Marie, I., Smith, E. & Prakash, A. (2002) Enhance-
ment and diversification o f IFN induc tion by IRF-7-mediated
positive feedback. J. Interferon Cytokine Res. 22, 87–93.
5. Bode, J., Benham, C., Knopp, A. & Mielke, C. (2000) Tran-
scriptional augmen tation: m odulation of gene expression by
scaffold/matrix-attached regions (S/MAR Elements). Crit. Rev.
Eukaryot. Gene Expr. 10, 73–90.
6. Tovey, M.G., Streuli, M., Gresser, I., Gugenheim, J., Blanchard,
B., Guymarho, J., Vignaux, F. & Gigou, M. (1987) Interferon
messenger RNA is pr oduced constitutively in the organs o f normal
individuals. Proc.NatlAcad.Sci.USA84, 5038–5042.
7. Nourbakhsh, M . & Hauser, H . (1997) The transcriptional silencer
protein NRF: a repressor of NF-jB enhancers. Immunobiology
198, 65–72.
8. Nourbakhsh, M. & Hauser, H. (1999) Constitutive silencing of
IFN-b promoter is mediated by NRF (NF-jB-repressing fac tor),
a nuclear inhibitor of NF-jB. EMBO J. 18, 6415–6425.
9. Weill, L., Shestakova, E. & B onnefoy, E. (2003) Transcription
factor YY1 binds to the murine beta interfe ron promoter and

regulates its transcriptional capacity with a dual activator/repres-
sor role. J. Virol. 77, 2903–2914.
10. Bode, J., Pucher, H.J. & Maass, K. (1986) Chromatin structure
and i nduction-d ependen t c onfo rmational changes of human
interferon-beta genes in a mouse host cell. Eur. J. Biochem. 158,
393–401.
11. Lomvardas, S. & Thanos, D. (2001) N ucleosome sliding via TBP
DNA binding in vivo. Cell 106, 685–696.
12. Parekh, B.S. & Maniatis, T. ( 1999) Virus infection leads to loca-
lized hype racetylation of histones H3 and H4 at the IFN-b pro-
moter. Mol. C e ll . 3, 125–129.
13. Bode, J., Hochkeppel, H.K. & Maass, K. (1982) Links between
effects of butyrate on histon e hyperacetylation and regulation of
interferon synthesis i n Namalva and FS-4 cell lines. J. Interferon
Res. 2, 159–166.
14. Shestakova, E., Bandu, M.T., Doly, J. & Bonnefoy, E. (2001)
Inhibition of histone deacetylation in duces constitutive derepres-
sion of the b eta interferon p romoter and confers a ntiviral activity.
J. Virol. 75, 3444–3452.
15. Wathelet, M .G., Lin, C.H., Parekh, B.S., Ronco, L.V., Howley,
P.M. & Maniatis, T. (1998) Virus infection induces the assembly
of coordinately activated transcription factors on the IFN-b
enhancer in vivo. Mol. Cell. 1, 507–518.
16. Navarro, L. & David, M. (1999) p38-dependent activation of
interferon regulatory factor 3 b y lipopolysaccharide. J. Biol.
Chem. 274, 35535–35538.
17. Fujita, T., Reis, L.F., Watanabe, N., Kimura, Y., Taniguchi,
T. & Vilcek, J . (1989b) Induction of the transcription factor
IRF-1 and interferon-beta mRNAs by cytokines and activators
of se cond- messenger pathways. Proc. Natl Acad. Sci. USA 86,

9936–9940.
18. Burke, D.C., Graham, C.F. & Lehman, J.M. (1978) Appearance
of interferon inducibility and sensitivity during differentiation of
murine teratocarcinoma cells in vitro. Cell 13, 243–248.
19. Harada, H., Willison, K., Sakakibara, J., Miyamoto, M., Fujita,
T. & Taniguchi, T. (1990) Absence of the type I IFN system in EC
3702 H. Yang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
cells: transcriptional activator (IRF-1) and repressor (IRF-2)
genes are developmentally regulated. Cell 63, 303–312.
20.Yang,H.,Lin,C.H.,Ma,G.,Baffi,M.O.&Wathelet,M.G.
(2003) Interferon regulatory factor-7 synergizes with other tran-
scription factors through multiple interactions with p300/CBP
coactivators . J. Biol. Chem. 278, 15495–15504.
21. Yang, H ., Lin, C.H., Ma, G ., Orr, M., Baffi, M.O. & Wathelet,
M.G. (2002) Transcriptional activity of i nterferon regulatory
factor (IRF )-3 depends on mu ltiple protein–protein interactions.
Eur J. Biochem. 269, 6142–6151.
22. Falvo, J.V., Parekh, B.S., Lin, C.H., Fraenkel, E. & Maniatis, T.
(2000) Assembly o f a function al beta interfe ron enh ance osome is
dependent o n AT F-2-c-ju n he terodime r or ientation. Mol. Cell.
Biol. 20, 4814–4825.
23. Thanos, D. & Maniatis, T . (1995) Virus induction of human IFN-
b gene expression re quires the assembly of an enhanceosome. Cell
83, 1091–1100.
24. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual. Cold Sp ring Harbor Laboratory
Press, Cold Spring Harbor, New York.
25. Gerritsen, M.E., Williams, A.J., Neish, A.S., Moore, S., Shi, Y. &
Collins, T. (1997) CREB-binding protein/p300 are transcriptional
coactivators o f p 65. Proc. Natl Acad. Sci. USA 94, 2927–2932.

26. Sato, M., Taniguchi, T. & Tanaka, N. (2001) The interferon sys-
tem and interfero n regulatory factor transcription factors – studies
from gene knockout mice . Cytokine Growth Factor Rev. 12,133–
142.
27. Bannister, A.J., Oehler, T., Wilhelm, D., Angel, P. & K ouzarides,
T. (1995) Stimulation of c-Jun a ctivity by C BP: c-Jun residues
Ser63/73 are required for CBP i nduced stimulation in vivo and
CBP binding in vitro. Oncogene 11, 2509–2514.
28. Kawasaki, H ., Song, J., Eckner, R., Ugai, H ., Chiu, R., Taira, K.,
Shi, Y., Jones, N. & Yokoyama, K.K. (1998) p300 a nd ATF-2 are
components of the DRF comple x, which regulates retinoic acid-
and E 1A-mediated transcription of the c-Jun gene in F9 cells.
Genes Dev. 12, 233–245.
29. Lee,J.S.,See,R.H.,Deng,T.&Shi,Y.(1996)AdenovirusE1A
downregulates cJun- and JunB-mediated transcription by
targeting their coactivator p300. Mol. Cell. B iol. 16, 431 2–
4326.
30. Merika, M., Williams, A.J., Chen, G., Collins, T. & Thanos, D .
(1998) Recruitment o f C BP/p 300 b y t he IF N b eta enhanceosome
is required for s ynergistic ac tivation of transcript ion. Mol. Cell 1,
277–287.
31.Fujita,T.,Nolan,G.P.,Ghosh,S.&Baltimore,D.(1992)
Independent modes of transcrip tional activation by the p50 and
p65 subunits of NF-kappa B . Genes Dev. 6, 775–787.
32. Thanos,D.&Maniatis,T.(1992)Thehighmobilitygroupprotein
HMGI(Y) is required for NF-jB- depend ent virus induction of
the human IFN-b gene. Cell 71 , 777–789.
33. Aulner, N., Monod, C., Man dicourt, G., Jullien, D ., Cuvier, O .,
Sall,A.,Janssen,S.,Laemmli,U.K.&Kas,E.(2002)TheAT-
hook protein D1 is essential for Drosophila melanogaster devel-

opment and i s implicated in position -effect variegation. Mol. Cell.
Biol. 22, 1218–1232.
34. Peters, K.L., Smith, H.L., Stark, G .R. & Sen, G.C. (2002) IRF-3-
dependent, NFjB- and JNK-independent activation of the 561
and IFN-beta genes in response to double-stranded RNA. Proc.
NatlAcad.Sci.USA99, 6322–6327.
35. Lin, R., Mamane, Y. & Hiscott, J. (1999) Structural and func-
tional analysis of in terfero n regulatory factor 3: localization o f the
transactivation and autoinhibitory domains. Mol. Cell. Biol. 19,
2465–2474.
36. Marie, I., Smith, E., Prakash, A. & Levy, D.E. (2000) Phos-
phorylation-induced dimerizationofinterferonregulatoryfactor7
unmasks DNA binding and a bipartite transactivation domain.
Mol. C ell . B i ol . 20, 8803–8814.
37. Lin, R., Mamane, Y. & Hiscott, J. (2000) Multiple regulatory
domains control IRF-7 activity in response to virus infection.
J. Biol. Chem. 275, 34320–34327.
38. Au, W.C., Yeow, W.S. & Pitha, P.M. (2001) Analysis of func-
tional domain s of inte rf eron regulatory fac tor 7 a n d its a ssociation
with IRF-3. Virology 280, 273–282.
39. Ackrill, A.M., Foster, G.R., Laxton, C.D., Flavell, D.M., Stark,
G.R. & Kerr, I.M. (1991) Inhibition of the cellular response to
interferons by pro ducts of the adenovirus type 5 E1A oncogene.
Nucleic Acids Res. 19, 4387–4393.
40. Lin, C.H., H are, B.J., Wagner, G., Harrison, S.C., Maniatis, T. &
Fraenkel, E. (2001) A small domain of CBP/p300 binds diverse
proteins: solution structure and functional studies. Mol. Cell 8,
581–590.
41. Shikama, N., Lyon, J. & La Thangue, N.B. (1997) The p300/CBP
family: integrating signals w ith trans cription factors an d chro -

matin. Trends Cell Biol. 7, 230–236.
42. Sato, M., Suemori, H., Hata, N ., Asagiri, M., Ogasawara, K.,
Nakao, K., Nakaya, T., Katsuki, M., Noguchi, S., Tanaka, N. &
Taniguchi, T. (2000) Distinct and essential roles of transcription
factors IRF-3 and IRF-7 in response to viruses for IF N-a/b gene
induction. Immunity 13, 539–548.
43. Hata,N.,Sato,M.,Takaoka,A.,Asagiri,M.,Tanaka,N.&
Taniguchi, T. (2001) Constitutive IFN -alpha/beta signal for effi -
cient I FN-a/b gene induction by virus. Biochem. Biophys. Res.
Commun. 285, 518–525.
44. Levy, D.E. & Garcia-Sastre, A. (2001) The virus battles: IFN
induction of the antiviral state and mechanisms of viral evasion.
Cytokine Growth Factor Rev. 12 , 143–156.
45. Ronco, L.V., Karpova, A .Y., Vidal, M. & Howley, P.M. (1998)
Human papillomavirus 16, E6 oncoprotein binds to interferon
regulatory factor-3 an d inhibits its transcriptional activity. Genes
Dev. 12, 2061–2072.
46. Zhu,F.X.,King,S.M.,Smith,E.J.,Levy,D.E.&Yuan,Y.(2002)
A Kaposi’s sarcoma-associated herpesviral protein inhibits virus-
mediated induction o f type I interferon by blocking IRF-7 phos-
phorylation a nd nuclear accumulation. Proc . Natl Acad. Sci. USA
99, 5573–5578.
47. Tanaka, N., Kawakami, T. & Taniguchi, T. (1993) Recognition
DNA sequenc es of in terferon regulatory factor 1 (IRF-1) and
IRF-2, regulators of cell growth and the interferon system. Mol.
Cell. Biol. 13, 4531–4538.
48. Lin, R., Genin, P., Mamane, Y. & Hiscott, J. (2000) Selective
DNA binding and association with th e CREB binding protein
coactivator contribu te to differential activation of a/b interferon
genes by interferon regulatory factors 3 and 7. Mol. Cell. Biol. 20,

6342–6353.
Ó FEBS 2004 Synergy in activation of the IFN-b gene (Eur. J. Biochem. 271) 3703