Eur J Biochem 271, 3693–3703 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04310.x Mechanism for transcriptional synergy between interferon regulatory factor (IRF)-3 and IRF-7 in activation of the interferon-b gene promoter Hongmei Yang1, Gang Ma1, Charles H Lin2,*, Melissa Orr1 and Marc G Wathelet1 Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA The interferon-b promoter has been studied extensively as 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 and NF-jB, and the coactivators p300/CBP play critical roles in the activation of this and other promoters It remains unclear, however, 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 factors to activate transcription First we showed in undifferentiated 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 IRF-7 was strongly affected by promoter context, with IRF-7 preferentially 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 was strongly dependent on the presence of p300/ 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 coactivators as architectural factors Specificity in transcriptional regulation is thought to derive in part from the combinatorial assembly of unique complexes of transcription factors at target promoters 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 (reviewed in [1]) Cells from vertebrate organisms respond to viral infection by activating antiviral enzymes and by modulating the expression levels of a set of cellular genes, some of which encode cytokines such as IFNs (reviewed in [2,3]) These cytokines signal the occurrence of an infection to other cells, allowing coordination of the adaptive response at the organismal level The IFN-b gene plays a crucial role in initiating and sustaining this response through its early direct transcriptional activation in infected cells and through its ability to enhance the induction by virus of the family of IFN-a genes (reviewed in [4]) The molecular basis for the regulation of IFN-b transcription has 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 transcription unit from the influence of other 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 cells [6] The uninduced state is maintained, at least in part, through the inhibitory effects of an NF-jB regulating factor (NRF; [7,8]), YinYang [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 cis-acting elements are called positive regulatory domains (PRDs) and are located between )99 and )55 relative to the transcription initiation site Virus infection results in the coordinate activation of 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 IIIPRD I (known as P31, )90 to )64) while the p50/p65 NF-jB dimer binds to PRD II ()66 to )55) (Fig 1A) VAF 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 low intrinsic affinity of IRF-3/7 Correspondence to M G Wathelet, Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576, USA Fax: +1 513 558 5738, Tel.: +1 513 558 4515, E-mail: marc.wathelet@uc.edu Abbreviations: CAT, chloramphenicol acetyl transferase; CREB, cAMP response element binding protein; CBP, CREB-binding protein; DOC, deoxycholate; GST, glutathione S-transferase; IFN, interferon; IRF, IFN regulatory factor; ISRE, IFN stimulated response element; NRF, NF-jB regulatory factor; PRD, positive regulatory domain; VAF, virus activated factor; WT, wild type *Present address: 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) Keywords: coactivator; interferon; IRF; synergy; virus Ó FEBS 2004 3694 H Yang et al (Eur J Biochem 271) A P31x2CAT in P19 cells Arbitrary Units B 18 16 14 12 10 IFNβCAT in P19 cells 14 12 10 Co SV pcDßA Co SV IRF-1 Co SV IRF-3 Co SV IRF-7 Co SV IRF-3/7 values 2 2 0 Co SV pcDßA Co SV IRF-1 Co SV IRF-3 Co SV IRF-7 Co SV IRF-3/7 values 1 4 Fig The IFN-b gene locus and promoter context dependence (A) Schematic representation of the human IFN-b gene locus, including flanking S/ MARs; the virus-responsive element (VRE) and the factors binding to it in the uninduced and virus-induced states are indicated (B) Activity of P31·2CAT and )110IFNbCAT in P19 cells P19 cells in 6-well plates were cotransfected with lg cytomegalovirus (CMV)-lacZ, lg reporter plasmid P31·2CAT (left panel) or )110IFNbCAT (right panel) and CMV-driven vectors directing the expression of the indicated transcription factors (HuIRF-1, lg; H6HuIRF-3, lg; F3HuIRF-7B, lg) CAT activity and b-galactosidase activity were measured in extracts of transfected cells infected with Sendai virus (SV) or mock-infected as control (Co) CAT activity was normalized to b-galactosidase activity to control for transfection efficiency and is expressed in arbitrary units, rather than fold induction, so that the relative strength of different reporters can be compared Fold induction can be computed from the values listed under the graph for this promoter (reviewed 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 transcriptional 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 factors 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] or lipopolysaccharide treatment additionally activate IRF-3 and/or IRF-7, and consequently the IFN-b gene Moreover, it is not understood why type I IFN genes can be activated by virus infection in most adult 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 ATF-2/c-Jun and NF-jB to activate IFN-b Ectopic expression of either IRF-3 or IRF7, but not IRF-1, was sufficient to allow virus-dependent activation of the IFN-b promoter in undifferentiated embryonic carcinoma cells These cells, as well as insect cells, were used to define the role played by each transcription factor and coactivator in activation of the IFN-b promoter We show 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- Ó FEBS 2004 Synergy in activation of the IFN-b gene (Eur J Biochem 271) 3695 scription factors was strongly dependent on the presence of p300 or CBP, even when these coactivators had only a modest effect on the transcriptional activity of each transcription factor alone Experimental procedures Plasmid constructs and sequence analysis Effector constructs for transient transfections of mammalian 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 expression of the chloramphenicol acetyl transferase (CAT) gene through the E1b TATA box, except the IFN-b promoter ()110 to +20) construct, which is driven by its own TATA box [15,20–23] Cell culture and transfections P19 cells were grown at 37 °C, 5% (v/v) CO2, in Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine serum, 50 mL)1 penicillin and 50 lgỈmL)1 streptomycin S2 cells were grown at 26 °C, in Schneider’s Drosophila medium containing 12% (v/v) fetal bovine serum, 50 mL)1 penicillin and 50 lgỈmL)1 streptomycin Transfections using the calcium phosphate coprecipitation technique were as described previously [24] P19 cells were seeded in 6-well plates (300 000 cells in mL), transfected the next day with 0.3 mL of a precipitate containing lg reporter, lg pCMV-lacZ and 1–3 lg of effector plasmid (with pcDbA added to a total of lg) for 18 h Cells were then washed three times with NaCl/Pi and further incubated with medium until harvested days after transfection Sendai virus was added for the last 18 h of transfection Sendai virus was obtained from SPAFAS (North Franklin, CT, USA) and used at 200 hemagglutinin unitsỈmL)1 S2 cells were seeded in 6-well plates (3 million cells in mL), transfected the next day 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 (with pPac added to a total of 5.75 lg), and harvested 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 transfection efficiency Variation in transfection efficiency between samples was normal Arbitrary units rather than fold activation was used in most Figures herein so that the relative strength of reporters can be compared Basal activity of a reporter displayed the most variation from experiment to experiment, presumably because the effect of small fluctuations is most visible on low values of CAT activity As a result, the net fold activation for the IFN-b promoter is different in different experiments Pull-down experiments The GST–p300/CBP fusions were described [25], as were the GST–ATF-2 and GST–c-Jun fusions [22] GST–IRF-3/7 were generated by subcloning previously described cDNA inserts [20,21] and verified by sequencing GST fusions were expressed in Escherichia coli BL21 and purified as recommended (Pharmacia), and dialyzed against phosphatebuffered saline/10% (v/v) glycerol In vitro translation in rabbit reticulocyte lysates was performed as recommended using the TnT kit (Promega), appropriately linearized pcDbA effector plasmids and T7 RNA polymerase 35 S-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 lgỈmL)1 ethidium bromide, 0.2% (v/v) NP-40 and 0.2% (w/v) BSA (binding buffer) for h at °C, followed by 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 quantified 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 arrangement 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 transcriptional 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 driving the expression of the CAT reporter through the E1b TATA box This artificial 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 cotransfected IRF both reporters had very little intrinsic activity and this activity was not significantly stimulated upon virus infection (Fig 1B) Cotransfection of IRF-1, a constitutive activator, stimulated each reporter to a similar extent and had no effect on their virus-inducibility Cotransfection of either IRF-3 or IRF-7 made both reporters virus-inducible, while cotransfection of both IRF-3 and IRF-7 had a synergistic effect, making both reporters strongly virusinducible (Fig 1B) Intriguingly, the effects of IRF-3 and IRF-7 were dramatically affected by context IRF-3 stimulated 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 stimulated virus-induced )110IFNbCAT activity only  ninefold, 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 context, while the ability 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 activation of the IFN-b gene and that there are interactions unique to the arrangement of regulatory elements in the promoter that favor the involvment of IRF-7 in its activation Ó FEBS 2004 3696 H Yang et al (Eur J Biochem 271) Benefits of using insect cells to reconstitute the activation of the IFN-b gene Transcription factor activities are selectively affected by coactivators 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 factor 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 cells 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-jB have been shown to be transcriptionally active in the Schneider S2 cell line [23] In contrast to IRF-1, both IRF-3 and IRF-7 require virusdependent phosphorylation of specific residues in their C-termini to display transcriptional activity These modifications cannot take place in S2 cells, as they lack the relevant kinase(s), but we have shown that 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) Interactions between individual transcription factors or between transcription factors and coactivators that are specific to the IFN-b promoter must account at least in part for the results obtained in P19 cells Therefore, we investigated these interactions both at the physical and functional levels First, we tested the ability of mammalian p300 and CBP, alone or in combination, to affect the activity of mammalian transcription factors in S2 cells (Fig 2) As described previously, IRF-3E7 did not activate transcription from an IFN stimulated response 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 contrast, IRF7Di displayed intrinsic transcriptional activity [20], which was further stimulated by either hp300 (approximately twofold) or mCBP (approximately fourfold) Coexpression of p300/CBP had little effect, if any, on IRF-1 transcriptional activity in S2 cells Thus, mCBP proved twice as active as hp300 for both IRF-7Di and IRF-3E7, consistent with the observation that both IRFs interact more 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 synergy was not observed with IRF-7Di B 60 Arbitrary Units 70 Arbitrary Units ISREx3CAT in S2 cells A 50 40 30 20 10 Vector IRF-1 hp300 IRF-7∆I mCBP 25 20 15 10 Vector hp300/mCBP PRDIVx6CAT in S2 cells IRF-7∆I IRF-3E7 Vector hp300 mCBP F3E7/F7 ∆ I hp300/mCBP PRDIIx3CAT in S2 cells D 40 35 2.5 2.0 1.5 1.0 0.5 0.0 30 IRF-3 E7 Arbitrary Units Arbitrary Units 3.0 35 Vector C P31x4CAT in S2 cells 40 -+ -+ Vector hp300 -+ -+ 30 25 20 15 10 ATF-2/c-Jun mCBP hp300/mCBP Vector + + + + NF-κB hp300 mCBP hp300/mCBP Fig Effects of hp300 and mCBP expression on the activity of transcription factors in S2 cells (A) Transcriptional activity of IRF-1, IRF-7Di and IRF-3E7 (0.5 lg) in the presence or absence of cotransfected hp300, mCBP and hp300/mCBP (1.5 lg) on the ISRE·3CAT reporter The value for vector alone was 0.24 and the value for IRF-3E7 without coactivator was 0.16 (B) Transcriptional activity of IRF-3E7 (1.5 lg) and IRF-7Di (2 lg), alone or in combination and in the presence or absence of cotransfected 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 or absence of cotransfected hp300, mCBP and hp300/mCBP (1.5 lg) on the PRDIV·6CAT reporter (D) Transcriptional activity of p50 (0.1 lg) and p65 (0.15 lg) in the presence or absence of cotransfected hp300, mCBP and hp300/mCBP (0.5 lg) on the PRDII·3CAT reporter The value for vector alone was 0.06 Ó FEBS 2004 Synergy in activation of the IFN-b gene (Eur J Biochem 271) 3697 hp300 mCBP 267 462 661 771 1069 1459 1892 2036 2231 2441 596 744 1571 1855 2010 2210 2370 IRF-1 IRF-3E7 IRF-7∆i ATF-2195 c-Jun nfkb1 (p50) RelA (p65) Fig Mapping of domains of hp300/mCBP interacting with transcription factors Summary of interaction studies between domains of the 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 following domains were used in pull-down experiments (with the amino acid coordinates 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, expressed as percentage of input bound, is indicated by different shades of gray and only interactions resulting in binding to more than 1.5% of input are shown IRF-3 and IRF-7 each bind with much higher affinity to the ISRE of IFN- and virus-inducible genes than to the P31 sequence within the IFN-b promoter [15] Accordingly, IRF-3 and 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 compared to mCBP, and the p300/ CBP combination stimulated activity to an intermediary level Expression of ATF-2/c-Jun in S2 cells resulted in increased activity of a PRDIV-driven reporter, and coexpression of p300 and/or CBP further stimulated it up to twofold (Fig 2C) By contrast, expression of the NF-jB dimer p50/p65 (known as nfkb1/RelA) led to a strong activation of a PRD II-driven reporter that, if anything, was slightly inhibited by coexpression of the p300/CBP coactivators Thus all the transcription factors known to bind the IFN-b gene promoter in virus-infected cells can be expressed 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 and p300/CBP [20,21] (summarized in Fig 3) Similarly, 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] Therefore, we conducted a systematic analysis of the domains within p300 and CBP that interact with mATF-2195 (the shorter murine activating form of ATF-2), c-Jun, p50, p65 and IRF-1, and the result of these experiments are summarized in Fig Binding of mATF2195 to GST-p300/CBP was undetectable in our standard assay conditions, but lowering the salt concentration from 150 to 75 mM salt allowed detection of relatively weak (binding £ 4% input) interactions with CBP-N, CBP-C2, p300-N, p300-M and p300-C2 Binding of c-Jun was stronger (up to 40% input) but mapped to the same domains Thus both ATF-2 and c-Jun can 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 very weak overall Binding 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 binding to p300 and CBP domains closely resembled that observed for IRF-7 [20], with relatively 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 virus-inducible; three copies of P31 are required to make a reporter strongly virus-inducible [15] Ó FEBS 2004 3698 H Yang et al (Eur J Biochem 271) 1.0 1.0 1.0 1.0 1.0 1.0 7.8 7.8 1.0 1.3 4.7 22.7 1.0 2.1 27.7 227.4 However, a single copy of the sequence encompassing the PRD IV and P31 sites, termed P431, confers significant virus-inducibility to 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 factors and the p300/CBP coactivators 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 presence However, the combination of transcription 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 Synergy was computed by dividing the 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 combination were added (Table 1) There was little synergy in 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 p300/CBP, however, very strong synergy was observed when all the transcription factors were combined (> 200-fold), and removing a single factor led to much lower levels of synergy Proteins binding to the IFN-b promoter interact weakly with each other Three forms of IRF-3 were produced 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 Wild type IRF-3 (IRF-3wt) interacted poorly, if at all, with the other proteins (Fig 4) By contrast, IRF-3E7, which partially mimics virus-activated IRF-3, interacted INPUT 3.4 6.9 116.8 1131.3 IRF-7∆i 0.9 1.0 8.2 56.3 IRF-7WT 2.9 2.7 28.8 34.6 IRF-3E7 1.0 0.9 1.8 2.6 IRF-3WT Fold induction P431·3CAT Vector IRF-3E7 IRF-7DI IRF-3E7/ IRF-7DI Synergy vs additive P431·3CAT Vector IRF-3E7 IRF-7DI IRF-3E7/IRF-7DI IRF-7WT IRF-7∆i IRF-7 388 IRF-1WT p50 p65 c-Jun Vector ATF-2/c-Jun Vector ATF-2/c-Jun ATF-2 p300/CBP mATF-2 c-Jun IRF-3WT IRF-3E7 IRF-3 328 GST Vector GST pull-downs IVT proteins Table Synergistic activation by ATF-2/c-Jun, IRF-3 and IRF-7 Transcriptional activity of the indicated combination of the transcription factors (0.5 lg each of ATF-2, c-Jun, IRF-3E7 and IRF-7Di) in the presence or absence of cotransfected p300/CBP (1.5 lg) on the P431·3CAT reporter Synergy was computed by dividing the 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 combination were added GST fusions Fig Physical interactions among transcription factors 35S-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 (p65) were incubated with the indicated GST fusions of ATF-2, c-Jun, IRF-3 and IRF-7 immobilized on glutathione sepharose Proteins retained on the GST fusions and 20% of the protein input were analyzed by SDS/PAGE and autoradiography; a representative experiment is shown weakly with all proteins tested Virus infection 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 indeed IRF-31–328 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-7Di and IRF-71–388, and found that IRF-71–388 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 binding to IRF-3 and IRF-7 was much weaker (Fig 4) Similarly, interactions with p50 or 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 and c-Jun, much weaker than their interactions with the p300/CBP coactivators 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 of the IFN-b promoter to be activated in S2 cells in response to various combinations of factors was investigated (Figs 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 of mammalian p300/CBP, on the Ó FEBS 2004 Synergy in activation of the IFN-b gene (Eur J Biochem 271) 3699 IFNβCAT in S2 cells IFNβCAT in S2 cells A 40 30 20 10 50 - + - + - + - + + + - + + - + + + + + + + + + + + + 10 120 90 60 - B r to ec + CBP + + p300/CBP - - C transcription of the )110IFNbCAT reporter (Fig 5A) Each transcription factor pair or IRF-1 could activate this reporter on their own To investigate synergy, their amount was titrated so that they would each minimally activate the reporter (< 1.5-fold for all apart from IRF-1, which was  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 effects, whether p300/CBP were present or not By contrast, IRF-3/IRF-7 with ATF-2/c-Jun or with NF-jB showed synergy that was entirely dependent on the presence of mammalian coactivators ( 5.3-fold and  3.3-fold, respectively) The ATF-2/c-Jun, IRF-1 and NFjB 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 NFjB combination strongly synergized ( 27.6-fold) but only in the presence of p300/CBP Thus, maximal activation was dependent on the simultaneous presence of the mammalian + IFNβCAT in S2 cells 50 Arbitrary Units All + IRF1 Fig IRF-3/IRF-7 but not IRF-1 synergize with ATF-2/c-Jun, p50/ p65 and p300/CBP in activation of the IFN-b promoter (A) Activity of the indicated combinations of transcription factors pairs (0.5 lg for ATF-2, c-Jun, IRF-3E7 and IRF-7Di; lg for IRF-1; 12 ng for p50 and 18 ng for p65) in the presence or absence of cotransfected p300/ CBP (1.5 lg of a : mix) on the )110IFNbCAT reporter (B) Threshold effect in activation of the )110IFNbCAT reporter by ATF2/c-Jun, p50/p65 and p300/CBP (All – IRFs, circles), in the presence of IRF-1 (All + IRF-1, triangles) or IRF-3E7/IRF-7Di (All + IRF3/7, squares); 8X corresponds to the amount of transcription factors used in (A), with or without lg of IRF-1 or of an IRF3/7 mix and 0.75 lg of CBP; 4X, 2X and 1X correspond to decrease of the amount used in 8X by factors of 2, and 8, respectively + 70 60 50 40 30 20 10 V All + IRF3/7 + IFNβCAT in S2 cells 30 All - IRFs + p300 Arbitrary Units 150 Fold Activation 20 IFNβCAT in S2 cells B 30 TFs 0.70 1.05 1.87 0.81 0.77 2.26 1.54 1.09 2.12 1.17 3.21 2.05 Vector p300/CBP 1 1 ATF-2/c-Jun IRF-1 IRF-3/IRF-7 NF-κB 40 l AT l F2 cJu n AJ -I R F-I R F7 F3 /7 p5 p6 -N Fk B -C BP p3 00 p/ C 50 A Arbitrary Units 60 60 Arbitrary Units A 40 30 20 10 Vector Vector +IRF7 0.1 ug All - IRF3 +IRF7 0.25 ug All + IRF3 0.25 ug +IRF7 0.5 ug All + IRF3 0.5 ug Fig Mechanism of synergistic activation of the IFN-b promoter in S2 cells (A) Activation of the )110IFNbCAT reporter by the transcription factors (TFs; 500 ng each of ATF-2, c-Jun, IRF-3E7 and IRF7Di, 100 ng p50 and 150 ng p65) in the presence or absence of 0.75 or 1.5 lg of the p300, CBP or p300/CBP coactivators The value in the absence of TFs was 0.09 (B) Activation of the )110IFNbCAT reporter by cotransfection with all the transcription factors [All; ATF2/c-Jun (1 lg), IRF-3E7/IRF-7Di (1 lg), 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 presence or absence of 0.25 or 0.5 lg of IRF-3 E7 and in the presence or absence of 0.1, 0.25 or 0.5 lg of IRF7Di, as indicated The value with vector alone was 0.17 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 equivalent of physical cooperativ- Ó FEBS 2004 3700 H Yang et al (Eur J Biochem 271) ity in the assembly of the components required for the function Cooperative assembly of transcription factors is expected to show a strong dependence on small changes in their concentrations near the threshold at which the complex can form The amount of transfected plasmids was serially increased by a factor of two over an eightfold range, and the experiment was performed in the presence or absence of IRF proteins (Fig 5B) Transfection of increasing amounts of ATF-2/c-Jun, NF-jB and CBP led to a linear increase in reporter activity (lower curve, circles) Remarkably, adding IRF-3/IRF-7 to this mix resulted in an exponential increase in reporter activity (upper curve, squares) At the two lowest amounts of transfected plasmids, the addition of IRF-3/IRF-7 had little effect on transcription Past that threshold, however, there was a sharp increase in transcriptional activity over the last two twofold increases in amounts of expression plasmids transfected, resulting in an approximately 115-fold increase in reporter activity over an eightfold increase in the amounts of transfected plasmids By contrast, when IRF-1 instead of IRF-3/IRF-7 was used (middle curve, triangles), reporter activity rose  5.6-fold over an eightfold increase 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 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 of reporter activation that was only modestly stimulated by the addition of IRF-3 However, in the presence of even small amounts of IRF-7, addition of IRF-3 resulted in a strong 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 presence of both IRF-3 and IRF-7 (Fig 6B,C), as observed in mammalian cells (Fig 1B; [15]) We have previously shown that reporters 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 IIIPRD 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, it was the combination of IRF-3 and IRF-7 that proved the most potent for both reporters Taken together, our data strongly suggest that maximal activation of the IFN-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 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 efficient than p300, and the combination of p300/CBP displayed an intermediary efficiency, as was the case for IRF-3/IRF-7 on the P31·4CAT reporter (Fig 2B) In Fig 6B, we tested the effect of removing individual factors Removal of either ATF-2 or p50 led to an increase in activity from the IFNbCAT reporter, suggesting c-Jun and p65 homodimers are stronger activators than the ATF-2/ c-Jun and p50/p65 heterodimers in this context By contrast, removal of either IRF-3 or IRF-7 led to a decrease in activity from the IFNbCAT reporter, and the decrease was more significant when IRF-7 was absent Discussion The current paradigm for specificity in transcriptional activation holds that the physiological concentration of transcription factors typically is such that a single factor does not activate transcription on its own The need for several factors to cooperate allows for a combinatorial principle to operate, which could account for specificity A PRDIII, PRDI and IRF binding sites B PRDIIIx10- & PRDIx7-CAT in P19 cells Arbitrary Units 40 30 20 10 Vector IRF-3 IRF-7 IRF-3/7 Vector IRF-3 IRF-7 IRF-3/7 PRDIIIx10CAT PRDIx7CAT 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 Fig 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 on the activity of PRDIII·10CAT or PRDI·7CAT in P19 cells uninfected (Co) or infected with Sendai virus (SV) Ó FEBS 2004 Synergy in activation of the IFN-b gene (Eur J Biochem 271) 3701 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 more potent when P31 is converted to such an ISRE By contrast, PRD II binds NF-jB with high affinity [31,32] Thus, in order to activate the IFN-b promoter, a physiological stimulus must not only activate transcription 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 factors activated 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 transcription factors in mammalian cells 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 remains noncooperative [23] We tested the 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, whether IRF-1 or IRF-3/7 were used (H.Yang and M G Wathelet, unpublished data) However, we note that D1, a Drosophila ortholog of HMG-I [33], is present in S2 cells in large amounts [E Kas (CNRS, UMR5099, Toulouse, 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-3E7/IRF-7Di strongly synergized with ATF-2/c-Jun, NF-jB and p300/CBP to activate the IFN-b promoter (Fig 5A) Presumably, the difference between the level of activation achieved with the IRF-3/7-containing set of factors vs that achieved 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 threshold effect, even with the IRF-3E7/IRF-7Di-containing set (Fig 5B), further suggests that the binding 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 physiological levels [34] Interestingly, no synergy was observed in the absence of either p300/CBP or IRF-3/IRF-7, suggesting that VAF serves 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 of 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 the IFN-b promoter if the arrangement of the PRDs allows them to occur The importance of these interactions was tested functionally 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 instead of IRF-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 account, at least 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 the presence of mammalian coactivator (Figs 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-Jun, IRF-7Di and NF-jB had intrinsic transcriptional activities that were only moderately stimulated by coexpression of the mammalian coactivators (Fig 2) Nevertheless, this combination of factors had little activity in the absence of coactivators but strongly synergized in their presence (Figs 6B.C) Thus, the ability of c-Jun, IRF7Di and RelA to interact with coactivators was 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 IFN-b promoter, coactivators not only serve as an adaptor between the general transcription machinery and the activators, but also act as a scaffold by stabilizing the formation of a nucleoprotein complex through simultaneous interactions with transcription factors The flexible nature of p300 and CBP may be crucial for accommodating the specific arrangement of activator proteins on the IFN-b promoter as well as on other complex gene regulatory elements [40] Such a scaffolding role for these coactivators has been hypothesized [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 so through different domains It is therefore somewhat puzzling that all the factors tested interacted most strongly with the N2 and C2 Ó FEBS 2004 3702 H Yang et al (Eur J Biochem 271) 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 dependent of its contacts with the coactivators [20] These observations could be reconciled considering that these domains in p300 and CBP are relatively large and it is thus possible for more than one factor to bind at once Furthermore, more than one molecule of coactivator is likely recruited to the 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 data) and VAF contains 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 the 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 embryonic stem cells or undifferentiated embryonic carcinoma cells [18,19] The data presented in Fig 1B suggest that in P19 cells (a pluripotential teratocarcinoma 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 endogenous levels of IRF-3 and IRF7 in P19 cells were apparently too low 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 Inactivation 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, in 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] These results are congruent with our observations in P19 and 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 the context of the IFN-b promoter favors its recruitment to the promoter even when 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and Mol Cell Biol 20, 6342–6353  ... activity of a transcription factor depends on the promoter context, which refers both to the specific arrangement of the cis-acting elements in the promoter and to the nature of the factors they bind... ATF-2 and c-Jun can 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. .. p300–C3(2210–2414); and p300–C(1571–2370) The intensity of binding, expressed as percentage of input bound, is indicated by different shades of gray and only interactions resulting in binding to more than 1.5% of