STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1

Biochem. J. (2015) 466, 511–524 (P in ed in G ea B i ain) doi:10.1042/BJ20140644 511 STAT2/IRF9 di ec s a p olonged ISGF3-like ansc ip ional esponse and an i i al ac i i y in he absence o STAT1 Ka a zyna Blaszczyk*1, Adam Olejnik*1, Hanna Nowicka*, Lilla Ozgyin†, Yi-Ling Chen‡, S e an Chmielewski*, Kaja Kos y ko*, Joanna Wesoly§, Balin Laszlo Balin †, Chien-Kuo Lee‡and Hans A.R. Bluyssen*2 *Depa men o Human Molecula Gene ics, Ins i u e o Molecula Biology and Bio echnology, Facul y o Biology, Adam Mickiewicz Uni e si y, Poznan, Poland †Uni e si y o Deb ecen/Medical Facul y, Depa men o Biochemis y and Molecula Biology Cen e o Clinical Genomics and Pe sonalized Medicine, Deb ecen, Hunga y ‡G adua e Ins i u e o Immunology, Na ional Taiwan Uni e si y College o Medicine, Taipei, Taiwan §Labo a o y o High Th oughpu Technologies, Ins i u e o Molecula Biology and Bio echnology, Facul y o Biology, Adam Mickiewicz Uni e si y, Poznan, Poland E idence is accumula ing o he exis ence o a signal ansduce and ac i a o o ansc ip ion 2 (STAT2)/in e e on egula o y ac o 9 (IRF9)-dependen , STAT1-independen in e e on alpha (IFNα) signalling pa hway. Howe e , no de ailed insigh exis s in o he genome-wide ansc ip ional egula ion and he biological implica ions o STAT2/IRF9-dependen IFNαsignalling as compa ed wi h in e e on-s imula ed gene ac o 3 (ISGF3). In STAT1-de eicien U3C cells s ably o e exp essing human STAT2 (hST2-U3C) and STAT1-de icien mu ine emb yonic ib oblas cells s ably o e exp essing mouse STAT2 (mST2- MS1KO) we obse ed ha he IFNα-induced exp ession o 2-5-oligoadenyla e syn hase 2 (OAS2) and in e e on-induced p o ein wi h e a icopep ide epea s 1 (I i 1) co ela ed wi h he kine ics o STAT2 phospho yla ion, and he p esence o a STAT2/IRF9 complex equi ing STAT2 phospho yla ion and he STAT2 ansac i a ion domain. Subsequen mic oa ay analysis o IFNα- ea ed wild- ype (WT) and STAT1 KO cells o e exp essing STAT2 ex ended ou obse a ions and iden i ied ∼120 known an i i al ISRE-con aining in e e on-s imula ed genes (ISGs) commonly up- egula ed by STAT2/IRF9 and ISGF3. The STAT2/IRF9-di ec ed exp ession p o ile o hese IFN-s imula ed genes (ISGs) was p olonged as compa ed wi h he ea ly and ansi- en esponse media ed by ISGF3. In addi ion, we iden i ied a g oup o ‘STAT2/IRF9-speci ic’ ISGs, whose esponse o IFNαwas ISGF3-independen . Finally, STAT2/IRF9 was able o igge an an i i al esponse upon encephalomyoca di is i us (EMCV) and esicula s oma i is Indiana i us (VSV). Ou esul s u he p o e ha IFNα-ac i a ed STAT2/IRF9 induces a p olonged ISGF3-like ansc ip ome and gene a es an an i i al esponse in he absence o STAT1. Mo eo e , he exis ence o ‘STAT2/IRF9-speci ic’ a ge genes p edic s a no el ole o STAT2 in IFNαsignalling. Key wo ds: al e na i e in e e on esponse pa hway, cy- okines/in e e on, hos –pa hogen in e ac ions, mic oa ay, STAT ansc ip ion ac o , signal ansduc ion. INTRODUCTION In e e ons (IFNs) a e a subse o cy okines ha media e cellula homoeos a ic esponses o i us in ec ion. IFNs ep esen a amily o molecules which can be di ided in o h ee main sub- amilies: Type I, Type II and Type III [1,2]. Type I IFNs p edominan ly consis o IFNαand IFNβsub ypes, Type II consis s o he single IFNγ ype, while Type III comp ises IFNλ1, IFNλ2and IFNλ3 [3]. All IFN ypes induce IFN-s imula ed gene (ISG) exp ession by phospho yla ing STAT1 and STAT2, membe s o he signal ansduce and ac i a o o ansc ip ion (STAT) amily, media ed by Janus kinases (JAKs). STAT1 homodime s acili a e ansc ip ional esponses o all ypes o IFN by di ec ly ac i a ing genes con aining he IFNγ-ac i a ed si e (GAS) DNA elemen [4]. Responses o Type I and Type III IFN also depend on STAT2 and he DNA-binding p o ein in e e on egula o y ac o (IRF) 9. They o m a he e o ime ic ansc ip ion complex wi h STAT1 e med in e e on-s imula ed gene ac o 3 (ISGF3) ha binds o he in e e on-s imula ed esponse elemen (ISRE) in ISG p omo e s [2,5,6]. In ISGF3, STAT2 con ibu es a po en ansac i a ion domain bu is unable o di ec ly con ac DNA, whe eas STAT1 s abilizes he complex by p o iding addi ional DNA con ac s [7]. As a componen o ISGF3, i is clea ha STAT2 plays an essen ial ole in he ansc ip ional esponses o IFN wi h a s ong dependence on STAT1. P e iously, we showed ha STAT2 is also capable o o ming homodime s when phospho yla ed in esponse o IFNα[7]. These STAT2 homodime s we e Abb e ia ions: CCL8, chemokine (C–C mo i ) ligand 8; CX3CL1, chemokine (C–X3–C mo i ) ligand 1; Ddx60, DEAD (Asp-Glu-Ala-Asp) box polypep ide 60; DUOX2, dual oxidase 2; EMCV, encephalomyoca di is i us; HA, haemagglu inin; HDACi, his one deace ylase inhibi o ; HERC5, HECT and RLD domain-con aining E3 ubiqui in p o ein ligase 5; hST2-U3C, U3C s ably o e exp essing human STAT2; I i 1, in e e on-induced p o ein wi h e a icopep ide epea s 1; IFI27, in e e on alpha-inducible p o ein; IFN, in e e on; IRF9-U3C, U3C s ably o e exp essing human IRF9; IRF, in e e on egula o y ac o ; ISG, in e e on-s imula ed gene; ISGF3, in e e on-s imula ed gene ac o 3; ISRE, IFN-s imula ed esponse elemen ; MEF, mu ine emb yonic ib oblas cells; Mig 1-MS1KO, MS1KO s ably o e exp essing Mig 1; Mig 1-U3C, U3C s ably o e exp essing Mig 1; MOI, mul iplici y o in ec ion; MS1KO, STAT1- de icien mu ine emb yonic ib oblas cells; mSTAT2-MS1KO, MS1KO s ably o e exp essing mouse STAT2; MX1, myxo i us (in luenza i us) esis ance 1, in e e on-inducible p o ein; NLS, nuclea localiza ion signal; OAS2, 2-5-oligoadenyla e syn hase 2; PKR, p o ein kinase, in e e on-inducible double- s anded RNA-dependen ac i a o ; qPCR, quan i a i e eal- ime PCR; qRT-PCR, quan i a i e e e se ansc ip ion–PCR; RIG-G, e inoic acid-induced gene G; RSAD2, adical S -adenosylme hionine domain-con aining 2; SOCS1, supp esso o cy okine signalling 1; STAT, signal ansduce and ac i a o o ansc ip ion; mSTAT2-MS1KO, MS1KO s ably o e exp essing mouse mSTAT2; U3C, TNFα, umou nec osis ac o α; VSV, esicula s oma i is Indiana i us; WT, wild- ype. 1These au ho s con ibu ed equally o his wo k. 2To whom co espondence should be add essed (email [email p o ec ed]). c The Au ho s Jou nal compila ion c 2015 Biochemical Socie y Biochemical Jou nal www.biochemj.o g © 2014 The Au ho (s) This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Licence (CC-BY) (h p://c ea i ecommons.o g/licenses/by/3.0/) which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. 512 K. Blaszczyk and o he s shown o in e ac wi h IRF9 and o m he ISGF3-like complex STAT2/IRF9 ha ac i a es ansc ip ion o ISRE-con aining genes in esponse o IFNα[7]. This p o ides e idence o he exis ence o STAT1-independen IFNαsignalling pa hways. In ag eemen wi h his, Hahm e al. showed ha i uses (like measles i us and lymphocy ic cho iomeningi is) e ade he immune sys em h ough a Type I IFN-media ed STAT2-dependen , bu STAT1- independen , mechanism [8]. Addi ionally, STAT2-dependency, bu no ha o STAT1, was shown by IRF7 exp ession du ing i al in ec ion [9]. On he con a y, IRF9 exp ession in esponse o IFNα equi ed bo h STAT1 and STAT2. Simila ly, IFNαinduc ion o he an i i al p o ein apolipop o ein B mRNA-edi ing enzyme, ca aly ic polypep ide-like 3G (A3G) and o he ISGs [(p o ein kinase, in e e on-inducible double-s anded RNA-dependen ac- i a o (PKR), ISG15 and myxo i us (in luenza i us) esis ance 1 (MX1)] was STAT1-independen , bu STAT2-dependen in mouse li e cells. Howe e , STAT1 signalling was unc ional and equi ed o IFNγ-induc ion o A3G in hese cells [10]. As was sugges ed by he au ho s, a po en ial mechanism esponsible o IFNα-induc ion o A3G could in ol e STAT2/IRF9-con aining complexes. In line wi h his, ch oma in immunop ecipi a ion (ChIP) analysis using p ime s speci ic o ISRE si es con i med he associa ion o STAT2 wi h he p omo e o an i i al genes induced in esponse o Dengue i us in STAT1-de icien mice [11]. Lou e al. [12] and Fink e al. [13] p o ided addi ional impo an p oo o he biological signi icance o STAT2/IRF9 complexes in he ansc ip ional egula ion o e inoic acid-induced gene G (RIG- G) and dual oxidase 2(DUOX2), espec i ely. Lou e al. showed ha he STAT2/IRF9 complex e ec i ely d i es ansc ip ion o he RIG-G gene in NB4 cells upon signalling c oss- alk be ween e inoic acid and IFNα, in a STAT1-independen manne [12]. On he o he hand, i was shown ha he la e an i i al gene DUOX2 was induced by an au oc ine/pa ac ine pa hway speci ically igge ed in ai way epi helial cells by syne gis ic ac ion o IFNβand umou nec osis ac o alpha (TNFα), and depending on STAT2/IRF9 bu no on STAT1 [13]. The e o e, e idence con inues o accumula e ha IFNαinduc ion o ISGs and biological ou comes can occu in a STAT2/IRF9-dependen , ISGF3-independen manne [14–16]. Howe e , no de ailed insigh exis s in o he genome-wide ansc ip ional egula ion and he biological implica ions o STAT2/IRF9-dependen IFNα signalling as compa ed wi h ISGF3. Ou esul s u he p o e ha an IFNα-media ed, STAT2/IRF9- dependen signalling pa hway can induce a p olonged ISGF3- like ansc ip ional esponse and gene a e an an i i al esponse analogous o ISGF3 in he absence o STAT1. Mo eo e , we p o ide e idence o he exis ence o ‘STAT2/IRF9-speci ic’ a ge genes, unco e ing a no el ole o STAT2 in IFNα signalling, and p o iding u he e idence ha IFNαsignalling can occu in a STAT2-dependen , STAT1-independen manne . EXPERIMENTAL Cell cul u e and eagen s Human ib osa coma 2 TGH [17] and STAT1-de icien U3C [18] cells we e gi s om D Sand a Pelleg ini (Ins i u e Pas eu , Pa is, F ance). U3A cells a e he s anda d model o STAT1- null cells [17], de i ed om a high- equency mu agenesis sc een. U3C cells we e selec ed om he same sc een and belong o he same complemen a ion g oup as U3A, designa ed U3 (D Sand a Pelleg ini, Ins i u e Pas eu , Pa is, F ance: pe sonal communica ion) [17]. Mu ine emb yonic ib oblas cells (MEF) and STAT1-de icien mu ine emb yonic ib oblas (MS1KO) we e desc ibed p e iously [19]. S able cell lines U3C s ably o e exp essing human STAT2 (hST2-U3C), U3C s ably o e exp essing Mig 1 (Mig 1-U3C) and U3C s ably o e exp essing human IRF9 (IRF9-U3C) we e es ablished in ou labo a o y by co- ans ec ing (using he calcium phospha e me hod [20]), U3C cells wi h he pcDNA6/TR (blas icidin- esis ance) plasmid oge he wi h he hSTAT2-3xHA-Mig 1, emp y Mig 1 o hIRF9-Mig 1 plasmid, espec i ely. Then, he cells we e pu on blas icidin (5 μg/ml) (In i oGen) selec ion medium, and speci ic clones we e selec ed based on GFP luo escence (de i ed om Mig 1 plasmid). MS1KO cells s ably o e exp essing mouse STAT2 (mSTAT2-MS1KO) o mouse mSTAT2 (mSTAT2-MS1KO) o Mig 1 (Mig 1-MS1KO) we e es ablished as ollows: i s he calcium phospha e me hod was used o ans ec HEK (human emb yonic kidney)-293T cells wi h mST2-Mig 1 o mSTAT2-Mig 1 plasmids, espec i ely, oge he wi h GAG-POL and ENV ec o s in a io 3:1:1. A e 48 h supe na an con aining e o i us was collec ed and used o ansduc ion o MS1KO cells as desc ibed be o e [21]. A e an addi ional 24 h, cells we e ans ec ed wi h pcDNA6/TR plasmid using Tu boFec ans ec ion eagen (Fe men as). Nex , he cells we e pu on blas icidin (4 μg/ml) selec ion medium and GFP posi i e clones we e chosen o u he cha ac e iza ion. All cell lines we e cul u ed in Dulbecco’s modi ied Eagle’s medium (DMEM, IITD PAN) supplemen ed wi h 10% e al bo ine se um (FBS) (PAA Labo a o ies) and 1 %L-glu amine, penicillin/s ep omycin (PAA Labo a o ies). The cells we e s imula ed wi h o wi hou 200 U/ml o ecombinan IFNα(Millipo e), human cells wi h human IFNα – IF007 and mouse cells wi h mouse IFNα– IF009. Plasmids and ans ec ion Human STAT2-3xHA-Mig 1, mouse STAT2-Mig 1, human IRF9-Mig 1 and mouse mSTAT2-3xHA-Mig 1 plasmids we e cons uc ed in he ollowing way: he ull-leng h cDNA sequence o IRF9 was cloned in o he XhoI and EcoRI es ic ion si es o he MigR1 plasmid [22]. The STAT2 and STAT2- TAD coding sequences (2769 bp and 2199 bp, espec i ely) combined wi h he human in luenza i us haemagglu inin (HA) epi ope (3xHA, 116 bp) we e sequen ially cloned in o he BglII and EcoRI es ic ion si es o Mig 1. The STAT2-Y690F plasmid was cons uc ed using he QuikChange si e-di ec ed mu agenesis ki (Agilen ). Human STAT2-3xHA-Mig 1 plasmid was used as a empla e and he ollowing p ime s we e designed o in oduce he poin mu a ion: Fo _hSTAT2_Y690F: CAGGAACGGAGGAAATTCCTGAAA-CACAGGCTC; Re _ hSTAT2_Y690F: GAGCCTGTGTTTCAG-GAATTTCCTCCGT TCCTG. Two ans ec ion me hods we e used: calcium phospha e me hod was used as desc ibed be o e [20], and Tu boFec ans ec ion eagen was used acco ding o he manu ac u e ’s desc ip ions (Fe men as). Immunop ecipi a ion and Wes e n blo ing To al cell lysa es we e p epa ed by lysing cells in lysis bu e [300 mM NaCl, 50 mM HEPES (pH 7.6), 1.5 mM MgCl2,10% glyce ol, 1%T i on X-100, 10 mM sodium py ophospha e, 20 mM NaF, 1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF and 1 mM Na3VO4]a 4 ◦C o 20 min. Lysa es we e quan i ied by he BCA me hod (The mo Scien i ic) and equal amoun s o samples we e esol ed by 8%SDS/PAGE, ollowed by ans e o PVDF memb ane (San a C uz) and Wes e n blo analysis wi h indica ed an ibodies. Fo immunop ecipi a ion o HA- agged human STAT2, o al cell lysa es we e subjec ed c The Au ho s Jou nal compila ion c 2015 Biochemical Socie y © 2014 The Au ho (s) This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Licence (CC-BY) (h p://c ea i ecommons.o g/licenses/by/3.0/) which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. STAT2/IRF9 di ec s an ISGF3-like ansc ip ome wi hou STAT1 513 o o e nigh incuba ion wi h 5 μg o an i-HA an ibody (05– 904; Millipo e) and 30 μl o P o ein G-Sepha ose beads (BioVision). The immunop ecipi a es we e washed acco ding o he manu ac u e ’s ins uc ions and p ocessed o Wes e n blo ing. To con ol o speci ici y, we addi ionally pe o med IP wi h an un ela ed an ibody (IgG) (no shown). P o eins we e immunode ec ed using α- ubulin (04-1117; Millipo e), phospho yla ed STAT2 (pSTAT1) (07-224; Millipo e), ISGF-3γp48 (sc-10793; San a C uz), human o al STAT2 ( STAT2) (sc-839; San a C uz), o al STAT1 ( STAT1) (sc- 346; San a C uz), phospho yla ed STAT1 (pSTAT1) (sc-7988- R; San a C uz), mouse o al STAT2 ( STAT2) [23] dilu ed in TBS-T con aining ei he 0.125%non- a milk o 1%BSA (BioShop). Nex , he ho se adish pe oxidase (HRP)-conjuga ed goa an i- abbi IgG seconda y an ibody (12-348; Millipo e) was applied and immuno eac i e bands we e isualized by enhanced chemiluminescence using he Lumina a Fo e HRP Subs a e (Millipo e) and de ec ed wi h he G:Box Sys em (Syngene). Quan i a i e e e se ansc ip ion-PCR (qRT-PCR) analysis To al RNA was p epa ed using he GeneMATRIX pu i ica ion ki (EURx) ollowing he manu ac u e ’s ins uc ions. To al RNA (500 ng) was subjec ed o e e se ansc ip ion and PCR ampli ica ion was pe o med in Maxima SYBR G een/ROX qRT-PCR Mas e Mix (Fe men as) on he Eco qRT-PCR Sys em (Illumina). Sequences o oligonucleo ides (Genomed) a e a ailable om H.A.R.B. on eques . The amoun o a ge gene in each sample was no malized o endogenous con ol ACT-β (CT). Da a we e ans o med as desc ibed p e iously [24]. Mic oa ay and da a analysis Fi s , human 2 TGH and hST2-U3C and mouse MEF wild- ype (WT) and mSTAT2-MS1KO cells we e ea ed wi h o wi hou IFNα o di e en imes: 0 h, 4 h, 8 h, 24 h. RNA om each sample was isola ed and labelled ia he Illumina® To alP epTM RNA Ampli ica ion Ki (Li e Technologies). S anda d Illumina Exp ession BeadChip HumanHT-12 4 o MouseRe -8 2 (Illumina) hyb idiza ion p o ocols we e used o ob ain he aw da a. Chips we e scanned using he HiScanSQ sys em (Illumina). The comple e da a o he Illumina Exp ession BeadChip analysis is a ailable a NCBI GEO, wi h he accession numbe GSE50007. The a e age gene exp ession signals om h ee ( o human cells) o wo ( o he mouse cells) independen biological expe imen s we e aken o s a is ical es ing. Backg ound sub ac ion and quan ile no maliza ion we e applied and genes signi ican ly (p- alue ⩽0.05) up- egula ed a leas 2- old in any o he IFNα- ea ed samples we e selec ed o u he analysis. S a is ically signi ican up- egula ed genes in di e en cell-line da a se s we e compa ed by Venn diag am analysis (h p://bioin ogp.cnb.csic.es/ ools/ enny/index.h ml) [25]. Iden i ica ion o o e lapping genes be ween human and mouse da a se s was based on ‘Gene ID and name’. Clus e analysis was pe o med using Genesis so wa e (h p:// genome. ug az.a /genesisclien /genesisclien _desc ip ion.sh ml) [26]. Fo hie a chical clus e ing he a e age linkage me hod was applied. Thus, induc ion a io o common up- egula ed genes be ween human 2 TGH and hST2-U3C o mouse MEF WT and MST2-MS1KOwas log2- ans o med and subjec ed o clus e analysis. The au oma ic gene clus e assignmen me hod was used o c ea e gene clus e s. Fo he common up- egula ed genes lis ed in Table 2, p omo e egions om −450 bp o +50 bp (in ela ion o he ansc ip ional s a si e) we e sea ched o he p esence o an ISRE sequence acco ding o he T ans ac da abase (PSCAN so wa e; h p://www.beaconlab.i /pscan) [27]. En ichmen in gene on ology (GO) ca ego ies was pe o med using Go illa so wa e (h p://cbl-go illa.cs. echnion.ac.il/) [28]. AP- alue o 10−3was used as a h eshold and Illumina gene lis s om HumanHT-12 4 o MouseRe -8 2 we e aken as a backg ound model. Nex , all he s a is ically signi ican and en iched GO ca ego ies we e analyzed by Re igo so wa e (h p:// e igo.i b.h /) [29]. To emo e edundan GO e ms, he allowed simila i y alue o 0.5 was used. Ch oma in immunop ecipi a ion ChIP was pe o med as desc ibed p e iously [30] wi h mino modi ica ions. B ie ly, cells we e ea ed wi h IFNα o 0 h and 24 h, ollowed by c oss-linking wi h DSG (Sigma) o 30 min and hen wi h o maldehyde (Sigma) o 10 min. A e ixa ion ch oma in was sonica ed wi h a Diagenode Bio up o Plus o gene a e 200–1000 bp agmen s. Ch oma in was immunop ecipi a ed wi h a p e-immune IgG (Millipo e, 12- 371B) o a polyclonal an ibody agains STAT2 (San a C uz, sc- 476X). Ch oma in–an ibody complexes we e p ecipi a ed wi h an i-IgA and an i-IgG pa amagne ic beads (Li e Technologies). A e six washing s eps, complexes we e elu ed and he c oss- links e e sed. DNA agmen s we e column pu i ied (Qiagen, MinElu e). DNA was quan i ied wi h a Qubi luo ome e (In i ogen). Immunop ecipi a ed DNA was quan i ied by quan i a i e PCR (qPCR) and no malized o alues ob ained a e ampli ica ion o unp ecipi a ed (inpu ) DNA. Sequences o oligonucleo ides (Genomed) a e a ailable on eques . An i i al assay An i i al assay was pe o med as desc ibed be o e [21,31] wi h modi ica ions. 2 TGH, U3C, hST2-U3C and Mig 1-U3C cells we e seeded on o 96-well pla es a 7 ×103cells/well. Nex day, cells we e p e ea ed wi h o wi hou 2- old se ial dilu ions o IFNα, s a ing om 250 U/ml o 24 h. Subsequen ly, encephalomyoca di is i us (EMCV) o VSV ( esicula s oma i is Indiana i us) a a mul iplici y o in ec ion (MOI) o 0.3 o 3 was added o he cells using se um- ee DMEM. Twen y hou s pos - in ec ion, he medium was emo ed and cells we e ixed wi h 10% o maldehyde solu ion o 20 min a oom empe a u e. A e ixa ion, cells we e isualized by c ys al iole s aining. Excess dye was emo ed by imme sing he pla e in wa e . RESULTS The ab oga ed IFNα esponse in STAT1 KO cells co ela es wi h diminished STAT2 phospho yla ion Fi s , we cha ac e ized IFNα esponses o he human 2 TGH (WT) and U3C (STAT1-de icien ) cell lines, and he mouse MEF (MEF WT) and MS1KO cells. Bo h human and mouse WT cells we e ea ed wi h IFNα o inc easing imes, which esul ed in a simila phospho yla ion pa e n o STAT1 and STAT2. Phospho yla ion o bo h p o eins inc eased a e 4 h o ea men and diminished o nea basal le els a e 8 and 24 h (Figu es 1A and 1B). Exp ession o STAT1 and STAT2 clea ly inc eased in ime in 2 TGH and MEF WT in esponse o IFNα. The exp ession o IRF9, on he o he hand, only inc eased in 2 TGH. The IFNα esponse in bo h U3C and MS1KO cells exhibi ed diminished STAT2 phospho yla ion, despi e he no mal exp ession o STAT2 and IRF9 p o eins (Figu es 1A and 1B). STAT2 phospho yla ion c The Au ho s Jou nal compila ion c 2015 Biochemical Socie y © 2014 The Au ho (s) This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Licence (CC-BY) (h p://c ea i ecommons.o g/licenses/by/3.0/) which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. 514 K. Blaszczyk and o he s Figu e 1 The IFNα esponse in STAT1 KO cells is ab oga ed (A,C) 2 TGH and U3C; (B,D) MEF WT and MS1KO we e ea ed wi h IFNα o indica ed imes. Fo (A)and(B), p o ein lysa es we e isola ed and analyzed by Wes e n blo analysis. To al STAT2 ( STAT2), phospho yla ed STAT2 (pSTAT2), o al STAT1 ( STAT1), phospho yla ed STAT1 (pSTAT1) and IRF9 we e analyzed using speci ic an ibodies. Equal loading was e i ied using an i- ubulin. Fo (C)and(D), o al RNA was ex ac ed and OAS2 and I i 1 ela i e old induc ion was quan i ied using qRT-PCR. S a is ical signi icance is p esen ed as compa ed wi h he non- ea ed con ol ( esul s a e means + −S.E.M.). S a is ical analysis was conduc ed using one-way ANOVA wi h Tukey’s pos hoc es . * P ⩽0.05, *** P ⩽0.01. in IFNα- ea ed human U3C cells was no de ec able (Figu e 1A), e en a e 1 h and 2 h o ea men (no shown). In mouse MS1KO cells diminished phospho yla ion o STAT2 could be de ec ed wi h mo e p olonged kine ics as compa ed wi h MEF WT cells (Figu e 1B). Exp ession o STAT2 and IRF9 did no inc ease o e ime in esponse o IFNα(Figu es 1A and 1B). Howe e , he IFNα-induced exp ession o he classical ISGs human 2- 5-oligoadenyla e syn hase 2 (OAS2) and mouse in e e on- induced p o ein wi h e a icopep ide epea s 1 (I i 1) s ill slowly inc eased o e ime, bu a a much lowe le el as compa ed wi h he WT cells (Figu es 1C and 1D). Toge he hese esul s show ha he dec ease in STAT2 phospho yla ion co ela ed wi h he diminu ion o OAS2 and I i 1 gene exp ession, sugges ing he in ol emen o STAT2 in IFNα-induc ion o he la e genes. STAT1 KO cells o e exp essing STAT2 ecapi ula e IFNα esponse To s udy he ole o STAT2 and IRF9 in he esidual IFNα- induced gene exp ession in he STAT1 KO cells, we nex gene a ed human and mouse STAT1 KO cells o e exp essing STAT2 (hST2-U3C and mST2-MS1KO, espec i ely) o emp y ec o (Mig 1-U3C and Mig 1-MS1KO, espec i ely). IFNα ea men o hST2-U3C and MST2-MS1KO o inc easing imes esul ed in high le els o STAT2 phospho yla ion, s ill being p esen a e 24 h (Figu es 2A and 2B). This co ela ed wi h he inc eased exp ession o IRF9 in hST2-U3C, bu no in MST2-MS1KO, cells (Figu es 2A and 2B). In e es ingly, unde hese condi ions, he IFNα-induced exp ession o OAS2 (in hST2-U3C) and I i 1 (in mST2-MS1KO) d ama ically inc eased as compa ed wi h he con ol cells (Mig 1-U3C and Mig 1-MS1KO, espec i ely) (Figu es 2C and 2D), wi h a maximum exp ession a e 24 h o IFNα ea men . In con as wi h he WT cells (Figu es 1A and 1B), he exp ession o hese genes in he human and mouse STAT1 KO cells o e exp essing STAT2 was p olonged, which co ela ed wi h he con inued p esence o phospho yla ed STAT2. In e es ingly, knocking down STAT1 exp ession in MEF WT, esul ed in a simila p olonged IFNα-induced exp ession pa e n o I i 1 and Oas2 as compa ed wi h con ol cells (da a no shown). This implies ha by inc easing le els o STAT2 in STAT1 KO he IFNα esponse can be es o ed. STAT2 and IRF9 in e ac and media e an IFNα esponse in he absence o STAT1 To p o e ha a STAT2/IRF9-con aining complex is esponsible o he IFNα esponse in he STAT1 KO cells o e exp essing STAT2, we pe o med addi ional expe imen s. Fi s , by using p o ein ex ac s om hST2-U3C cells ea ed wi h IFNα o inc easing imes in combina ion wi h an i-HA an ibodies o immunop ecipi a e STAT2, we we e able o obse e speci ic STAT2/IRF9complex o ma ione ena e 24ho IFNα ea men (Figu e 3A; inpu con ol is shown in Figu e 2A). In e es ingly, he STAT2/IRF9 complex could al eady be de ec ed in he absence o IFNα ea men (lane 1, Figu e 3A), and was no a ec ed by inc eased STAT2 phospho yla ion. On he o he hand, he phospho yla ion kine ics o STAT2 co ela ed wi h he p olonged exp ession pa e n o OAS2 (Figu es 2A and 2C). We also checked he le el o ISG exp ession in esponse o IFNαin wo di e en clones o hST2-U3C wi h a ying STAT2 mRNA le els. In hST2-U3C, he STAT2 mRNA le el was 75- old highe han in Mig 1-U3C con ol, whe eas in hST2-U3Ca he e was a 30- old di e ence (Figu e 3B). This co ela ed wi h he di e ence in exp ession o OAS2 in hese wo cell lines in esponse o IFNα, being 9- old highe in hST2-U3C (46- old) as opposed o hST2-U3Ca (5- old), when compa ed wi h un ea ed cells (Figu e 3B). In addi ion o mST2-MS1KO cells, we gene a ed a MS1KO s able cell line o e exp essing a C- e minally unca ed o m o mSTAT2 (mST2-MS1KO), which lacks he ans- ac i a ion domain o STAT2 and ac s as a dominan nega i e. As shown in Figu e 3(C), he le els o STAT2 in mST2-MS1KO cells co ela ed wi h he high induc ion o mouse I i 1. mST2- MS1KO acili a ed no signi ican induc ion o he mouse I i 1 gene in esponse o IFNα. Subsequen ly, we in es iga ed in mo e de ail he ole o IRF9 in he IFNα esponse in he absence o STAT1. We gene a ed a U3C cell line s ably o e exp essing IRF9 (IRF9-U3C). In e es ingly, OAS2 exp ession inc eased only c The Au ho s Jou nal compila ion c 2015 Biochemical Socie y © 2014 The Au ho (s) This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Licence (CC-BY) (h p://c ea i ecommons.o g/licenses/by/3.0/) which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. STAT2/IRF9 di ec s an ISGF3-like ansc ip ome wi hou STAT1 515 Figu e 2 The IFNα esponse in STAT1 KO cells is ecapi ula ed by inc easing STAT2 le els (A,C) hSTAT2-U3C and Mig 1-U3C; (B,D) mST2-MS1KO and Mig 1-MS1KO, we e ea ed wi h IFNα o he indica ed imes. Fo (A)and(B), p o ein lysa es we e isola ed and analyzed by Wes e n blo analysis o exp ession o STAT2, pSTAT2, STAT1, pSTAT1 and IRF9. Equal loading was e i ied using an i- ubulin. In (C,D), o al RNA was ex ac ed and OAS2 and I i 1 ela i e old induc ion was quan i ied using qRT-PCR. S a is ical signi icance is p esen ed as compa ed wi h he non- ea ed con ol ( esul s a e means+ −S.E.M.). S a is ical analysis was conduc ed using one-way ANOVA wi h Tukey’s pos hoc es . * P ⩽0.05, ** P ⩽0.01. Figu e 3 STAT2 and IRF9 complex and media e an IFNα esponse in he absence o STAT1 (A) The in e ac ion be ween STAT2 and IRF9 was analyzed by immunop ecipi a ion. hSTAT2-U3C we e ea ed wi h IFNα o he indica ed imes. Cell lysa es we e immunop ecipi a ed wi h an i-HA an ibody ollowed by Wes e n blo ing wi h IRF9, STAT2 and pSTAT2 an ibodies. (B) Two di e en clones o hST2-U3C (hST2-U3Ca and hST2-U3C) a ying in hSTAT2 exp ession le el and hei con ol Mig 1-U3C; (C)mST2-MS1KO, mST2-MS1KO and hei con ol Mig 1-MS1KO; (D) Mig 1-U3C, IRF9-U3C and hST2-U3C; (E) hST2-U3C ansien ly ans ec ed wi h Mig 1-IRF9 (500 ng); (F) U3C cells ansien ly ans ec ed wi h STAT2-Y690F o STAT2 plasmid (2.5 μg) we e all ea ed wi h o wi hou 200 U/ml IFNα o 8 h (B–E)o 24h(F). To al RNA was ex ac ed and OAS2, I i 1, STAT2 o IRF9 ela i e old induc ions we e quan i ied using qRT-PCR. S a is ical signi icance is p esen ed as compa ed wi h he non- ea ed con ol ( esul s a e means + −S.E.M.). S a is ical analysis was conduc ed using one-way ANOVA wi h Tukey’s pos hoc es excep in (E) whe e a S uden ’s - es , wo- ailed, was used. * P ⩽0.05, ** P ⩽0.01. c The Au ho s Jou nal compila ion c 2015 Biochemical Socie y © 2014 The Au ho (s) This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Licence (CC-BY) (h p://c ea i ecommons.o g/licenses/by/3.0/) which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. 516 K. Blaszczyk and o he s Figu e 4 STAT2/IRF9 and ISGF3 egula e exp ession o a common se o ISGs wi h di e en kine ics (A) 2 TGH and hST2-U3C o (B) MEF WT and mST2-MS1KO we e ea ed wi h IFNα o 0 h, 4 h, 8 h and 24 h and subjec ed o mic oa ay analysis. Common up- egula ed genes we e selec ed by compa ing ansc ip omes o indi idual cell lines. S a is ically signi ican up- egula ed genes in human (A) and mouse (B) cell-line da a se s we e compa ed by Venn diag am analysis. A e age exp ession p o iles o common up- egula ed genes be ween (C) 2 TGH and hST2-U3C and (D) MEF WT and mST2-MS1KO a e displayed in cen oid iew. Exp ession alues a e shown as log2 a io; e o ba s =S.D. 3- old as compa ed wi h Mig 1-U3C cells a e 8 h o IFNα ea men (Figu e 3D). Howe e , hST2-U3C cells ansien ly ans ec ed wi h IRF9 showed a 10- old inc ease in OAS2 gene exp ession in compa ison wi h he hST2-U3C IFNα- ea ed cells and a 57- old inc ease in con as wi h Mig 1-U3C cells (Figu e 3E). Finally, we compa ed exp ession o IFIT2 and OAS2 in U3C cells ansien ly ans ec ed wi h STAT2 o he y osine mu an STAT2Y690F (mu an o m o STAT2 ha canno be phospho yla ed on y osine). U3C-ST2 showed a 10- old inc ease upon IFN ea men , whe eas U3C-ST2Y690F exhibi ed no esponse, implying ha he STAT2/IRF9-media ed IFNα- esponse is dependen on STAT2 phospho yla ion. Toge he , hese esul s poin o he impo ance o he STAT2/IRF9 complex in he p olonged IFNα esponse in he absence o STAT1 and sugges an ISGF3-like unc ion. STAT2/IRF9 and ISGF3 egula e exp ession o a common se o ISGs wi h di e en kine ics To cha ac e ize IFNα-media ed ansc ip ional esponses and iden i y he genes being egula ed by STAT2/IRF9 in ela ion o ISGF3, we pe o med mic oa ay expe imen s compa ing human and mouse STAT1 KO cells o e exp essing STAT2 wi h hei WT coun e pa s ea ed wi h IFNα o 4 h, 8 h and 24 h. A e quali y check and da a analysis, we only ocused on he up- egula ed genes. By compa ing he exp ession p o iles o hST2-U3C wi h 2 TGH, we iden i ied 303 up- egula ed genes in hST2-U3C o which 117 we e in common wi h 2 TGH (Figu e 4A). Simila ly, by compa ing he exp ession p o iles o mST2-MS1KO wi h MEF-WT, we iden i ied 295 up- egula ed genes wi h 126 genes commonly induced be ween he wo cell lines (Figu e 4B). To cha ac e ize hese commonly up- egula ed genes in mo e de ail, i s we pe o med hie a chical clus e analysis (based on a e age linkage clus e ing o a ios) compa ing human 2 TGH wi h hST2- U3C and mouse MEF-WT wi h mST2-MS1KO (Figu es 5A and 5B, espec i ely). S ikingly, among he commonly induced genes in bo h human and mouse cell lines many known ISGs could be ecognized, including IFITs, IFIs, ISGs, OASs, MX, adical S- adenosylme hionine domain-con aining (RSAD2) and HECT and RLD domain-con aining E3 ubiqui in p o ein ligase 5 (HERC5). In gene al, he induc ion le el o hese genes was lowe in he STAT1 KO cells o e exp essing STAT2 as opposed o WT cells. The cen oid iew, ep esen ing he a e age gene exp ession pa e n in human (Figu e 4C) and mouse (Figu e 4D) cells un eiled a p olonged p o ile in hST2-U3C and MST2-MS1KO cells in esponse o IFNα. In con as , in he WT cells, his was ea ly and ansien . In o de o alida e he mic oa ay da a, qRT- PCR was pe o med o a selec ion o hese genes. Indeed, IFIT1, IFIT2, IFIT3, ISG15 and MX1 exhibi ed a p olonged IFNα- induced exp ession p o ile in hST2-U3C as compa ed wi h he 2 TGH cells (no shown). The same was ue o he exp ession o Mx2, I i 3, Isg15, Oas1b and RSAD2 when compa ed wi h MST2-MS1KO e sus MEF-WT (no shown). Collec i ely, ou esul s e eal ha STAT2/IRF9 and ISGF3 egula e exp ession o a common se o ISGs, howe e , wi h a di e en kine ics. STAT2/IRF9 and ISGF3-media ed ansc ip ional esponses p edic unc ional o e lap Nex , GO en ichmen was pe o med on he commonly up- egula ed genes in human and mouse WT and STAT1 KO cells o e exp essing STAT2 (Table 1). In e es ingly, based on he log10 P- alue pa ame e , he ca ego ies ha we e highly o e ep esen ed in bo h species displayed i s main in ol emen in h ee g oups: (1) ‘ esponse o i us’ (whi e) including GO ca ego ies such as de ence esponse o egula ion o i al ep oduc ion; (2) ‘ esponse o s imulus’ (ligh g ey) including esponse o cy okine o bio ic s imulus ca ego ies; and (3) ‘mul i-o ganism p ocesses’ (da k g ey) including esponse o s ess and o ganic subs ance. We subsequen ly examined he op-20 commonly up- egula ed genes in 2 TGH e sus hST2- U3C de i ed om he ‘ esponse o he i us’ ca ego y based on he 24 h exp ession p o ile o hST2-U3C. Indeed, hese genes included well known ISGs wi h an i i al unc ions such as IFIT1, c The Au ho s Jou nal compila ion c 2015 Biochemical Socie y © 2014 The Au ho (s) This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Licence (CC-BY) (h p://c ea i ecommons.o g/licenses/by/3.0/) which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. STAT2/IRF9 di ec s an ISGF3-like ansc ip ome wi hou STAT1 517 Figu e 5 STAT2/IRF9- and ISGF3-media ed ansc ip ional esponses p edic unc ional o e lap Clus e analysis o common up- egula ed genes be ween (A) 2 TGH and hST2-U3C o (B) MEF WT and mST2-MS1KO. To al RNA om IFNα- ea ed cell lines was analyzed using Illumina Human HT-12 4 (A) o MouseRe 8 2 (B) mic oa ays. Fo mic oa ay analysis, backg ound sub ac ion and quan ile no maliza ion we e used, genes wi h a io ⩾2and P ⩽0.05 we e conside ed as up- egula ed. log2 a ios om up- egula ed genes we e clus e ed using a e age linkage me hod. c The Au ho s Jou nal compila ion c 2015 Biochemical Socie y © 2014 The Au ho (s) This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Licence (CC-BY) (h p://c ea i ecommons.o g/licenses/by/3.0/) which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. 518 K. Blaszczyk and o he s Table 1 Gene on ology en ichmen Common up- egula ed genes om human and mouse mic oa ay expe imen s we e aken o gene on ology en ichmen analysis using Go illa and Re igo so wa e. Gene on ology e ms we e g ouped as ollow: op eigh e ms classi ied as ‘ esponse o i us’ (whi e backg ound), nex i e we e ca ego ized as ‘ esponse o s imulus’ (ligh g ey backg ound) and las six as ‘mul i-o ganism p ocesses’ (da k-g ey backg ound) based on he log10 P - alues. F equency sco es we e he pe cen age o p o eins in UniP o which we e anno a ed wi h a GO e m in he GOA da abase, i.e. a highe equency deno es a mo e gene al e m. e m_ID Desc ip ion Human Mouse equency log10 p- alue equency log10 p- alue GO:0006952 de ense esponse 0.92% -25.3 0.92% -18.0 GO:0035455 esponse o in e e on-alpha 0.00% -11.2 0.00% -4.8 GO:0050896 esponse o s imulus 10.88% -10.9 10.88% -4.3 GO:0050792 egula ion o i al ep oduc ion 0.01% -9.7 0.01% -7.0 GO:0035456 esponse o in e e on-be a 0.00% -9.1 0.00% -11.6 GO:0032020 ISG15-p o ein conjuga ion 0.00% -6.1 0.00% -8.7 GO:0048002 an igen p ocessing and p esen a ion o pep ide an igen 0.04% -5.5 0.04% -7.1 GO:0019884 an igen p ocessing and p esen a ion o exogenous an igen 0.01% -4.6 0.01% -4.0 GO:0034097 esponse o cy okine s imulus 0.06% -30.7 0.06% -8.0 GO:0009607 esponse o bio ic s imulus 0.71% -25.3 0.71% -20.4 GO:0002376 immune sys em p ocess 0.75% -24.6 0.75% -17.3 GO:0002252 immune e ec o p ocess 0.05% -23.1 0.05% -16.8 GO:0042221 esponse o chemical s imulus 1.88% -11.6 1.88% -3.9 GO:0051704 mul i-o ganism p ocess 4.28% -17.9 4.28% -19.4 GO:0010033 esponse o o ganic subs ance 0.36% -15.0 0.36% -5.2 GO:0006950 esponse o s ess 4.21% -13.9 4.21% -9.4 GO:0043901 nega i e egula ion o mul i-o ganism p ocess 0.01% -12.7 0.01% -6.0 GO:0043900 egula ion o mul i-o ganism p ocess 0.02% -12.5 0.02% -8.9 GO:2000241 egula ion o ep oduc i e p ocess 0.03% -7.5 0.03% -4.6 IFIT2, IFIT3, in e e on alpha-inducible p o ein (IFI)27, IFI44, IFI44L, OAS1, OAS2, OASL, ISG15, MX1 and RSAD2 (Table 2). Using Pscan we con i med he p esence o a classical ISRE in he p omo e o all o hese genes (Table 2). BioMa om Ensemble successi ely allowed us o iden i y mouse homologues o hese 20 human genes. Fo six o hese genes we ound mo e han one mouse homologue, including OASL and IFI27 (Table 2, indica ed by **), whe eas no mouse homologue was iden i ied o HERC5. I i 27l1, I i44l and DEAD (Asp-Glu-Ala-Asp) box polypep ide 60 (Ddx60) mouse gene p obes we e no p esen on he mouse beadchip a ay (Table 2, indica ed by *). On he o he hand, he p obe o mouse I i 1 ailed on he a ay (Table 2, indica ed by ***) al hough ou qPCR expe imen s showed compa able esul s o human IFIT1 (da a no shown). All o he iden i ied mouse homologues also con ained a classical ISRE sequence in hei p omo e , which co ela ed wi h a simila exp ession pa e n as compa ed wi h hei human equi alen s (Table 2). Pe o ming ChIP-qPCR on hST2-U3C ea ed wi h o wi hou IFNαand using an ibodies agains STAT2 o IgG clea ly showed enhanced binding o STAT2 o he ISRE o he IFI27, MX1, OAS2, IFIT1, IFIT3 and ISG15 genes, in an IFNα-dependen manne (Figu e 6). Toge he wi h he clus e analysis, his s ongly implied unc ional o e lap be ween STAT2/IRF9 and ISGF3 in human and mouse cells, especially o he po en ial o gene a ing an IFNα-induced an i i al esponse. STAT2/IRF9 egula es exp ession o ISRE-independen ISGs Compa ing he exp ession p o iles o hST2-U3C wi h 2 TGH also iden i ied 186 genes speci ically up- egula ed in hST2-U3C cells (Figu e 4A). Table 3 illus a es he op en o hese genes, o which he exp ession o chemokine (C-C mo i ) ligand 8 (CCL8) and chemokine (C-X3-C mo i ) ligand 1 (CX3CL1) was con i med by qRT-PCR in hST2-U3C and 2 TGH a e IFNα ea men (Figu e 7). As shown in Figu e 7(A), he exp ession o CCL8 and CX3CL1 depended on bo h STAT2 and IRF9, bu was absen om WT cells. Indeed, hei IFNα-induced exp ession co ela ed wi h he STAT2 le els in hST2-U3C and hST2-U3Ca. Mo eo e , hST2- U3C cells ansien ly ans ec ed wi h IRF9 showed inc eased exp ession o CCL8 and CX3CL1 in compa ison wi h he hST2- U3C in esponse o IFNα(Figu e 7B). De ailed p omo e analysis o hese genes did no iden i y a classical ISRE mo i , implying a di e en mode o egula ion. This sugges s ha STAT2/IRF9 also egula es exp ession o ISRE-independen ISGs. STAT2/IRF9 media es a simila an i i al esponse agains EMCV and VSV i us as ISGF3 To p o ide u he e idence o he unc ional o e lap be ween STAT2/IRF9 and ISGF3 in he an i i al esponse, we pe o med a se ies o an i i al assays on 2 TGH, U3C, hST2-U3C and c The Au ho s Jou nal compila ion c 2015 Biochemical Socie y © 2014 The Au ho (s) This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Licence (CC-BY) (h p://c ea i ecommons.o g/licenses/by/3.0/) which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. STAT2/IRF9 di ec s an ISGF3-like ansc ip ome wi hou STAT1 519 Table 2 The op-20 commonly up- egula ed an i i al genes in 2 TGH e sus hST2-U3C Cells we e un ea ed o s imula ed wi h IFNα o 4 h, 8 h and 24 h. Exp ession a ios (o ea ed e sus un ea ed con ol) we e calcula ed as means om h ee (human) and wo (mouse) epea s. Genes we e selec ed om he ‘ esponse o he i us’ GO ca ego y (Table 1). Mouse homologues (indica ed as he pe cen age homology wi h he human gene) we e iden i ied using Ensemble BioMa . P: posi ion o he i s nucleo ide in he p edic ed ISRE sequence in ela ion o he ansc ip ional s a si e. S: consensus ISRE ma ching sco e ( om 0 o 1), wi h 1 ep esen ing 100% iden i y. Human Mouse Gene 2 TGH hST2-U3C P omo e Mouse homologes % homolog MEF WT mST2-MS1KO P omo e 4h 8h 24h 4h 8h 24h ISRE Sequence P S 4h 8h 24h 4h 8h 24h ISRE Sequence P S IFI27* 7.6 19.8 38.0 6.2 37.4 179.7 GAGTTTCAGTTTCCT -24 0.92 I i27l1* 47% - - - - - - AAGTTTCGATTTCCC 29 0.90 I i27l2a* 44% - - - - - - TAGTTTCCATTTCAT -319 0.85 I i27l2b* 48% - - - - - - GAGTCTCTCTTGCTC -30 0.78 CXCL10 36.5 129.8 3.5 8.7 57.7 96.5 AGGTTTCACTTTCCA -184 0.88 Cxcl10 68% 57.2 22.7 13.1 3.1 7.0 13.4 AAGTTTCACTTTCCA -215 0.92 RSAD2 343.7 364.4 29.4 20.9 48.2 44.0 AGGTTTCAGTTTCCC -35 0.90 Rsad2 83% 109.0 71.6 53.6 33.3 74.8 1801.5 GAGTTTCTGTTTTCT -110 0.90 IFIT2 90.3 32.0 3.5 35.7 39.9 26.6 CAGTTTCACTTTCCT -7 0.96 I i 2 62% 8.3 4.1 1.9 1.7 1.6 2.3 CAGGATCCTTTTCTG -341 0.74 IFIT1*** 50.7 45.1 16.8 24.8 34.3 26.0 TAGTTTCACTTTCCC -1 0.98 I i 1*** 53% - - - - - - CAGTTTCACTTTCCA -107 0.96 2010002M12Rik* 51% - - - - - - CAGTTTCACTTTCCA -53 0.96 Gm14446* 55% - - - - - - AGGTTTCATTTTCTG -26 0.86 OAS2 24.7 45.0 18.7 7.3 20.6 24.3 CACTTTCACTTTCCT -17 0.88 Oas2 60% 68.3 112.6 147.7 1.0 1.0 88.4 GAGTTTCGATTTCCT -79 0.87 IFI44L* 43.5 245.1 140.6 1.0 7.3 21.1 TAGTTTCACTTTCCC -61 0.98 I i44l* 21% - - - - - - CATTTTCATTTTACT -195 0.79 CCL5 2.2 4.2 2.6 1.9 5.5 19.1 CAGTTTCAGTTTCCC -187 0.98 Ccl5 80% 1.3 1.2 1.6 1.0 1.2 2.4 CAGTTTTCTTTTCCA -153 0.83 IFIT3 100.3 75.1 10.7 19.0 29.0 18.6 CAGTTTCGGTTTCCC -79 0.94 I i 3 49% 13.0 11.9 10.7 39.1 70.0 101.2 AAGTTTCACTTTCCT -159 0.93 I830012O16Rik 50% - - - - - - AAGTTTCACTTTCCT -191 0.93 HERC5** 29.4 90.0 14.7 5.0 19.9 16.9 CAGTTTCCTTTTCCT -126 0.91 ----** ---- - - - - - - - - - IFI44 14.6 38.2 17.4 3.5 8.8 12.8 GAGTTTCAGGTTTCT -63 0.82 I i44 54% 1.1 1.0 1.0 1.0 1.0 0.8 GAGTTTCAGTTTTCG -9 0.93 OASL 36.3 45.1 8.2 7.1 15.6 10.5 GAGTTTCGATTTTTC -16 0.88 Oasl1 70% 109.7 58.7 44.6 7.4 17.8 40.2 TAGTTTCTCTTTTGT -159 0.90 Oasl2 48% 20.8 20.3 26.1 19.8 47.6 89.9 TGGTTTTGTTTTTGT -247 0.73 ISG15 22.3 34.8 30.8 2.9 6.6 9.4 CAGTTTCATTTCTGT -114 0.90 Isg15 62% 17.3 16.4 17.1 7.9 13.6 24.9 CGGTTTCCTTTTCCT -80 0.87 BST2 3.7 10.0 13.8 1.4 2.6 6.5 CAGTTTCGGTTTCCT -108 0.91 Bs 2 36% 3.6 3.6 3.1 5.2 13.8 22.0 CAGTTTCATTTTCCT -167 0.95 DDX58 16.4 16.2 4.8 4.1 6.4 5.7 CAGTTTTCTTTTCCG -118 0.85 Ddx58 77% 6.1 5.4 4.4 6.8 9.9 13.6 CAGTTTCGATTTCCT -1 0.90 DHX58 14.7 44.8 18.6 2.4 4.3 5.6 CAGTTTCAGTTTCCA -1 0.94 Dhx58 79% 14.5 16.6 16.7 13.7 31.3 59.0 CAGTTTCATTTCTAG -1 0.91 ISG20 2.6 4.6 2.5 1.7 3.7 5.2 CAGTTTTGGTTTCCC -183 0.86 Isg20 82% 3.3 2.1 1.8 1.6 2.5 4.9 TAGTTTCAGTTTCTG -311 0.91 DDX60* 7.2 13.6 7.4 2.5 5.3 4.8 TAGTTTCGTTTCCCT -78 0.87 Ddx60* 75% - - - - - - TAGTTTCGGTTTCTC -23 0.90 OAS1 17.7 28.4 11.0 2.6 5.7 4.6 TGGTTTCGTTTCCTC 8 0.83 Oas1g 60% 5.5 26.4 34.0 2.4 20.7 937.4 CAGTTTCCATTTCCC -35 0.93 Oas1a 59% 131.8 92.4 135.8 1.0 1.0 42.9 CAGTTTCCATTTCCC -22 0.93 MX1 16.0 31.9 19.0 1.5 2.9 4.0 CGGTTTCATTTCTGC -32 0.88 Mx2 74% 40.5 31.2 30.5 31.9 127.9 766.7 AAGTTTCAATTCTCC -69 0.89 Mx1 41% 24.5 13.7 9.6 3.4 1.5 47.1 CGGTTTCAATTCTCC -69 0.89 * - No mouse p obe on a ay ** - No mouse homolog gene ***- Mouse p obe ailu e Mig 1-U3C cells (Figu e 8). The cells we e i s p e ea ed wi h 2- old se ial dilu ions o IFNα o 24 h and subsequen ly in ec ed wi h ei he EMCV o VSV wi h MOI =0.3 (Figu es 8A and 8B) o 3 (Figu es 8C and 8D) o each i us. Indeed, we could obse e a es o ed an i i al esponse in hST2-U3C cells, as compa ed wi h 2 TGH, due o he o e exp ession o STAT2. U3C cells showed no an i i al p o ec ion as well as he Mig 1-U3C con ol cells e en when ea ed wi h he lowe i us concen a ion o MOI =0.3. In conclusion, STAT2/IRF9 media es a simila an i i al esponse agains EMCV and VSV i us as ISGF3. DISCUSSION P e iously, we showed ha STAT2 homodime s in e ac wi h IRF9 (STAT2/IRF9) o ac i a e ansc ip ion o ISRE con aining ISGs in esponse o IFNα[7]. Indeed, e idence is accumula ing o he exis ence o a STAT1-independen IFNαsignalling pa hway, whe e STAT2/IRF9 can subs i u e o he ole o ISGF3 [14–16]. He e, we p o ide u he insigh in o he genome- wide ansc ip ional egula ion and he biological implica ions o STAT2/IRF9-dependen IFNαsignalling as compa ed wi h ISGF3. By compa ing he imely IFNα esponse o human and mouse WT cells, we obse ed an ea ly and ansien cha ac e ha co ela ed wi h he phospho yla ion kine ics o he ISGF3 componen s STAT1 and STAT2 and he p esence o IRF9 (Figu e 1). The exp ession o he classical ISGs OAS2 and I i 1 ollowed his pa e n, con i ming he ansien ISGF3-dependen IFNα- esponse displayed in many di e en cell ypes [6]. As expec ed, in STAT1 KO cells his ISGF3-dependen IFNα- esponse was se e ely ab oga ed, highligh ing he impo ance o STAT1 [32]. Howe e , IFNα-induced STAT2 phospho yla ion c The Au ho s Jou nal compila ion c 2015 Biochemical Socie y © 2014 The Au ho (s) This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Licence (CC-BY) (h p://c ea i ecommons.o g/licenses/by/3.0/) which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.