Functional Relevance of the Conserved DNA-binding Domain of STAT2

Several distinct type I interferon (IFN)-inducible STAT2-containing complexes have been identified. For the IFN-stimulated gene factor 3 (ISGF3), STAT1 and IRF-9 mediate IFN-stimulated response element (ISRE) binding, whereas STAT2 provides a potent transactivational domain. ISGF3-independent STAT2-containing complexes, specifically STAT2:1 and STAT2:3, bind a γ-activated sequence (GAS)-like element, yet the contribution of each STAT to DNA binding is unknown. Moreover, the contribution of these ISGF3-independent STAT2-containing complexes to IFN-inducible responses is not defined. Accordingly, we generated mutant cDNAs, targeting the DNA-binding domain in STAT2. These cDNAs were introduced by transfection into U6A cells lacking STAT2, resulting in a panel of cell lines expressing mutant STAT2 proteins. Studies assessed the sensitivity of U6A cells reconstituted with intact STAT2 (U6A-2) and cells expressing mutant STAT2s (U6A-2E426A,E427A (EE-AA), U6A-2V453I, U6A-2V454I, U6A-2V454A, U6A-2V453I,V454I(VV-II), U6A-2N458A) to IFN-inducible responses. Our data reveal that none of the mutations in the STAT2 DNA-binding domain affected IFN-inducible ISGF3 activation, and only the VV-II mutation restricted antiviral and growth inhibitory responses to IFN. Indeed, U6A-2VV-II cells are refractory to these IFN-inducible biological activities and also exhibit impaired IFN-inducible GAS-driven transcriptional activation and subsequent gene expression. Chromatin immunoprecipitation assays revealed that the VV-II mutation in STAT2 does not abrogate, but reduces the DNA binding activity of STAT2:1 heterodimers. Taken together, these data suggest a role for the conserved DNA-binding domain of STAT2 specific to the activity of ISGF3-independent STAT2-containing complexes. Several distinct type I interferon (IFN)-inducible STAT2-containing complexes have been identified. For the IFN-stimulated gene factor 3 (ISGF3), STAT1 and IRF-9 mediate IFN-stimulated response element (ISRE) binding, whereas STAT2 provides a potent transactivational domain. ISGF3-independent STAT2-containing complexes, specifically STAT2:1 and STAT2:3, bind a γ-activated sequence (GAS)-like element, yet the contribution of each STAT to DNA binding is unknown. Moreover, the contribution of these ISGF3-independent STAT2-containing complexes to IFN-inducible responses is not defined. Accordingly, we generated mutant cDNAs, targeting the DNA-binding domain in STAT2. These cDNAs were introduced by transfection into U6A cells lacking STAT2, resulting in a panel of cell lines expressing mutant STAT2 proteins. Studies assessed the sensitivity of U6A cells reconstituted with intact STAT2 (U6A-2) and cells expressing mutant STAT2s (U6A-2E426A,E427A (EE-AA), U6A-2V453I, U6A-2V454I, U6A-2V454A, U6A-2V453I,V454I(VV-II), U6A-2N458A) to IFN-inducible responses. Our data reveal that none of the mutations in the STAT2 DNA-binding domain affected IFN-inducible ISGF3 activation, and only the VV-II mutation restricted antiviral and growth inhibitory responses to IFN. Indeed, U6A-2VV-II cells are refractory to these IFN-inducible biological activities and also exhibit impaired IFN-inducible GAS-driven transcriptional activation and subsequent gene expression. Chromatin immunoprecipitation assays revealed that the VV-II mutation in STAT2 does not abrogate, but reduces the DNA binding activity of STAT2:1 heterodimers. Taken together, these data suggest a role for the conserved DNA-binding domain of STAT2 specific to the activity of ISGF3-independent STAT2-containing complexes. Type I interferons (IFNs) 1The abbreviations used are: IFN, interferon; STAT, signal transducer and activator of transcription; ISGF3, IFN-stimulated gene factor 3; ISRE, interferon-stimulated response element; GAS, γ-activated sequence; pIRE, palindromic interferon response element; IRF, interferon regulatory factor; EMCV, encephalomyocarditis virus; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation.1The abbreviations used are: IFN, interferon; STAT, signal transducer and activator of transcription; ISGF3, IFN-stimulated gene factor 3; ISRE, interferon-stimulated response element; GAS, γ-activated sequence; pIRE, palindromic interferon response element; IRF, interferon regulatory factor; EMCV, encephalomyocarditis virus; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation. elicit antiviral, antiproliferative, and immunomodulatory effects in target cells by activating specific, cognate cell surface receptors (1.Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3343) Google Scholar, 2.Brierley M.M. Fish E.N. J. Interferon Cytokine Res. 2002; 22: 835-845Crossref PubMed Scopus (159) Google Scholar, 3.Platanias L.C. Fish E.N. Exp. Hematol. 1999; 27: 1583-1592Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). This results in the activation of the receptor-associated Janus kinases (Jaks), Tyk2 and Jak1, the phosphorylation of multiple signaling elements, and the engagement of numerous signaling cascades, including signal transducer and activator of transcription (STAT) pathways (4.Schindler C. Shuai K. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (713) Google Scholar, 5.Muller U. Steinhoff U. Reis L.F. Hemmi S. Pavlovic J. Zinkernagel R.M. Aguet M. Science. 1994; 264: 1918-1921Crossref PubMed Scopus (1963) Google Scholar, 6.Colamonici O. Yan H. Domanski P. Handa R. Smalley D. Mullersman J. Witte M. Krishnan K. Krolewski J. Mol. Cell. Biol. 1994; 14: 8133-8142Crossref PubMed Google Scholar). The Jak-STAT pathways are essential for the transcriptional activation of many IFN-stimulated genes. In particular, STAT2 is a critical component of IFN signaling (7.Leung S. Qureshi S.A. Kerr I.M. Darnell Jr., J.E. Stark G.R. Mol. Cell. Biol. 1995; 15: 1312-1317Crossref PubMed Google Scholar, 8.Park C. Li S. Cha E. Schindler C. Immunity. 2000; 13: 795-804Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). IFN-inducible activation of STAT2 leads to the formation of two distinct types of STAT2-containing DNA binding complexes: IFN-stimulated gene factor 3 (ISGF3) and ISGF3-independent STAT2:1 and STAT2:3 heterodimers (9.Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10.Li X. Leung S. Qureshi S. Darnell Jr., J.E. Stark G.R. J. Biol. Chem. 1996; 271: 5790-5794Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 11.Fu X.Y. Kessler D.S. Veals S.A. Levy D.E. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8555-8559Crossref PubMed Scopus (332) Google Scholar). ISGF3-independent STAT2-containing complexes translocate into the nucleus and bind γ-activated sequence (GAS)-like palindromic IFN-response element (pIRE) (10.Li X. Leung S. Qureshi S. Darnell Jr., J.E. Stark G.R. J. Biol. Chem. 1996; 271: 5790-5794Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 12.Ghislain J.J. Wong T. Nguyen M. Fish E.N. J. Interferon Cytokine Res. 2001; 21: 379-388Crossref PubMed Scopus (48) Google Scholar). Alternatively, STAT2:1 heterodimers may associate with the DNA-binding adaptor protein, IFN regulatory factor 9 (IRF-9), to form the ISGF3 complex that binds the IFN-stimulated response element (ISRE) (1.Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3343) Google Scholar, 13.Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3330) Google Scholar). Together, ISGF3 and ISGF3-independent STAT2-containing complexes mediate transcriptional activation of IFN-sensitive genes (ISGs), including double-stranded RNA-activated protein kinase (PKR), 2′,5′-oligoadenylate (OAS), and IRF-1, involved in mediating the biologic effects of IFNs (10.Li X. Leung S. Qureshi S. Darnell Jr., J.E. Stark G.R. J. Biol. Chem. 1996; 271: 5790-5794Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 14.Goodbourn S. Didcock L. Randall R.E. J. Gen. Virol. 2000; 81: 2341-2364Crossref PubMed Scopus (843) Google Scholar, 15.Clemens M.J. Elia A. J. Interferon Cytokine Res. 1997; 17: 503-524Crossref PubMed Scopus (512) Google Scholar, 16.Rebouillat D. Hovanessian A.G. J. Interferon Cytokine Res. 1999; 19: 295-308Crossref PubMed Scopus (178) Google Scholar). Within the ISGF3 complex, STAT2 contributes its potent transcriptional activation domain, whereas STAT1 and IRF-9 mediate DNA binding (17.Qureshi S.A. Leung S. Kerr I.M. Stark G.R. Darnell Jr., J.E. Mol. Cell. Biol. 1996; 16: 288-293Crossref PubMed Scopus (146) Google Scholar, 18.Bluyssen H.A. Levy D.E. J. Biol. Chem. 1997; 272: 4600-4605Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 19.Paulson M. Pisharody S. Pan L. Guadagno S. Mui A.L. Levy D.E. J. Biol. Chem. 1999; 274: 25343-25349Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The carboxyl-terminal transactivation domain of STAT2 also contributes to the transcriptional activation potential of ISGF3-independent complexes (10.Li X. Leung S. Qureshi S. Darnell Jr., J.E. Stark G.R. J. Biol. Chem. 1996; 271: 5790-5794Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). However, the role of STAT2 in ISGF3-independent STAT2:1 heterodimers is not fully defined and the contribution of each STAT protein to DNA binding remains unknown. In earlier studies we described the preferential binding of STAT2:1 heterodimers to a palindromic GAS-like sequence, suggesting that within this complex, STAT2 contributes to sequence-specific DNA binding activity (12.Ghislain J.J. Wong T. Nguyen M. Fish E.N. J. Interferon Cytokine Res. 2001; 21: 379-388Crossref PubMed Scopus (48) Google Scholar). Indeed, several residues in STATs that contribute to DNA binding, namely residues corresponding to arginine 378 (Arg378), valine-threonine (Val426-Thr427), histidine 431 (His431) and asparagine 460 (Asn460) in STAT1, glutamates 434 and 435 (Glu434-Glu435) and valines 461 and 462 (Val461-Val462) in STAT3 and valines 466 and 467 (Val466-Val467) in STAT5, are conserved in STAT2 (20.Horvath C.M. Wen Z. Darnell Jr., J.E. Genes Dev. 1995; PubMed Scopus Google Scholar, J. L. C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, E. O. Darnell Jr., J.E. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, X. U. D. Darnell Jr., J.E. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, the of and to DNA revealed that each STAT protein with DNA of the X. U. D. Darnell Jr., J.E. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. 1998; PubMed Scopus Google Scholar). Taken together, these data suggest in ISGF3-independent STAT2-containing complexes, STAT2 may contribute to DNA binding by of its DNA-binding domain. In the we that IFN-inducible ISGF3-independent STAT2-containing complexes contribute to IFN-inducible biologic responses in target we that in cells IFN-inducible ISGF3 activation is is to IFN-inducible transcriptional activation and by GAS-like gene elements, by resulting in antiviral and responses. a panel of STAT2 we that specific residues in the DNA-binding domain of STAT2 binding activity and transcriptional activation of and and U6A were Stark were in with and in the antiviral assays specific by L. and were STAT2 and STAT1 were IRF-9 by STAT2 the STAT2 gene C. Schindler STAT2 to the The were and and and and and and The STAT2 were introduced into U6A cells by transfection the and were and in growth and were with IFN for and in phosphorylation described J. S. L.C. Fish E.N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). described J. S. L.C. Fish E.N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). for IFN-inducible antiviral activity encephalomyocarditis been cells were with the of IFN for with for The of the is a A. I.M. S. S. Fish E.N. L.C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). were in a of in the of the of IFN and were for assessed described S. Fish E.N. D. C. M. L.C. 1997; Google Scholar). were with a and S. J. P. R. Fish E.N. L.C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google a gene L. E. D. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google described A. Levy D.E. M. Fish E.N. L.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and were to and with IFN for were described (9.Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). of protein and IFN cells were described (9.Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). For of with for to of the of the and the GAS-like palindromic element (pIRE) were (12.Ghislain J.J. Wong T. Nguyen M. Fish E.N. J. Interferon Cytokine Res. 2001; 21: 379-388Crossref PubMed Scopus (48) Google Scholar, M. Fish E.N. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. D. P. J. J. PubMed Scopus Google Scholar). The are: ISRE, and pIRE, For in these were with Chromatin were with IFN for assays were to the of to of and of of by to the M. C. Li Z. Wen Z. Biol. 2002; PubMed Scopus Google to the and to the gene The were and and and DNA cells and used and were in a and a DNA and cells were with IFN for were and and the to the The DNA of in the of and for for were the DNA I and the and were used for The in a of of each and of The were and and and and and were for each and and target were for each of DNA-binding of DNA binding activity of STAT1 been and studies have residues in STAT1 that are critical for DNA binding activity E. O. Darnell Jr., J.E. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, X. U. D. Darnell Jr., J.E. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. P. U. Genes Dev. 17: PubMed Scopus Google Scholar). a of the of STAT1 to and we the STAT1 and the protein to the DNA-binding domain and residues involved in mediating DNA This to several residues that for mediating DNA binding and for the of the DNA-binding domain in the and that with the and with the of the DNA This a with that contributes to the of this this by a is the and is a to DNA and specific of the DNA are also for DNA binding within and for the of these residues is conserved in STAT2 and and we that of to may DNA binding by the and of This mutation may the and mutation of to may the of the and and with the DNA to mutation the critical of this mutation of to may have a DNA binding by specific to the of the conserved DNA-binding domain of STAT2, the mutations were introduced into the STAT2 (EE-AA), and cDNAs the STAT2 were introduced by transfection into U6A cells lacking STAT2 to a panel of cell lines expressing these mutant STAT2 proteins. intact STAT2 also introduced into U6A the cell in a IFN of GAS-driven earlier we that of cells with IFN in ISGF3-independent STAT2-containing complexes (9.Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12.Ghislain J.J. Wong T. Nguyen M. Fish E.N. J. Interferon Cytokine Res. 2001; 21: 379-388Crossref PubMed Scopus (48) Google Scholar). a for we U6A cells reconstituted with intact STAT2 (U6A-2) with of IFN not The data that is for of IFN-inducible STAT2 studies for IFN-inducible transcriptional activation were and the results a in Indeed, a of IFN is to GAS-driven transcriptional activity in cells not Accordingly, to the of ISGF3-independent STAT2-containing complexes, a of IFN used in subsequent in the STAT2 DNA-binding IFN-inducible STAT of the STAT2 into U6A to of STAT proteins. of STAT1 and STAT2, with the of cells expressing the form of STAT2, in STAT2 are that the mutations introduced into STAT2 have not affected the of STAT2 to IFN of were cells were by and with the form of STAT2. The data reveal that mutations in the DNA-binding domain of STAT2 not with IFN-inducible phosphorylation of STAT2 The of IFN-inducible STAT1 phosphorylation also in the Studies have that STAT2 is for STAT1 suggesting is a activation of STAT the IFN (7.Leung S. Qureshi S.A. Kerr I.M. Darnell Jr., J.E. Stark G.R. Mol. Cell. Biol. 1995; 15: 1312-1317Crossref PubMed Google Scholar, S.A. Leung S. Kerr I.M. Stark G.R. Darnell Jr., J.E. Mol. Cell. Biol. 1996; 16: 288-293Crossref PubMed Scopus (146) Google Scholar). However, STAT1 activation by IFN been in cells K. M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). to the mutations in the DNA-binding domain of STAT2 affected the of STAT1 to IFN STAT1 phosphorylation in in cells expressing the STAT2 in U6A cells lacking STAT2, the of STAT1 phosphorylation to that in cell the VV-II STAT2 IFN-inducible mutations in the DNA-binding domain of STAT2 have affected the biological of IFN-inducible we IFN-inducible antiviral activity in the U6A cells lacking STAT2 are to the antiviral effects of IFN cells expressing intact STAT2 to IFN with the antiviral U6A-2V453I, U6A-2V454I, U6A-2V454A, and cells are also to a antiviral response IFN However, cells expressing the VV-II STAT2 mutant to a IFN-inducible antiviral response were also for IFN-inducible growth inhibitory responses the cell U6A cells not exhibit a growth inhibitory response to IFN In IFN of cells expressing intact STAT2 results in growth expressing the and mutant of STAT2, exhibit of IFN-inducible growth to results in the antiviral cells expressing the VV-II STAT2 mutant are impaired in to to the growth inhibitory effects of IFN Together, these data suggest that the VV-II mutation signal that IFN-inducible antiviral and growth inhibitory responses are the VV-II of STAT2 IFN-inducible IFN-inducible transcriptional activation in cells expressing the mutant STAT2 and gene assays were were introduced into the cell types by a IFN activity In U6A IFN-stimulated activity by and In cells expressing the intact of the mutant of STAT2, IFN-inducible activity In cells expressing the mutant of STAT2, IFN-inducible GAS-driven activity However, IFN-stimulated activity by the element in cells expressing the VV-II mutant form of STAT2. in the DNA-binding of STAT2 IFN-inducible STAT DNA in the STAT2 mutations with STAT complex formation and DNA binding, a of were antiviral, growth inhibitory and gene assays only the VV-II mutation in STAT2 IFN we STAT complex formation and DNA binding in U6A-2VV-II cells and in two that to IFN, namely and the we Within the ISGF3 complex, STAT2 with STAT1 its domain and with IRF-9 by of its domain, residues M. M.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). we that mutations to STAT2 not IFN-inducible phosphorylation of STAT1 STAT2 we to the effects of these in STAT2 the formation of ISGF3 complexes and to bind were with IFN for were with ISRE, and by The in IFN of U6A cells lacking STAT2 to in ISGF3 In cells expressing STAT2, intact mutant IFN-inducible complex formation is STAT2 IRF-9 the of the ISGF3 the of ISGF3 to the IFN of cells expressing the and mutant of STAT2 results in of with that for U6A cells expressing intact STAT2. IFN of cells expressing the VV-II STAT2 mutant results in of the formation and DNA binding activity of ISGF3-independent STAT2:1 heterodimers. a binding we have a pIRE, a GAS-like element the STAT binding sequence the preferential binding for STAT2:1 heterodimers (12.Ghislain J.J. Wong T. Nguyen M. Fish E.N. J. Interferon Cytokine Res. 2001; 21: 379-388Crossref PubMed Scopus (48) Google Scholar). and STAT2:1 complexes bind this DNA The results in that IFN of U6A cells leads to formation and DNA binding to this The of the DNA-binding complex were in IFN of and expressing intact STAT2, results in and STAT2:1 complex formation and binding the to the STAT2:1 complex, affected the mobility of the and complexes. the to associate with STAT2 in the ISGF3 and a we that the of STAT2 with STAT1 and its with DNA is distinct ISGF3 and STAT2:1 complexes. for the of the complex is its in the of cell U6A IFN of cells expressing mutant of STAT2 in the formation of and complexes suggesting that the mutations in STAT2 have not the of ISGF3-independent STAT2-containing heterodimers to bind In the of and STAT2:1 complexes are in cells expressing the VV-II STAT2 the binding activity of STAT2:1 complexes is The VV-II STAT2 the DNA of STAT2-containing to the DNA binding activity of IFN-inducible STAT2-containing complexes in the ISGF3 DNA binding, a specific for sequence of the were to a of the this GAS-like not to the STAT2:1 sequence, this element by only and been to bind the STAT2:1 in (10.Li X. Leung S. Qureshi S. Darnell Jr., J.E. Stark G.R. J. Biol. Chem. 1996; 271: 5790-5794Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). expressing the and mutant of STAT2 to IFN-inducible STAT activation, transcriptional and antiviral and responses. these cells were not in IFN of and cells expressing intact STAT2 in binding chromatin ISGF3 activation by in that the VV-II mutation in STAT2 affected IFN-inducible ISGF3 the of transcription factor binding to that ISGF3 and DNA binding are in the U6A-2VV-II IFN-inducible binding chromatin DNA also of these complexes are IFN of cells the VV-II mutant form of STAT2 with cells expressing intact STAT2, of binding chromatin are in U6A-2VV-II cells whereas the VV-II mutation in STAT2 does not DNA binding of ISGF3 to is the DNA binding activity of STAT2-containing heterodimers to The VV-II STAT2 the IFN-inducible of the cells expressing the VV-II STAT2 mutant are to with IFN, a of a IFN gene assessed specific for the and genes and GAS-driven gene for the The data IFN cells expressing the VV-II mutant form of STAT2 of and gene with cells In U6A gene is IFN of U6A cells of gene to a In cells a of IFN that this gene is by STAT1 and STAT3 and and by only a in U6A-2VV-II of the of DNA in to the DNA binding activity of STAT2-containing heterodimers to the VV-II mutation in STAT2 also IFN-inducible GAS-driven gene expression. activation of STAT2 is for IFN response in target STAT2 in the of ISGF3 complexes (7.Leung S. Qureshi S.A. Kerr I.M. Darnell Jr., J.E. Stark G.R. Mol. Cell. Biol. 1995; 15: 1312-1317Crossref PubMed Google Scholar, 8.Park C. Li S. Cha E. Schindler C. Immunity. 2000; 13: 795-804Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, S.A. Leung S. Kerr I.M. Stark G.R. Darnell Jr., J.E. Mol. Cell. Biol. 1996; 16: 288-293Crossref PubMed Scopus (146) Google yet is the role of STAT2 in ISGF3-independent complexes. STAT2 a conserved DNA-binding domain, the activity of this domain not been In this we for STAT2 to IFN-inducible biological responses that are of ISGF3 studies suggest that STAT2 a with STAT1 that bind to chromatin and transcriptional activation that contributes to IFN-inducible biological responses. Our with the U6A cells that STAT2 that STAT2 is a critical for IFN in target In the of STAT2, cells are to the antiviral and growth inhibitory effects of IFN and IFN-inducible ISGF3 DNA binding, and transcriptional activation were in cells expressing of the STAT2 mutations in DNA-binding domain. Moreover, these STAT2 mutations not with the formation of IFN-inducible ISGF3-independent STAT2:1 heterodimers. the mutations that we introduced into STAT2 not fully by mutant STAT2 the VV-II mutation affected a in IFN-inducible binding resulting in GAS-driven transcriptional activation that in antiviral and growth inhibitory responses to IFN. This STAT2 mutation gene of the U6A-2VV-II cells exhibit IFN-stimulated gene with In U6A cells we also expression. Studies have in to STAT2:1 and complexes bind the GAS-like element within the of the gene (9.Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10.Li X. Leung S. Qureshi S. Darnell Jr., J.E. Stark G.R. J. Biol. Chem. 1996; 271: 5790-5794Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Our that STAT1 and STAT3 may contribute to its transcriptional activation, STAT2 is for expression. of the of STAT1 to DNA valines and in STAT2 for mediating DNA binding X. U. D. Darnell Jr., J.E. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. 1998; PubMed Scopus Google Scholar). These residues not mediate DNA but for the DNA-binding The to mutations were to by of the of a were not to the of this of the STAT2 protein in the of DNA binding transcriptional The VV-II mutation impaired the of STAT2, within ISGF3-independent STAT2-containing complexes, to mediate IFN-inducible responses. the results not suggest a in the DNA binding for the VV-II mutant with intact STAT2 the data that binding of IFN-inducible STAT2-containing complexes that the VV-II mutant STAT2 to is with complexes intact STAT2 Moreover, the VV-II mutation in STAT2 affected IFN-inducible GAS-driven transcriptional activation, in that this of STAT2 is for DNA The are that STAT2:1 complexes with chromatin DNA in a distinct with the in EMSA, and that is a in STAT2, residues in the DNA-binding with that is critical for these Studies the DNA binding activity of STATs have that residues in the DNA-binding domain of STAT may have distinct For residues and are critical for binding but are for the DNA binding activity of (20.Horvath C.M. Wen Z. Darnell Jr., J.E. Genes Dev. 1995; PubMed Scopus Google Scholar, L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of to in STAT3 DNA binding but does not have (20.Horvath C.M. Wen Z. Darnell Jr., J.E. Genes Dev. 1995; PubMed Scopus Google Scholar). However, of the corresponding residues in DNA binding and also growth of the complexes J. L. C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). that binding activity is not for into the nucleus T. A. P. U. Genes Dev. 17: PubMed Scopus Google Scholar). Indeed, mutations in STAT1 and DNA binding but not STAT1 C. J. 2000; 19: PubMed Google Scholar, M. Kerr I.M. J. 2001; PubMed Scopus Google Scholar). Our data suggest that the VV-II mutation in STAT2 not STAT2 In the and were that of STAT2-containing complexes into the nucleus intact in cell including cells expressing the VV-II STAT2 data that IFN-inducible STAT2-containing complexes the VV-II mutation the nucleus and bind Moreover, we have that is by the VV-II In the ISGF3 complex, STAT2 contributes its potent transactivation domain and role in mediating transcription (17.Qureshi S.A. Leung S. Kerr I.M. Stark G.R. Darnell Jr., J.E. Mol. Cell. Biol. 1996; 16: 288-293Crossref PubMed Scopus (146) Google Scholar, 18.Bluyssen H.A. Levy D.E. J. Biol. Chem. 1997; 272: 4600-4605Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The carboxyl-terminal STAT2 transactivation domain with a of and including and are for transcriptional activation M. Pisharody S. Pan L. Guadagno S. Mui A.L. Levy D.E. J. Biol. Chem. 1999; 274: 25343-25349Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, M. C. E. Levy D.E. Biol. 2002; PubMed Scopus Google Scholar). the of STAT is that a in the DNA-binding domain the STAT protein with Indeed, we that in cells expressing the VV-II mutant form of STAT2, IFN-inducible transcription and gene were this mutation not transcriptional activation by with that is that the VV-II mutation in STAT2 affected the transactivation domain and the transcriptional activation and gene to the binding we to These data that the DNA-binding domain of STAT2 and its role is specific to the activity of ISGF3-independent STAT2-containing complexes. of the of STAT1 to DNA revealed that STAT DNA DNA-binding (20.Horvath C.M. Wen Z. Darnell Jr., J.E. Genes Dev. 1995; PubMed Scopus Google Scholar, J. L. C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In with these suggest that in STAT2:1 STAT2 bind Indeed, the data suggest within ISGF3-independent STAT2-containing complexes, the of this DNA-binding domain of STAT2 is to mediate with GAS-like elements, the transcriptional activation of a of The of is to the contribution of this of to IFN-inducible responses.

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