title: Functional relevance of the conserved DNA-binding domain of STAT2.
abstract: 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 gamma-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.
authors.affiliation: Division of Cell & Molecular Biology, Toronto General Research Institute, University Health Network and Department of Immunology, University of Toronto, Toronto, Ontario M5G 2M1, Canada.
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Brierley
authors.forename: Eleanor N
authors.initials: EN
authors.lastname: Fish
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D019281:Dimerization; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D007372:Interferons / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D050795:STAT2 Transcription Factor; D013997:Time Factors; D015534:Trans-Activators / Q000737:chemistry* / Q000502:physiology; D015533:Transcriptional Activation; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
chemical_list: D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D050795:STAT2 Transcription Factor; C494084:STAT2 protein, human; D015534:Trans-Activators; D014443:Tyrosine; D012313:RNA; D004247:DNA; D007372:Interferons; D008156:Luciferases