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 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), U6A2N458A) 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.