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.
Highlights
Type I interferons (IFNs)1 elicit antiviral, antiproliferative, and immunomodulatory effects in target cells by activating specific, cognate cell surface receptors [1,2,3]
We showed that our mutations to STAT2 did not affect IFN-inducible tyrosine phosphorylation of STAT1 or STAT2 (Fig. 2), we undertook experiments to examine the effects of these residue changes in STAT2 on the formation of IFN-stimulated gene factor 3 (ISGF3) complexes and their ability to bind IFN-stimulated response element (ISRE)
Considerable attention has focused on STAT2 in the context of ISGF3 complexes [7, 8, 17], yet little is understood about the role of STAT2 in ISGF3-independent complexes
Summary
Type I interferons (IFNs) elicit antiviral, antiproliferative, and immunomodulatory effects in target cells by activating specific, cognate cell surface receptors [1,2,3] This results in the activation of the receptor-associated Janus kinases (Jaks), Tyk and Jak, the phosphorylation of multiple signaling elements, and the engagement of numerous signaling cascades, including signal transducer and activator of transcription (STAT) pathways (4 – 6). The crystal structures of STAT1:1 and STAT3:3 homodimers bound to DNA revealed that each STAT protein interacts with DNA on opposite sides of the double helix [23, 24] Taken together, these data suggest that, in ISGF3-independent STAT2-containing complexes, STAT2 may contribute to DNA binding by virtue of its putative DNAbinding domain. STAT2 DNA-binding Domain in IFN Signaling intact, it is possible to disrupt IFN-inducible transcriptional activation and ISG expression mediated by GAS-like gene elements, effected by STAT2:1-DNA interactions, resulting in blunted antiviral and antiproliferative responses. Using a panel of STAT2 mutants, we provide evidence that specific residues in the putative DNA-binding domain of STAT2 influence STAT2:1-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
