The interferon-inducible Stat2:Stat1 heterodimer preferentially binds in vitro to a consensus element found in the promoters of a subset of interferon-stimulated genes.

Regulated expression of type I interferon (IFN)-stimulated genes (ISG) requires the binding of the signal transducer and activator of transcription (Stat) complexes to enhancer elements located in the ISG promoters. These enhancer elements include the IFN-stimulated response element (ISRE) and the palindromic IFN-gamma activation site (GAS) element. Regulated expression of ISRE containing ISG depends on IFN-stimulated gene factor 3 (ISGF3), a heterodimer involving Stat1 and Stat2 in association with a DNA-binding adapter protein, p48/IFN regulatory factor-9 (IRF-9). Several GAS binding Stat complexes involving Stat1, Stat3, Stat4, and Stat5 have been described, but their contribution to GAS-dependent ISG expression remains to be established. We and others previously identified an IFN-alpha-inducible Stat2:1 heterodimer that exhibits binding to the GAS element of the IRF-1 gene. These previous studies raise the possibility that Stat2:1 may participate in the transcriptional activation of the subset of ISG containing GAS elements. To address this question, we performed a PCR-assisted binding site selection procedure to define the Stat2:1 recognition sequence. The data reveal that Stat2:1 preferentially binds to a palindromic sequence similar to the consensus GAS element found in the promoter of several ISG. Our results establish that in addition to the classic complex formation involving Stat2, Stat1, and p48 associations, Stat2:1 heterodimers are formed in response to IFN treatment that may play an important role in the transcriptional regulation of certain ISG.

Application of genomic DNA affinity chromatography identifies multiple interferon-alpha-regulated Stat2 complexes.

Interferon-alpha (IFN-alpha)-induced signal transduction is mediated by the phosphorylation-activation of the signal transducer and activator of transcription (STAT) proteins Stat1, Stat2, and Stat3. Previous studies have shown that these activated STATs dimerize to form four distinct STAT complexes which translocate to the nucleus and activates transcription by binding to specific promoter elements. The interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1 heterodimers in association with a DNA-binding protein, p48, that binds to the interferon stimulated response element. Homo-and heterodimers of Stat1 and Stat3 bind to the palindromic interferon response element (pIRE). In this report we demonstrate the utility of a biochemical procedure that we have developed, based on genomic DNA affinity chromatography, for the identification of IFN-alpha-induced STAT complexes. Using this approach, we identified ISGF3-independent Stat2-containing STAT complexes. Results from the analysis of Stat2 complexes in the electrophoretic mobility shift assay were consistent with genomic DNA affinity chromatography results and identified a Stat2:1 complex that binds with low affinity to the pIRE of the interferon regulatory factor-1 gene. Immunoprecipitation studies of Stat2 revealed an IFN-alpha dependent co-precipitation of both Stat1 and Stat3. Taken together, our results suggest that IFN-alpha activates, in addition to ISGF3, other Stat2-containing STAT complexes, one of which binds to an element related to the interferon regulatory factor-1 pIRE.