Assembly of a functional beta interferon enhanceosome is dependent on ATF-2-c-jun heterodimer orientation

Heterodimeric transcription factors, including the basic region-leucine zip per (bZIP) protein ATF-2-c-jun, are well-characterized components of an enh anceosome that mediates virus induction of the human beta interferon (IFN-b eta) gene. Here we report that within the IFN-beta enhanceosome the ATF-2-c -jun heterodimer binds in a specific orientation, which is required for ass embly of a complex between ATF-2-c-jun and interferon regulatory factor 3 ( IRF-3). We demonstrate that correct orientation of the ATF-2-c-jun binding site is required for virus induction of the IFN-beta gene and for IRF-3-dep endent activation of a composite ATF-2-c-jun-IRF site in the IFN-beta promo ter. We also show that in vitro the DNA-bound ATP-2-c-jun heterodimer adopt s a fixed orientation upon the binding of IRF-3 at an adjacent site in the IFN-beta enhancer and that the DNA-binding domain of IRF-3 is sufficient to mediate this effect. In addition, we show that the DNA-binding domain of A TF-2 is necessary and sufficient for selective protein-protein interactions with IRF-3. Strikingly, in vivo chromatin immunoprecipitation experiments with IFN-beta reporter constructs reveal that recruitment of IRF-3 to the I FN-beta promoter upon virus infection is dependent on the orientation of th e ATP-2-c-jun heterodimer binding site. These observations demonstrate func tional and physical cooperativity between the bZIP and IRF transcription fa ctor families and illustrate the critical role of heterodimeric transcripti on factors in formation of the IFN-beta enhanceosome.