The POU transcription factor Oct-1 represses virus-induced interferon A gene expression.

Alpha interferon (IFN-alpha) and IFN-beta are able to interfere with viral infection. They exert a vast array of biologic functions, including growth arrest, cell differentiation, and immune system regulation. This regulation extends from innate immunity to cellular and humoral adaptive immune responses. A strict control of expression is needed to prevent detrimental effects of unregulated IFN. Multiple IFN-A subtypes are coordinately induced in human and mouse cells infected by virus and exhibit differences in expression of their individual mRNAs. We demonstrated that the weakly expressed IFN-A11 gene is negatively regulated after viral infection, due to a distal negative regulatory element, binding homeoprotein pituitary homeobox 1 (Pitx1). Here we show that the POU protein Oct-1 binds in vitro and in vivo to the IFN-A11 promoter and represses IFN-A expression upon interferon regulatory factor overexpression. Furthermore, we show that Oct-1-deficient MEFs exhibit increased in vivo IFN-A gene expression and increased antiviral activity. Finally, the IFN-A expression pattern is modified in Oct-1-deficient MEFs. The broad representation of effective and potent octamer-like sequences within IFN-A promoters suggests an important role for Oct-1 in IFN-A regulation.

Preferential binding sites for interferon regulatory factors 3 and 7 involved in interferon-A gene transcription.

Transcription of the murine interferon-A4 (IFN-A4) gene is mediated by a virus responsive element (VRE-A4) located in the promoter proximal [-120 to -43] region. VRE-A4 contains four DNA modules (A to D) which cooperate for maximal IFN-A4 activation following virus infection. The differential expression between the highly expressed IFN-A4 and the weakly inducible IFN-A11 gene promoters is essentially due to point mutations within the C and D modules of the virus-responsive element VRE-A11. We now demonstrate that in murine L929 and human 293 cells, transcription factors IRF-3 and IRF-7, which are potent activators of virus-induced type I IFN transcription, differentially affect IFN-A4 and IFN-A11 promoter activities. Using electrophoretic mobility shift assays and DNase I footprinting data, our studies demonstrate that the AB modules correspond to a preferential site for IRF-7, whereas the C module is preferentially recognized by IRF-3. Furthermore, transfection of reporter constructs driven by four copies of different GAAANN hexameric motifs found within VRE-A4 indicates that the NN residues of these hexameric sequences define the preferential binding sites for IRF-3 or IRF-7. Together, these experiments clarify the molecular basis for differential expression of IFN-A genes following virus infection by delineating the sequence requirements for IRF association with the virus responsive elements of the IFN-A genes.

The virus-induced factor VIF differentially recognizes the virus-responsive modules of the mouse IFNA4 gene promoter.

Maximal activation of murine infection-A4 (IFNA4) gene transcription following viral infection requires the presence of four cooperating DNA sequences (denoted A to D), which make up the virus responsive element VRE-A4. The B, C, and D modules, when tandemized, form binding sites for the virus-induced factor (VIF), a multiprotein complex that is detected early after viral infection in the nuclei of mouse L929 cells. We now demonstrate that IFN regulatory factor-3 (IRF-3) is a component of VIF and that VIF is different from the previously identified virus-activated complexes containing IRF-3 and coactivators of transcription, such as CREB binding protein (CBP) or p300. We also show that the C module is critical for both IRF-3-mediated and virus-induced transcription of the murine IFNA4 gene. Consistently, DNase I footprinting experiments and EMSA performed with increasing amounts of recombinant GST-IRF-3(DBD) fusion proteins demonstrate that cooperativity between the modules facilitate the binding of IRF-3 and recruitment of transcription coactivators on the IFNA4 promoter. These results indicate that VIF differentially recognizes the virus-responsive modules of VRE-A4 and further actualize our previous model concerning the differential expression of murine IFNA genes.