Complex formation of the interferon (IFN) consensus sequence-binding protein with IRF-1 is essential for murine macrophage IFN-gamma-induced iNOS gene expression

This study describes the role of the interferon (IFN) consensus sequence-binding protein (ICSBP or IRF-8) in iNOS gene expression by murine macrophages. An ICSBP binding site in the iNOS promoter region (-923 to -913) was identified using an electrophoretic mobility shift assay and chromatin co-immunoprecipitation. Overexpression of ICSBP greatly enhanced IFN-gamma-induced iNOS promoter activation in RAW264.7 cells, and IFN-gamma-induced iNOS promoter activation was abolished in ICSBP-/- macrophages. Furthermore, transduction of retrovirus-ICSBP in ICSBP-/- macrophages rescued IFN-gamma-induced iNOS gene expression. However, transduction of retrovirus-ICSBP in the absence of IFN-gamma activation did not induce iNOS expression in either RAW264.7 cells or ICSBP-/- macrophages. Interestingly, ICSBP alone transduced into ICSBP-/- macrophages did not bind to IFN-stimulated response element site (-923 to -913) of the iNOS promoter region, although following activation with IFN-gamma, a DNA.protein complex was formed that contains ICSBP and IRF-1. Co-transduction of ICSBP with IRF-1 clearly induces nitric oxide production. In addition, interleukin-4 inhibits IFN-gamma-induced iNOS gene expression by attenuating the physical interaction of ICSBP with IRF-1. Complex formation of ICSBP with IRF-1 is essential for iNOS expression, and interleukin-4 attenuates the physical interaction of ICSBP with IRF-1 resulting in the inhibition of INOS gene expression.     

Selective DNA binding and association with the CREB binding protein coactivator contribute to differential activation of alpha/beta interferon genes by interferon regulatory factors 3 and 7

Recent studies implicate the interferon (IFN) regulatory factors (IRF) IRF-3 and IRF-7 as key activators of the alpha/beta IFN (IFN-alpha/beta) genes as well as the RANTES chemokine gene. Using coexpression analysis, the human IFNB, IFNA1, and RANTES promoters were stimulated by IRF-3 coexpression, whereas the IFNA4, IFNA7, and IFNA14 promoters were preferentially induced by IRF-7 only. Chimeric proteins containing combinations of different IRF-7 and IRF-3 domains were also tested, and the results provided evidence of distinct DNA binding properties of IRF-3 and IRF-7, as well as a preferential association of IRF-3 with the CREB binding protein (CBP) coactivator. Interestingly, some of these fusion proteins led to supraphysiological levels of IFN promoter activation. DNA binding site selection studies demonstrated that IRF-3 and IRF-7 bound to the 5'-GAAANNGAAANN-3' consensus motif found in many virus-inducible genes; however, a single nucleotide substitution in either of the GAAA half-site motifs eliminated IRF-3 binding and transactivation activity but did not affect IRF-7 interaction or transactivation activity. These studies demonstrate that IRF-3 possesses a restricted DNA binding site specificity and interacts with CBP, whereas IRF-7 has a broader DNA binding specificity that contributes to its capacity to stimulate delayed-type IFN gene expression. These results provide an explanation for the differential regulation of IFN-alpha/beta gene expression by IRF-3 and IRF-7 and suggest that these factors have complementary rather than redundant roles in the activation of the IFN-alpha/beta genes.