Differential Regulation of Human Interferon A Gene Expression by Interferon Regulatory Factors 3 and 7

Differential expression of the human interferon A (IFN-A) gene cluster is modulated following paramyxovirus infection by the relative amounts of active interferon regulatory factor 3 (IRF-3) and IRF-7. IRF-3 expression activates predominantly IFN-A1 and IFN-B, while IRF-7 expression induces multiple IFN-A genes. IFN-A1 gene expression is dependent on three promoter proximal IRF elements (B, C, and D modules, located at positions −98 to −45 relative to the mRNA start site). IRF-3 binds the C module of IFN-A1, while other IFN-A gene promoters are responsive to the binding of IRF-7 to the B and D modules. Maximal expression of IFN-A1 is observed with complete occupancy of the three modules in the presence of IRF-7. Nucleotide substitutions in the C modules of other IFN-A genes disrupt IRF-3-mediated transcription, whereas a G/A substitution in the D modules enhances IRF7-mediated expression. IRF-3 exerts dual effects on IFN-A gene expression, as follows: a synergistic effect with IRF-7 on IFN-A1 expression and an inhibitory effect on other IFN-A gene promoters. Chromatin immunoprecipitation experiments reveal that transient binding of both IRF-3 and IRF-7, accompanied by CBP/p300 recruitment to the endogenous IFN-A gene promoters, is associated with transcriptional activation, whereas a biphasic recruitment of IRF-3 and CBP/p300 represses IFN-A gene expression. This regulatory mechanism contributes to differential expression of IFN-A genes and may be critical for alpha interferon production in different cell types by RIG-I-dependent signals, leading to innate antiviral immune responses.The immediate cellular response to virus infection is characterized by the transcriptional activation of type I interferon (IFN) genes, which are involved in the host antiviral defense program (35, 79). Multiple alpha interferon (IFN-α) subtypes and the single IFN-β subtype exhibit antiviral and immunomodulatory activities, which culminate in the maturation of antigen-presenting cells and T-cell activation (79). IFN-α subtypes, although signaling through the same cell surface receptor, display distinct biological effects (16, 33, 82, 84), suggesting that qualitative and quantitative differences in IFN-α production during viral infection may affect the formation, magnitude, and duration of innate and adaptive antiviral immune responses.The interferon A (IFN-A) multigenic family consists of 13 functional members located in the ifn locus on human chromosome 9, together with the single IFN-B gene (7, 13). Analysis of human IFN-A gene expression patterns in Sendai virus-infected plasmacytoid dendritic cells (pDCs), monocytes, and monocyte-derived dendritic cells (mDCs) has demonstrated that each cell population expresses predominantly IFN-A1, with multiple species of IFN-A expressed in pDCs (32), whereas IFN-A genes are expressed at similar levels in pDCs infected by influenza virus (10). Thus, differential expression of human IFN-A genes varies depending on virus and cell type, in agreement with previous in vivo studies of mice (1, 4, 11, 12, 24, 31, 37). IFN-A/B gene transcription is triggered via distinct signaling pathways that converge on the activation of interferon regulatory factor 3 (IRF-3) and IRF-7 (26), although other IRFs are also involved in cell-specific regulation of the late phases of IFN-A gene transcription (76). In pDCs, Toll-like receptor (TLR)-mediated signaling by TLR7/8/9-dependent pathways is critical for rapid and high-level expression of IFN-A/B genes mediated by IRF-7 (26-28, 30, 34, 36). TLR3 that is predominantly expressed in immature mDCs and TLR-4 essentially operating in mDCs and monocyte/macrophage cells require TANK-binding kinase 1 (TBK1), IRF-3, and IRF-7 (25). In conventional dendritic cells and other cell types such as fibroblasts or epithelial cells, RNA virus infection stimulates IFN-α expression upon ligand stimulation of the mitochondrion-associated RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), which leads to the activation of TBK1 and IκB-related kinase ɛ (IKKɛ) (29, 34, 44, 55, 66, 86). DNA virus-dependent signaling through DNA-dependent activator of IRFs requires TBK1 and IRF-3 to induce type I IFN gene expression (78).Phosphorylation of specific C-terminal serine/threonine residues of IRF-3 and IRF-7 by TBK1 and/or IKKɛ is essential for inducing IFN-A/B gene expression (14, 75). Thus, IRF-3 and IRF-7 display similarities regarding their virus-induced phosphorylation, dimerization, and nuclear transport (23, 42, 43, 51). These factors differ in their expression levels and their interaction with DNA and transcriptional coactivators CBP and p300 (62, 63, 69, 77). IRF-3 is constitutively expressed in all cell types, whereas the expression of IRF-7 is constitutive in lymphoid cells and certain dendritic cell subsets but is also induced by IFN-α/β produced during viral infection (73). Most of the data on the individual roles of IRF-3 and IRF-7 in differential IFN-A gene regulation derive from in vitro studies of mice. They indicate that maximal virus-induced transcription of the mouse IFN-A4 gene requires the presence of three IRF elements located in the virus-responsive element A4 (VRE-A4), delimited to a region (positions −120 to −40) of the IFN-A4 gene promoter (2, 5). IRF-3 and IRF-7 display differential specificity and affinity for these IRF elements (B, C, and D modules) and determine the expression levels of the IFN-A4 gene following virus infection (9). Among these three modules, the C module specifically binds IRF-3 but is a weak site for IRF-7 interaction; the B and D modules are primarily required for IRF-7 binding and transactivation. The mouse IFN-A11 gene is poorly induced by virus infection because of nucleotide substitutions that disrupt both the C and D modules in the promoter, thus preventing IRF-3 and IRF-7 binding and activation (8, 56, 57). It was also suggested that IRF-3 ensures the initial activation of IFN-A4 and IFN-B genes in mouse embryonic fibroblasts and that in a second step, it participates in the amplification of the IFN-A4 and IFN-B genes and transactivation of other IFN-A genes, in cooperation with IRF-7 (50). All together, these data suggest that subtle differences in the cognate binding sites present in target promoters determine the magnitude of the IFN-A gene response to IRF-3 or IRF-7.In this study, we analyzed the relative contribution of IRF-3 and IRF-7 to virus-induced expression of human IFN-A subtypes. Real-time reverse transcription-quantitative PCR (qPCR) data, together with chromatin immunoprecipitation (ChIP) assays and promoter activation analyses, demonstrate that differential recruitment of IRF-3 and/or IRF-7 to the IFN-A promoter modules dictates the relative levels of IFN-A gene activation.