IRF9: a mode switch of interferon-induced transcription

Interferons (IFN) are peptide mediators (cytokines) of the immune system. They are released in infected organisms. Whereas type I interferons (IFN-I) are made by most, if not all infected cells, the interferon- (IFN is released by T cells, or ontologically related cells such as NK and NKT cells. Type I IFN are mainly antiviral cytokines, the main task of IFN is to activate macrophages. Macrophages are effector cells that ingest and kill microorganisms. To activate macrophages for enhanced antimicrobial activity IFN must stimulate intracellular signals that reprogram the genome to transcribe a large set of genes de novo. The corresponding products allow the macrophage to progress from a resting into an activated immunological state. Gene expression in response to IFN is induced by transcription factors belonging to the signal transducer and activator of transcription (STAT) family. In their canonical signaling pathways the IFN receptor complexes with their associated Janus protein tyrosine kinases (Jaks) respond to ligand binding by phosphorylating STAT transcription factors on tyrosine, thus causing it them to dimerize, translocate to the nucleus and activate transcription. In canonical pathways IFN-I cause formation of transcription factor ISGF3, a STAT1-STAT2 heterodimer associated with a third subunit, the interferon regulatory factor 9 (IRF9). By contrast, IFN causes assembly of the gamma-interferon-activated factor (GAF), a STAT1 homodimer. However, several reports in the recent literature suggest noncanonical STAT complexes participating in the transcriptional response to IFN Our recent results suggest an important role for IRF9 as subunit of these noncanonical transcription factors. The proposed experiments aim at clarifying how noncanonical complexes containing IRF9 exert influence on IFN-induced transcription and to identify their target genes as well as their interacting (STAT) proteins. We will thus provide a mechanistic explanation for the impact of IRF9 particularly on IFN responses which do not require IRF9 in the canonical pathway. In addition we will investigate antimicrobial immune responses in IRF9-deficient macrophages and mice. The aim of these experiments is to clarify to what facet of IFN-mediated immunity requires the presence of IRF9. Together our findings will increase the understanding of an essential immune mechanism for the clearance of microbial infections.

First-line defense against microorganisms requires the activation of macrophages for enhanced microbial killing. This is effected by IFNg, a cytokine belonging to the larger family of interferons (IFN) that includes the type I IFN (IFN-I, mainly IFNa and IFNb), IFNg or type II IFN and the IFNl or type III IFN. The different IFN types bind to different cell surface receptors that all reprogram the transcriptome of their target cells via JAK-STAT signal transduction. According to the prevailing view, the IFN-I and IFNg receptors with their associated Janus kinases (JAKs) produce activated STAT1 and STAT2 that heterodimerize and associate with a third subunit, IRF9, to form transcription factor ISGF3. The binding site for ISGF3 in target gene promoters is the ISRE sequence element. Contrasting IFN-I and IFNl, IFNg causes its receptor complex to activate STAT1 alone. STAT1 dimerizes to form transcription factor GAF. The promoter binding site for GAF is a sequence element designated GAS. Our work, funded by the FWF through project P 30992-B28, corrects and modifies the current understanding of IFN signaling in significant ways. First, we show that immune homeostasis includes low-level expression of interferon-induced genes, mediated by a complex of STAT2 and IRF9. This complex is preassembled independently of IFN receptor signaling and localized in the cell nucleus. Treatment with interferon causes a switch towards gene control by the complete ISGF3 complex, which forms on DNA. Second, ISGF3 shows a large participation in the response to IFNg, revising the concept of IFNg receptor signaling. The participation of ISGF3 in gene control is shown via an IRF9 subunit requirement for the the expression of a large fraction of interferon-induced genes and by IFNg-stimulated association of the complete ISGF3 complex with control regions of (promoters) of interferon-induced genes. In further experiments we determined the requirement for the STAT2/IRF9 or ISGF3 complexes for the generation of an open chromatin configuration at interferon-induced genes. The role of STAT2/IRF9 complexes in this process is minor, if any, but ISGF3 is required for chromatin opening at many genes, including some that are not directly stimulated to be expressed by interferon. Consistent with gene expression data, the ISGF3 requirement for chromatin opening was seen when transcription control was exerted by IFN-I and IFNg receptor signals. Taken together our data identify the STAT2/IRF9 'light' version of the ISGF3 complex as a mediator of immune homeostasis and reveal an important role of the complete ISGF3 complex as regulator of IFN-induced gene transcription.