Investigations of the DNA-binding activity and gene regulatory properties of IRF3, IRF5, and IRF7 homodimers

Abstract

The innate immune response is an essential component of the mammalian immune system that responds rapidly to pathogens. This response to pathogens is initiated by the detection of pathogen associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs). PRR signaling activates antipathogen gene programs via transcription factors (TFs) such as the interferon regulatory factors (IRFs). IRF3, IRF5, and IRF7 (IRF3/5/7) are key signal-dependent TFs that have overlapping, yet distinct, roles in the mammalian response to pathogens. To examine the role that DNA-binding specificity plays in delineating IRF3/5/7-specific gene regulation, we used protein-binding microarrays (PBMs) to characterize the DNA binding of IRF3/5/7 homodimers. We identified both common and dimer-specific DNA binding sites, and show that DNA-binding differences can translate into dimer-specific gene regulation. Central to the antiviral response, IRF3/5/7 regulate type I interferon (IFN) genes. We show that IRF3 and IRF7 bind to many interferon-stimulated response element (ISRE)-type sites in the virus-response elements (VREs) of IFN promoters. However, strikingly, IRF5 does not bind the VREs, suggesting evolutionary selection against IRF5 homodimer binding. Mutational analysis identified a a critical specificity-determining residue that inhibits IRF5 binding to the ISRE-variants present in the IFN gene promoters. Integrating PBM and reporter gene data we find that both DNA-binding affinity and affinity-independent mechanisms determine the transcriptional activation ability of DNA-bound IRF dimers, suggesting that DNA-based allostery plays a role in IRF binding site function. To assay the sequence determinants of IRF-dependent transcriptional regulation, we propose using a modified massively parallel reporter assay (MPRA). The proposed MPRA leverages unique molecular identifiers to improve the accuracy of reporter gene quantitation. This work provides new insights into the role and limitations of DNA-binding affinity in delineating IRF3/5/7-specific gene expression and lays groundwork for further understanding the complexities of IRF-dependent transcriptional regulation of innate immune genes.