title: Complex Regulation Pattern of IRF3 Activation Revealed by a Novel Dimerization Reporter System.
abstract: Induction of type I IFN (IFN-I) is essential for host antiviral immune responses. However, IFN-I also plays divergent roles in antibacterial immunity, persistent viral infections, autoimmune diseases, and tumorigenesis. IFN regulatory factor 3 (IRF3) is the master transcription factor that controls IFN-I production via phosphorylation-dependent dimerization in most cell types in response to viral infections and various innate stimuli by pathogen-associated molecular patterns (PAMPs). To monitor the dynamic process of IRF3 activation, we developed a novel IRF3 dimerization reporter based on bimolecular luminescence complementation (BiLC) techniques, termed the IRF3-BiLC reporter. Robust induction of luciferase activity of the IRF3-BiLC reporter was observed upon viral infection and PAMP stimulation with a broad dynamic range. Knockout of TANK-binding kinase 1, the critical upstream kinase of IRF3, as well as the mutation of serine 386, the essential phosphorylation site of IRF3, completely abolished the luciferase activity of IRF3-BiLC reporter, confirming the authenticity of IRF3 activation. Taken together, these results demonstrated that the IRF3-BiLC reporter is a highly specific, reliable, and sensitive system to measure IRF3 activity. Using this reporter system, we further observed that the temporal pattern and magnitude of IRF3 activation induced by various PAMPs are highly complex with distinct cell type-specific characteristics, and IRF3 dimerization is a direct regulatory node for IFN-α/β receptor-mediated feed-forward regulation and crosstalk with other pathways. Therefore, the IRF3-BiLC reporter has multiple potential applications, including mechanistic studies as well as the identification of novel compounds that can modulate IRF3 activation.
authors.affiliation: Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275, China;
authors.forename: Zining
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275, China;
authors.forename: Jingyun
authors.initials: J
authors.lastname: Ji
authors.affiliation: Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275, China;
authors.forename: Di
authors.initials: D
authors.lastname: Peng
authors.affiliation: Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Suzhou Institute of Systems Medicine, Suzhou, Jiangsu 215123, China; and Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095.
authors.forename: Feng
authors.initials: F
authors.lastname: Ma
authors.affiliation: Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Suzhou Institute of Systems Medicine, Suzhou, Jiangsu 215123, China; and Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095.
authors.forename: Genhong
authors.initials: G
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275, China; Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Suzhou Institute of Systems Medicine, Suzhou, Jiangsu 215123, China; and fqin1@foxmail.com.
authors.forename: F Xiao-Feng
authors.initials: FX
authors.lastname: Qin
mesh_terms: D064113:CRISPR-Cas Systems; D002460:Cell Line; D015536:Down-Regulation; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism*; D007370:Interferon Type I / Q000276:immunology*; D016328:NF-kappa B / Q000378:metabolism; D000069452:Pathogen-Associated Molecular Pattern Molecules / Q000276:immunology; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D053633:Receptor, Interferon alpha-beta / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014777:Virus Diseases / Q000276:immunology
chemical_list: C507964:IFNAR1 protein, human; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D007370:Interferon Type I; D016328:NF-kappa B; D000069452:Pathogen-Associated Molecular Pattern Molecules; D053633:Receptor, Interferon alpha-beta; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human