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.

A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation

“…2C, 2D). As a complementary approach, we also used IRF3-BiLC reporter system developed in our laboratory to more accurately assess the kinetics of IRF3 dimer formation in A549 cells (37). Indeed we found that FBXO17 knockdown enhanced IRF3-dimer luciferase activities induced by SeV, intracellular poly(I:C), and poly(dA:dT), respectively (Supplemental Fig.…”

STING-dependent paracriny shapes apoptotic priming of breast tumors in response to anti-mitotic treatment

“…Transcriptional induction of NOXA by type I IFN may result from the activation of IFN-stimulated response elements in the regulatory region of the human PMAIP1 gene 22 . ChIP assay indeed revealed recruitment of IRF3 to the PMAIP1 promoter in paclitaxel or TNFα/IFNα-treated cells ( Supplementary Fig.…”

The innate sensor ZBP1-IRF3 axis regulates cell proliferation in multiple myeloma

“…Our genome-wide and sequential ChIP assays demonstrate and validate extensive cooccupancy by IRF3 and IRF4 in the myeloma regulatory genome including at the superenhancer of IRF4, with genes involved in cell cycle control being amongst the targets of the IRF3-IRF4 synergy. IRF3 binds chromatin either as a homodimer [46][47][48] or in some cases as heterodimer in association with IRF7 49,50,48 . Similarly, IRF4 may function as a homodimer or in a heterodimeric association with PU.1 or IKZF1 [51][52][53] ; whether IRF3 and IRF4 heterodimerize with each other in myeloma cells remains to be investigated.…”