Transcriptional Profiling of Interferon Regulatory Factor 3 Target Genes: Direct Involvement in the Regulation of Interferon-Stimulated Genes

Abstract

Ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated after virus infection and functions as a key activator of the immediate-early alpha/beta interferon (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-3 (IRF-3 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-3. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-3 5D. Among the genes upregulated by IRF-3 were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-3 directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-␤ enhanceosome, IRF-3 is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.

IRF-3-dependent and augmented target genes during viral infection

“…Activated IRF-3 is sufficient to induce genes such as ISG54 and cig5/ viperin and they were shown previously to be induced by a constitutively active mutation of IRF-3. 21 In contrast, IRF-3 is required but not sufficient to induce expression of the IFN-b gene. 9,20,21 IRF-3 independent and augmented direct response genes Viral infection also stimulated the expression of direct response genes in the absence of IRF-3 (Supplementary Table 10).…”

“…21 In contrast, IRF-3 is required but not sufficient to induce expression of the IFN-b gene. 9,20,21 IRF-3 independent and augmented direct response genes Viral infection also stimulated the expression of direct response genes in the absence of IRF-3 (Supplementary Table 10). Although expression of this set of genes was independent of IRF-3, evaluation of the comparative levels of gene expression in the WT or KO cells revealed two distinct classes.…”

“…44 Other augmented genes include a number of IFN-stimulated genes such as ISG15, in which either activated IRF-3 or activated STAT complexes are sufficient to induce expression. 21,23 It is not uncommon for a gene to be regulated independently by distinct transcription factors. By comparative analysis of RNA expression in the presence or absence of IRF-3 we have identified IRF-3 augmented direct response genes.…”

“…One approach evaluated the effects of a constitutively active IRF-3 mutant. 21 Serine amino acids were replaced with aspartic acid to mimic serine phosphorylation and the active IRF-3 conformation. Only a handful of genes were found to be induced by overexpression of the constitutive IRF-3 mutant and this group did not include IFN.…”

Mitotic progression following DNA damage enables pattern recognition within micronuclei

“…CRISPR-Cas9 knockout of either cytosolic cGAS or STING reduced activation of downstream IRF3 target genes ISG54, ISG56 and the NF-κB target CCL5 post-IR (Fig 3a and Extended data 3a) 17, 18 . Attenuated STAT1 activation and IFITM1 induction was also evident in cGAS-STING knockouts (Fig 3b and Extended data 3c).…”