IRF1 is a context-dependent homeostatic gatekeeper of basal immunity and antiviral readiness

Interferon regulatory factor 1 (IRF1) plays a pivotal role in interferon (IFN) signaling. Here, we dissect the impact of IRF1 on gene transcription regulation in HeLa cells, by targeted knockout (KO) or overexpression of IRF1. IRF1 KO partially diminished IFN-γ but not IFN-β induced gene regulation. IRF1 KO did show a homeostatic role in basal transcript abundance, including increasing the abundance of antiviral gene transcripts, apparently through increased expression of other IRF genes. IRF1 overexpression induced potent antiviral protection, which is mediated by secretion of type I IFN proteins, particularly of IFN-α subtypes, which expression is driven by IRF1. This paracrine effect was confirmed by transcriptomics, cytokine profiling, and mass spectrometry. Surprisingly, antiviral protection was observed also in JAK1 KO or ruxolitinib-treated cells but not in type I IFN receptor KO cells, suggesting the involvement of noncanonical signaling pathways. Hierarchical clustering of RNA-seq data revealed distinct IFN-independent gene clusters activated or repressed by IRF1, including pathways related to adaptive immunity and T cell function. Using protein-binding microarrays and predictive modeling, we generated an energy-normalized binding matrix for IRF1, enabling sequence-specific prediction of promoter-binding affinities beyond classical consensus motifs. This approach allows estimation of IRF1-binding potential across diverse genomic contexts as validated for the IFIT2 gene promoter by a reporter assay. Evaluating the biological significance of our study, we show that IRF1 abundance varies by 10,000-fold between cell lines, with positive correlations of IRF1 with the abundance of gene transcripts involved in antiviral and immune-driving activities.