Epigenetic Regulation of Inflammatory NF-κB Target Genes by IFN-γ via IRF1

Abstract The regulation of inflammatory gene expression involves complex interactions between transcription factors (TFs), signaling pathways and epigenetic chromatin-mediated mechanisms. This study investigated mechanisms by which by IFN-γ-mediated priming augments TLR-induced expression of NF-κB target genes in primary human monocytes. IFN-γ priming enhanced the expression of signature inflammatory genes such as IL6, TNF, IL1B, and CXCL10 when monocytes were exposed to various TLR agonists. RNA-seq analysis identified genes synergistically activated by IFN-γ and LPS, which were enriched in inflammatory pathways. Similar synergistic activation was observed with the TLR1/2 agonist PAM3CYS, suggesting a shared regulatory mechanism. ATAC-seq analysis revealed that TLR ligands induce IRF1 TF activity independently of IFN-γ. JAK1/2 inhibitor (iJAK) treatment reduced IRF1 expression and protein levels, especially in IFN-γ-treated monocytes, but not in LPS-stimulated monocytes, suggesting LPS-induced IRF1 may compensate for loss of IFN-γ-induced IRF1. We applied CRISPR-Cas9 to knock out IRF1 in primary human monocytes and found loss of IRF1 abrogates synergistic activation of key inflammatory genes, suggesting a pivotal role for IRF1. This genetic data was corroborated by IRF1 CUT&RUN data showing resistance of IRF1 binding to JAK inhibition under (IFN-γ + LPS) costimulated conditions, and co-occupancy of IRF1 binding sites by NF-κB. This study enhances our understanding of inflammatory gene regulation, highlighting IRF1 as a key player and a potential therapeutic target for inflammatory diseases. Competing Interest Statement The authors have declared no competing interest.