The IRF regulatory network in innate immune training of macrophages - Macrophages play vital roles as sentinel cells of the immune system to coordinate immune responses in response to pathogen exposure. Macrophage responses are shaped by their exposure history. We and other have shown that when exposed to pathogens or cytokines, macrophages remodel the epigenome through formation of de novo enhancers. This epigenomic reprogramming produces innate immune “training” and alters responses to subsequent stimuli. “Trained” macrophages may have increased capacity to fight infection, but when dysregulated, may also produce unwanted inflammation. Thus, it is critical to develop a mechanistic understanding of how epigenomic remodeling and innate immune training occur. We have recently described how the transcription factor NF-κB produces de novo enhancers in a stimulus-specific manner (Science, 372, pp.1349-1353). Here, we extend those findings to study the roles of the interferon- regulatory factors (IRFs) and STATs, focusing on IRF1, IRF3, IRF7, ISGF3 and GAF. Their regulation is dynamic and interdependent and they all bind the same DNA sequence. Thus, how their functional specificity is regulated is not intuitive. Our studies focus on bone-marrow-derived and tissue-resident macrophages. HYPOTHESIS: IRF/STAT family members remodel the epigenome and chromosome organization of macrophages in a combinatorial manner; each factor plays a distinct role, working sequentially to reprogram gene expression responses to subsequent stimuli. In Aim 1 we will dissect the roles of innate immune IRFs in de novo enhancer formation, using H3K4me1 ChIP- seq and ATAC-seq after stimulation with a systematic panel of ligands and genetic knockouts. Based on our preliminary results we hypothesize that IRF1 is a critical pioneer factor. We will test this hypothesis in vitro and in vivo with peritoneal macs and Kupffer cells In Aim 2 we will dissect how IRF/STATE chromatin remodeling affects later gene expression in response to subsequent endotoxin challenge. We distinguish between persistently activated genes, potentiated gene responses, and tolerized genes, and identify distinct functions by IRFs in these regulatory strategies. In Aim 3 we will connect the de novo enhancers of Aim 1 and the gene expression training of Aim 2, by leveraging recent advances from the 4D Nucleome Consortium to predict how IRF-dependent de novo enhancers mediate large-scale chromosomal remodeling to move immune genes closer to or away from nuclear speckles, so-called transcription factories. We will test these predictions with CRISPR-edited IRF enhancers.
