IRF1 cooperates with ISGF3 or GAF to form innate immune de novo enhancers

Macrophages exposed to immune stimuli reprogram their epigenomes to alter their subsequent functions. Endotoxin exposure causes widespread nucleosome remodeling and formation of thousands of de novo enhancers. However, it remains unclear how signal-dependent transcription factors collaborate to remodel the epigenome. Here, we dissected the regulatory logic by which the network of interferon regulatory factors (IRFs) catalyzes the opening of chromatin and the formation of de novo enhancers. We found that endotoxin-activated IRF3 is directly responsible for the induction of few de novo enhancers but is broadly required indirectly through activation of type I interferon-induced ISGF3. However, ISGF3 has limited capacity to initiate enhancer formation by itself – it must cooperate with IRF1, particularly at locations where the chromatin is less accessible. At these locations, IRF1 is required for the initial opening of chromatin, with ISGF3 extending accessibility and promoting the deposition of H3K4me1, marking poised enhancers. Because IRF1 expression must be induced by NFκB, IRF-regulated enhancers require that both branches of the innate immune signaling network, IRF3 and NFκB, are activated. However, type II interferon, typically produced by T-cells, may also induce IRF1 via the STAT1 homodimer GAF. We show that in this context, IRF1 is also responsible for opening inaccessible chromatin sites that can then be exploited by GAF to form de novo enhancers. Our results reveal how combinatorial logic gates of IRF1-ISGF3 or IRF1-GAF restrict immune epigenomic memory formation to macrophages exposed to pathogen or IFNγ-secreting T cells, but not bystander macrophages exposed only to transient type I interferon. Human monocyte-derived macropahges were generated with MCSF. After 7 days for maturation in MCSF macrophages were left untimulated or stimulated with either LPS 100ng/mL, interferon-gamma 100ng/mL, interferon-beta 10u/mL, or Pam3CSK4 100ng/mL. After 8 hours stimulation cells were isolated and CUT&Tag for mono-mehtylated Lysine 4 on Histone 3 (H3K4me1) was performed.