Summary Synergistic activation of inflammatory cytokine genes by interferon-γ (IFN-γ) and Toll-like receptor (TLR) signaling is important for innate immunity and inflammatory disease pathogenesis. Enhancement of TLR signaling, a previously proposed mechanism, is insufficient to explain strong synergistic activation of cytokine production in human macrophages. Rather, we found that IFN-γ induced sustained occupancy of transcription factors STAT1, IRF-1 and associated histone acetylation at promoters and enhancers at the TNF, IL6 and IL12B loci. This priming of chromatin did not activate transcription, but greatly increased and prolonged recruitment of TLR4-induced transcription factors and RNA polymerase II to gene promoters and enhancers. Priming sensitized cytokine transcription to suppression by Jak inhibitors. Genome-wide analysis revealed pervasive priming of regulatory elements by IFN-γ, and linked coordinate priming of promoters and enhancers with synergistic induction of transcription. Our results provide a synergy mechanism whereby IFN-γ creates a primed chromatin environment to augment TLR-induced gene transcription.

Disruption of the interaction of bromo and extra terminal (BET) proteins with acetylated histones using small molecule inhibitors suppresses Myc-driven cancers and TLR-induced inflammation in mouse models. The predominant mechanism of BET inhibitor action is to suppress BET-mediated recruitment of positive transcription elongation factor b (pTEFb) and thus transcription elongation. We investigated the effects of BET inhibitor I-BET151 on transcriptional responses to TLR4 and TNF in primary human monocytes and also on responses to cytokines IFN-γ, IFN-γ, IL-4 and IL-10 that activate the JAK-STAT signaling pathway and are important for monocyte polarization and inflammatory diseases. I-BET151 suppressed TLR4- and TNF-induced IFN responses by diminishing both autocrine IFN-β expression and transcriptional responses to IFN-β. I-BET151 inhibited cytokine-induced transcription of STAT targets in a gene-specific manner without affecting STAT activation or recruitment. This inhibition was independent of Myc or other upstream activators. Interferon-stimulated gene transcription is regulated primarily at the level of transcription initiation. Accordingly we found that I-BET151 suppressed the recruitment of transcriptional machinery to the CXCL10 promoter and an upstream enhancer. Our findings suggest that BET inhibition reduces inflammation partially through suppressing cytokine activity and expand the understanding of the inhibitory and potentially selective immunosuppressive effects of inhibiting BET proteins.