1. Physiological circulating steroid levels are often pulsatile (ultradian) in nature. Utilizing the glucocorticoid receptor (GR) signaling system, we uncovered striking spatiotemporal relationships between receptor loading, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We found that hormone-induced DHSs were enriched within +/-50 kb of GR-responsive genes and displayed a broad spectrum of lifetimes upon hormone withdrawal. These lifetimes dictate the strength of the DHS interactions with gene targets and contribute to gene regulation from a distance. Our results demonstrate that pulsatile and constant hormone stimulations induce unique, treatment-specific patterns of gene and regulatory element activation. These modes of activation have implications for corticosteroid function in vivo and for steroid therapies in various clinical settings. 2. Through live, single-cell imaging, we monitored both the dynamics of nuclear factor kB (NF-kB) signaling and inflammatory cytokine transcription in macrophages exposed to the bacterial product lipopolysaccharide (LPS). Our analysis revealed a previously uncharacterized positive feedback loop involving induction of the expression of Rela, which encodes the RelA (p65) NF-kB subunit. This positive feedback loop rewired the regulatory network when cells were exposed to LPS above a distinct concentration. Paradoxically, this rewiring of NF-kB signaling in macrophages (a myeloid cell type) required the transcription factor Ikaros, which promotes the development of lymphoid cells. Mathematical modeling and experimental validation showed that the RelA positive feedback overcame existing negative feedback loops and enabled cells to discriminate between different concentrations of LPS to mount an effective innate immune response only at higher concentrations. We conclude that this switching in the relative dominance of feedback loops (feedback dominance switching) may be a general mechanism in immune cells to integrate opposing input signals.
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