Partner-regulated interaction of IFN regulatory factor 8 with chromatin visualized in live macrophages

Abstract

IFN regulatory factor (IRF) 8 is a transcription factor that directs macrophage differentiation. By fluorescence recovery after photobleaching, we visualized the movement of IRF8-GFP in differentiating macrophages. Recovery data fitted to mathematical models revealed two binding states for IRF8. The majority of IRF8 was highly mobile and transiently interacted with chromatin, whereas a small fraction of IRF8 bound to chromatin more stably. IRF8 mutants that did not stimulate macrophage differentiation showed a faster recovery, revealing little interaction with chromatin. A macrophage activation signal by IFN-␥͞LPS led to a global slowdown of IRF8 movement, leading to increased chromatin binding. In fibroblasts where IRF8 has no known function, WT IRF8 moved as fast as the mutants, indicating that IRF8 does not interact with chromatin in these cells. However, upon introduction of IRF8 binding partners, PU.1 and͞or IRF1, the mobility of IRF8 was markedly reduced, producing a more stably bound component. Together, IRF8 -chromatin interaction is dynamic in live macrophages and influenced by partner proteins and immunological stimuli.real-time mobility ͉ transcription factor ͉ fluorescence recovery after photobleaching G ene expression in immune cells is controlled by binding of transcription factors to chromatin targets. A classic view is that active transcription factors stably bind to the chromatinized promoter to drive transcription, a view mostly derived from biochemical studies in vitro. With the advent of live cell technologies, the views on the behavior of transcription factors are changing. Studies by fluorescence recovery after photobleaching (FRAP) of many nuclear proteins show that they are highly mobile and only transiently interact with chromatin (1, 2). Proteins showing rapid mobility include general transcription factors, chromatin modifiers, and DNA replication factors (3-9). Some DNA-specific transcription factors, such as nuclear hormone receptors, are also highly mobile in live nuclei (10-12). In addition, Stat1 (signal transducer and activator of transcription 1), a transcription factor that regulates IFN responses, is shown to be mobile before and after translocation into the nucleus (13), although E2F and retinoblastoma protein appear to be more stationary (14). In contrast, core histones, stable components of chromatin, are essentially immobile, showing little recovery after photobleaching (15). A consensus emerging from these studies is that FRAP recovery primarily reflects interactions of nuclear proteins with chromatin and the surrounding genomic DNA (hereafter referred to as chromatin) (16). Nevertheless, photobleaching technologies are still in their early phase of application, and mechanisms regulating FRAP mobility are not fully understood. FRAP mobility might reflect an intrinsic chromatin-binding property of a protein. However, the mobility may be influenced by other factors, such as soluble molecular constituents and structural components of the nucleus. In addition, most transcripti...

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“…It has been well shown that IFN-g binding to its receptor triggers signal transducers and activators of transcription 1 (STAT1) phosphorylation and dimerization. The dimerized STAT1 then translocates to the nucleus and binds directly to the IRF8 promoter element (the GAS element) to activate IRF8 transcription (37,38,48). Because IFN-g up-regulates Fas expression in SW620 cells, it is reasonable to assume that the function of STAT1 in the IFN-g receptor-initiated signaling pathway is not altered.…”

IRF8 inhibits C/EBPα activity to restrain mononuclear phagocyte progenitors from differentiating into neutrophils

“…4e-g), which is consistent with the results in 293T cells. Of note, neither IRF8 K79E nor IRF8 R289E induces monocyte differentiation-related IRF8 target genes 22,29 , suggesting that the modes of IRF8 action in promoting monocyte differentiation and inhibiting neutrophil differentiation are indeed distinct.…”

“…IRF8 K79E is defective in DNA binding, while IRF8 R289E cannot interact with PU.1 and other IRFs 28 . Neither mutant is capable of inducing macrophage and DC differentiation 10,28,29 . Interestingly, IRF8 R289E still blocked the transactivation by C/EBPa, whereas IRF8 K79E lost this inhibitory effect, suggesting the involvement of DBD in the inhibition of C/EBPa activity.…”

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“…Mass spectrometry was performed in the Proteomics and Mass Spectrometry Facility of National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. GST pull-down assays and bifluorescence complementation analysis were performed as described (18,19).…”