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Sustained oscillations of NF-kappaB produce distinct genome scanning and gene expression profiles.

ABSTRACT: NF-kappaB is a prototypic stress-responsive transcription factor that acts within a complex regulatory network. The signaling dynamics of endogenous NF-kappaB in single cells remain poorly understood. To examine real time dynamics in living cells, we monitored NF-kappaB activities at multiple timescales using GFP-p65 knock-in mouse embryonic fibroblasts. Oscillations in NF-kappaB were sustained in most cells, with several cycles of transient nuclear translocation after TNF-alpha stimulation. Mathematical modeling suggests that NF-kappaB oscillations are selected over other non-oscillatory dynamics by fine-tuning the relative strengths of feedback loops like IkappaBalpha. The ability of NF-kappaB to scan and interact with the genome in vivo remained remarkably constant from early to late cycles, as observed by fluorescence recovery after photobleaching (FRAP). Perturbation of long-term NF-kappaB oscillations interfered with its short-term interaction with chromatin and balanced transcriptional output, as predicted by the mathematical model. We propose that negative feedback loops do not simply terminate signaling, but rather promote oscillations of NF-kappaB in the nucleus, and these oscillations are functionally advantageous.

SUBMITTER: Sung MH

PROVIDER: S-EPMC2747007 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Sustained oscillations of NF-kappaB produce distinct genome scanning and gene expression profiles.

Sung Myong-Hee MH Salvatore Luigi L De Lorenzi Rossana R Indrawan Anindya A Pasparakis Manolis M Hager Gordon L GL Bianchi Marco E ME Agresti Alessandra A

PloS one 20090929 9

NF-kappaB is a prototypic stress-responsive transcription factor that acts within a complex regulatory network. The signaling dynamics of endogenous NF-kappaB in single cells remain poorly understood. To examine real time dynamics in living cells, we monitored NF-kappaB activities at multiple timescales using GFP-p65 knock-in mouse embryonic fibroblasts. Oscillations in NF-kappaB were sustained in most cells, with several cycles of transient nuclear translocation after TNF-alpha stimulation. Mat ...[more]

PMID: 19787057

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Project description:BackgroundThe nuclear factor-kappaB (NF-kappaB) family of transcription factors plays a role in a wide range of cellular processes including the immune response and cellular growth. In addition, deregulation of the NF-kappaB system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF-kappaB activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-kappaB-induced genes is regulated by differential dynamics of single NF-kappaB subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-kappaB activation have been observed in response to the cytokine tumor necrosis factor alpha (TNFalpha), little is known about the occurrence of oscillations in response to bacterial infections.ResultsTo quantitatively assess NF-kappaB dynamics we generated human and murine monoclonal cell lines that stably express the NF-kappaB subunit p65 fused to GFP. Furthermore, a high-throughput assay based on automated microscopy coupled to image analysis to quantify p65-nuclear translocation was established. Using this assay, we demonstrate a stimulus- and cell line-specific temporal control of p65 translocation, revealing, for the first time, oscillations of p65 translocation in response to bacterial infection. Oscillations were detected at the single-cell level using real-time microscopy as well as at the population level using high-throughput image analysis. In addition, mathematical modeling of NF-kappaB dynamics during bacterial infections predicted masking of oscillations on the population level in asynchronous activations, which was experimentally confirmed.ConclusionsTaken together, this simple and cost effective assay constitutes an integrated approach to infer the dynamics of NF-kappaB kinetics in single cells and cell populations. Using a single system, novel factors modulating NF-kappaB can be identified and analyzed, providing new possibilities for a wide range of applications from therapeutic discovery and understanding of disease to host-pathogen interactions.

Project description:BackgroundThe cytokine tumor necrosis factor (TNF) initiates tissue inflammation, a process mediated by the NF-kappaB transcription factor. In response to TNF, latent cytoplasmic NF-kappaB is activated, enters the nucleus, and induces expression of inflammatory and anti-apoptotic gene expression programs. Recently it has been shown that NF-kappaB displays two distinct activation modes, monophasic and oscillatory, depending on stimulus duration. Characterization of temporal expression patterns for the NF-kappaB network and determination of those genes under monophasic- or oscillatory control has not been experimentally addressed.ResultsTo identify the kinetics of NF-kappaB-dependent gene expression and determine whether these two types of NF-kappaB translocation modes control distinct gene programs, a detailed kinetic analysis of a validated microarray data set was performed on 74 unique NF-kappaB-dependent genes in response to TNF. Hierarchical clustering identified distinct expression profiles termed the "Early", "Middle", "Late" response groups, peaking 1, 3, and 6 h after stimulation, respectively. These expression patterns were validated by Quantitative Real Time PCR (Q-RT-PCR) and NF-kappaB binding was demonstrated by chromatin immunoprecipitation (ChIP) assays. Each response group was mapped to its molecular function; this analysis indicated that the Early group encodes cytokines or negative regulators of the IKK-NF-kappaB pathway, and the Late group encodes cell surface receptors, adhesion molecules and signal adapters. That similar coordinated sequential cascades of gene expression were also seen in response to stimulation by the cytokine IL-1, and expression patterns observed in MRC-5 fibroblasts indicated that the epithelial NF-kappaB program is relatively stimulus- and cell type-independent. Bioinformatic analysis of the Early and Late gene promoters indicates that although both groups contain similar patterns of NF-kappaB-binding sites, only the Early gene promoters contain NF-kappaB-binding sites located in phylogenetically conserved domains. Stimulation protocols designed to produce either monophasic or oscillatory NF-kappaB activation modes showed that the oscillatory mode is required only for expression of the Late genes.ConclusionThis analysis provides important insights into the TNF-regulated genetic response program in epithelial cells, where NF-kappaB controls sequential expression patterns of functionally distinct genes that depend on its oscillatory activation mode.

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Sustained oscillations of NF-kappaB produce distinct genome scanning and gene expression profiles.

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Sustained oscillations of NF-kappaB produce distinct genome scanning and gene expression profiles.

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