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

Sung MH, Salvatore L, De Lorenzi R, Indrawan A, Pasparakis M, Hager GL, Bianchi ME, Agresti A - PLoS ONE (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. sungm@mail.nih.gov

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.

Show MeSH
Real time monitoring of GFP-p65 using GFP knock-in MEF and live cell microscopy allows accurate quantification of single cell dynamics of endogenous p65.(A) The population average time course of nuclear NF-κB (red) can show strongly damped oscillation even when the individual cells (black, 20 out of 1000 shown) have sustained oscillatory dynamics. Thousand hypothetical sine waves were generated to have a period slightly varying from 2 hours (15% S.D. in cycle frequency) and linearly decreasing amplitude. The late peaks become unsynchronized, making the average profile appear constant. (B) The knock-in mice have the endogenous p65 locus replaced by GFP-p65 and have wild-type phenotype. (C) A typical time series of a single living cell treated with 10 ng/ml TNF-α and imaged overnight. The low GFP level required special image acquisition setup. The quantification of the GFP intensity data is shown in the time course plot in (D). (D) The time lapse image analysis procedure is shown schematically. The nuclear:total ratio of GFP-p65 plotted is for the cell in (C), where the labeled arrows correspond to the images. The ratio was obtained as the mean nuclear intensity divided by mean cellular intensity (see Methods). The periodogram on right has a single sharp peak and indicates that the estimated period is ∼1.5 hours for this cell.
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pone-0007163-g001: Real time monitoring of GFP-p65 using GFP knock-in MEF and live cell microscopy allows accurate quantification of single cell dynamics of endogenous p65.(A) The population average time course of nuclear NF-κB (red) can show strongly damped oscillation even when the individual cells (black, 20 out of 1000 shown) have sustained oscillatory dynamics. Thousand hypothetical sine waves were generated to have a period slightly varying from 2 hours (15% S.D. in cycle frequency) and linearly decreasing amplitude. The late peaks become unsynchronized, making the average profile appear constant. (B) The knock-in mice have the endogenous p65 locus replaced by GFP-p65 and have wild-type phenotype. (C) A typical time series of a single living cell treated with 10 ng/ml TNF-α and imaged overnight. The low GFP level required special image acquisition setup. The quantification of the GFP intensity data is shown in the time course plot in (D). (D) The time lapse image analysis procedure is shown schematically. The nuclear:total ratio of GFP-p65 plotted is for the cell in (C), where the labeled arrows correspond to the images. The ratio was obtained as the mean nuclear intensity divided by mean cellular intensity (see Methods). The periodogram on right has a single sharp peak and indicates that the estimated period is ∼1.5 hours for this cell.

Mentions: NF-κB is a classical example of a transcription factor subject to negative feedback loops. NF-κB regulates numerous cell signaling processes and its activity is controlled in part by the level of nuclear translocation. In resting cells, the predominant dimer p65∶p50 exists mostly as a cytoplasmic complex bound to its inhibitor IκB proteins. Numerous upstream signals induce degradation of the IκB proteins following phosphorylation by the IκB kinase complex (IKK). This release from latency in the cytoplasm allows active NF-κB to translocate into the nucleus and activate expression of target genes, including several feedback genes [4]. One such target is the Nfkbia gene which codes for IκBα. The re-synthesis of IκBα acts as strong negative feedback, causing the inactivation of NF-κB and its re-localization to the cytoplasm. However, previous biochemical and genetic investigations have indicated that the propensity to oscillate, driven by induction of IκBα, is counteracted by mechanisms that modulate and dampen the oscillation [4], [5], [6], [7]. Observations at least from cell populations show rapid down-regulation of NF-κB after activation. This has led to the view that NF-κB signaling is biphasic, i.e. its activity rises rapidly and is strongly attenuated by feedback inactivation, and an oscillatory behavior after the first cycle of NF-κB activity might be insignificant or even deleterious for cellular function. However, the inherent limitation of kinetic studies using cell populations [1], [3], [8], [9] is that transient activities that are not synchronized from cell to cell cannot be detected even by the most quantitative assays (Fig. 1A) [10], [11].


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

Sung MH, Salvatore L, De Lorenzi R, Indrawan A, Pasparakis M, Hager GL, Bianchi ME, Agresti A - PLoS ONE (2009)

Real time monitoring of GFP-p65 using GFP knock-in MEF and live cell microscopy allows accurate quantification of single cell dynamics of endogenous p65.(A) The population average time course of nuclear NF-κB (red) can show strongly damped oscillation even when the individual cells (black, 20 out of 1000 shown) have sustained oscillatory dynamics. Thousand hypothetical sine waves were generated to have a period slightly varying from 2 hours (15% S.D. in cycle frequency) and linearly decreasing amplitude. The late peaks become unsynchronized, making the average profile appear constant. (B) The knock-in mice have the endogenous p65 locus replaced by GFP-p65 and have wild-type phenotype. (C) A typical time series of a single living cell treated with 10 ng/ml TNF-α and imaged overnight. The low GFP level required special image acquisition setup. The quantification of the GFP intensity data is shown in the time course plot in (D). (D) The time lapse image analysis procedure is shown schematically. The nuclear:total ratio of GFP-p65 plotted is for the cell in (C), where the labeled arrows correspond to the images. The ratio was obtained as the mean nuclear intensity divided by mean cellular intensity (see Methods). The periodogram on right has a single sharp peak and indicates that the estimated period is ∼1.5 hours for this cell.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747007&req=5

pone-0007163-g001: Real time monitoring of GFP-p65 using GFP knock-in MEF and live cell microscopy allows accurate quantification of single cell dynamics of endogenous p65.(A) The population average time course of nuclear NF-κB (red) can show strongly damped oscillation even when the individual cells (black, 20 out of 1000 shown) have sustained oscillatory dynamics. Thousand hypothetical sine waves were generated to have a period slightly varying from 2 hours (15% S.D. in cycle frequency) and linearly decreasing amplitude. The late peaks become unsynchronized, making the average profile appear constant. (B) The knock-in mice have the endogenous p65 locus replaced by GFP-p65 and have wild-type phenotype. (C) A typical time series of a single living cell treated with 10 ng/ml TNF-α and imaged overnight. The low GFP level required special image acquisition setup. The quantification of the GFP intensity data is shown in the time course plot in (D). (D) The time lapse image analysis procedure is shown schematically. The nuclear:total ratio of GFP-p65 plotted is for the cell in (C), where the labeled arrows correspond to the images. The ratio was obtained as the mean nuclear intensity divided by mean cellular intensity (see Methods). The periodogram on right has a single sharp peak and indicates that the estimated period is ∼1.5 hours for this cell.
Mentions: NF-κB is a classical example of a transcription factor subject to negative feedback loops. NF-κB regulates numerous cell signaling processes and its activity is controlled in part by the level of nuclear translocation. In resting cells, the predominant dimer p65∶p50 exists mostly as a cytoplasmic complex bound to its inhibitor IκB proteins. Numerous upstream signals induce degradation of the IκB proteins following phosphorylation by the IκB kinase complex (IKK). This release from latency in the cytoplasm allows active NF-κB to translocate into the nucleus and activate expression of target genes, including several feedback genes [4]. One such target is the Nfkbia gene which codes for IκBα. The re-synthesis of IκBα acts as strong negative feedback, causing the inactivation of NF-κB and its re-localization to the cytoplasm. However, previous biochemical and genetic investigations have indicated that the propensity to oscillate, driven by induction of IκBα, is counteracted by mechanisms that modulate and dampen the oscillation [4], [5], [6], [7]. Observations at least from cell populations show rapid down-regulation of NF-κB after activation. This has led to the view that NF-κB signaling is biphasic, i.e. its activity rises rapidly and is strongly attenuated by feedback inactivation, and an oscillatory behavior after the first cycle of NF-κB activity might be insignificant or even deleterious for cellular function. However, the inherent limitation of kinetic studies using cell populations [1], [3], [8], [9] is that transient activities that are not synchronized from cell to cell cannot be detected even by the most quantitative assays (Fig. 1A) [10], [11].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. sungm@mail.nih.gov

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.

Show MeSH