Limits...
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
Perturbations of NF-κB oscillations by inhibiting either shuttling or IκBα re-synthesis cause opposite defects in characteristic genome-scanning activity of p65.(A) The line FRAP protocol. The line FRAP assay allowed photodamage-free in vivo analysis of the dim cells by limiting the laser scan area to a line. The white line is the scanning path of the confocal microscope. Segment EF represents the bleaching region (5 µm). Segment AB represents the background while CD is used to correct for the bleaching during imaging in the double normalization procedure (see Methods). This example shows a GFP-p65 knock-in cell co-treated with TNF-α and CHX for 2.5 hrs. In this condition, p65 mobility is the lowest (see panel B). (B) This image represents the line FRAP output from the cell in (A). The x axis is the laser scanning path. Each repetition of the line scanning appears as a line along the time axis. The bleached region is visible as a solid green rectangle. The dark area below the bleached region is indicative of the slow recovery. (C) Line FRAP experiments were performed at the specified times on cells treated with TNF-α (top panel) and on cells co-treated with TNF-α and LMB or CHX (bottom left and right panels). The recovery curves represent fitting of the raw data by bi-exponential curves and *** indicates a statistically significant difference (p<0.001) according to a permutation test (see Methods). The reference profile for all comparisons was that from the first p65 cycle after TNF-α (red curve, top panel). The arrows indicate the shift of the curves over time.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2747007&req=5

pone-0007163-g005: Perturbations of NF-κB oscillations by inhibiting either shuttling or IκBα re-synthesis cause opposite defects in characteristic genome-scanning activity of p65.(A) The line FRAP protocol. The line FRAP assay allowed photodamage-free in vivo analysis of the dim cells by limiting the laser scan area to a line. The white line is the scanning path of the confocal microscope. Segment EF represents the bleaching region (5 µm). Segment AB represents the background while CD is used to correct for the bleaching during imaging in the double normalization procedure (see Methods). This example shows a GFP-p65 knock-in cell co-treated with TNF-α and CHX for 2.5 hrs. In this condition, p65 mobility is the lowest (see panel B). (B) This image represents the line FRAP output from the cell in (A). The x axis is the laser scanning path. Each repetition of the line scanning appears as a line along the time axis. The bleached region is visible as a solid green rectangle. The dark area below the bleached region is indicative of the slow recovery. (C) Line FRAP experiments were performed at the specified times on cells treated with TNF-α (top panel) and on cells co-treated with TNF-α and LMB or CHX (bottom left and right panels). The recovery curves represent fitting of the raw data by bi-exponential curves and *** indicates a statistically significant difference (p<0.001) according to a permutation test (see Methods). The reference profile for all comparisons was that from the first p65 cycle after TNF-α (red curve, top panel). The arrows indicate the shift of the curves over time.

Mentions: We used a non-invasive optimized line FRAP technique [23] (Fig. 5A, B) in conjunction with real time monitoring, and found that p65 mobility during peak of nuclear translocation did not change from early to late cycles of TNF-α induced oscillations (Fig. 5C, top panel). This result implies that p65 in the late translocation cycles is capable of diffusing on and interacting with the genome as effectively as the first p65 molecules activated after TNF-α. This is remarkable given the numerous feedback and post-translational mechanisms that act upon p65 to attenuate its activity following stimuli.


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)

Perturbations of NF-κB oscillations by inhibiting either shuttling or IκBα re-synthesis cause opposite defects in characteristic genome-scanning activity of p65.(A) The line FRAP protocol. The line FRAP assay allowed photodamage-free in vivo analysis of the dim cells by limiting the laser scan area to a line. The white line is the scanning path of the confocal microscope. Segment EF represents the bleaching region (5 µm). Segment AB represents the background while CD is used to correct for the bleaching during imaging in the double normalization procedure (see Methods). This example shows a GFP-p65 knock-in cell co-treated with TNF-α and CHX for 2.5 hrs. In this condition, p65 mobility is the lowest (see panel B). (B) This image represents the line FRAP output from the cell in (A). The x axis is the laser scanning path. Each repetition of the line scanning appears as a line along the time axis. The bleached region is visible as a solid green rectangle. The dark area below the bleached region is indicative of the slow recovery. (C) Line FRAP experiments were performed at the specified times on cells treated with TNF-α (top panel) and on cells co-treated with TNF-α and LMB or CHX (bottom left and right panels). The recovery curves represent fitting of the raw data by bi-exponential curves and *** indicates a statistically significant difference (p<0.001) according to a permutation test (see Methods). The reference profile for all comparisons was that from the first p65 cycle after TNF-α (red curve, top panel). The arrows indicate the shift of the curves over time.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747007&req=5

pone-0007163-g005: Perturbations of NF-κB oscillations by inhibiting either shuttling or IκBα re-synthesis cause opposite defects in characteristic genome-scanning activity of p65.(A) The line FRAP protocol. The line FRAP assay allowed photodamage-free in vivo analysis of the dim cells by limiting the laser scan area to a line. The white line is the scanning path of the confocal microscope. Segment EF represents the bleaching region (5 µm). Segment AB represents the background while CD is used to correct for the bleaching during imaging in the double normalization procedure (see Methods). This example shows a GFP-p65 knock-in cell co-treated with TNF-α and CHX for 2.5 hrs. In this condition, p65 mobility is the lowest (see panel B). (B) This image represents the line FRAP output from the cell in (A). The x axis is the laser scanning path. Each repetition of the line scanning appears as a line along the time axis. The bleached region is visible as a solid green rectangle. The dark area below the bleached region is indicative of the slow recovery. (C) Line FRAP experiments were performed at the specified times on cells treated with TNF-α (top panel) and on cells co-treated with TNF-α and LMB or CHX (bottom left and right panels). The recovery curves represent fitting of the raw data by bi-exponential curves and *** indicates a statistically significant difference (p<0.001) according to a permutation test (see Methods). The reference profile for all comparisons was that from the first p65 cycle after TNF-α (red curve, top panel). The arrows indicate the shift of the curves over time.
Mentions: We used a non-invasive optimized line FRAP technique [23] (Fig. 5A, B) in conjunction with real time monitoring, and found that p65 mobility during peak of nuclear translocation did not change from early to late cycles of TNF-α induced oscillations (Fig. 5C, top panel). This result implies that p65 in the late translocation cycles is capable of diffusing on and interacting with the genome as effectively as the first p65 molecules activated after TNF-α. This is remarkable given the numerous feedback and post-translational mechanisms that act upon p65 to attenuate its activity following stimuli.

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