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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.

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Periodic cycles in TNF-α induced NF-κB oscillations are sustained in most cells.(A) 79% of the cells (n = 95) had sustained oscillations in nuclear p65. Ten temporal profiles from TNF-α treated cells are shown, with eight oscillating (left 4 columns) and two non-oscillating (last column) cells. The lower panel is from control time lapse data where cells were followed by time lapse microscopy without TNF-α treatment. The red line at ratio 1 indicates the state where the nuclear and the total mean intensities are the same. (B) The distribution of the period identified by Fourier analysis (see Methods).
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pone-0007163-g002: Periodic cycles in TNF-α induced NF-κB oscillations are sustained in most cells.(A) 79% of the cells (n = 95) had sustained oscillations in nuclear p65. Ten temporal profiles from TNF-α treated cells are shown, with eight oscillating (left 4 columns) and two non-oscillating (last column) cells. The lower panel is from control time lapse data where cells were followed by time lapse microscopy without TNF-α treatment. The red line at ratio 1 indicates the state where the nuclear and the total mean intensities are the same. (B) The distribution of the period identified by Fourier analysis (see Methods).

Mentions: Figure 1 (C, center plot in D) shows an example of a time lapse experiment after TNF-α stimulation where we quantify the nuclear:total ratio of GFP-p65. The long-term dynamics of p65 in individual single cells ranged from 1∼2 cycles to dramatic oscillations with 6∼7 cycles. A minority of cells had a single cycle with subsequent minor and irregular fluctuations (8 oscillating cells and 2 non-oscillators are shown in Fig. 2A). Most cells showed multiple striking peaks; later peaks had gradually decreasing amplitudes but were clearly discernible. Often the p65 flux out of the nucleus between the periodic cycles was so efficient as to reduce the nuclear level to a basal state. We subjected the time course data from 95 cells to Fourier analysis (Fig. 1D) and found that the majority of the cells (79%) oscillated with a somewhat variable period with a median value of 2.2 hours (n = 75, Fig. 2B). Cells that were not treated with TNF-α had no nuclear translocation of p65 as expected (Fig. 2A bottom panel). Although we classified the time courses into two categories by the presence/absence of a strong periodic component, we cannot rule out the possibility that even the ‘non-oscillating’ cells may have weak oscillations which can nevertheless mediate some signaling effect.


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)

Periodic cycles in TNF-α induced NF-κB oscillations are sustained in most cells.(A) 79% of the cells (n = 95) had sustained oscillations in nuclear p65. Ten temporal profiles from TNF-α treated cells are shown, with eight oscillating (left 4 columns) and two non-oscillating (last column) cells. The lower panel is from control time lapse data where cells were followed by time lapse microscopy without TNF-α treatment. The red line at ratio 1 indicates the state where the nuclear and the total mean intensities are the same. (B) The distribution of the period identified by Fourier analysis (see Methods).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747007&req=5

pone-0007163-g002: Periodic cycles in TNF-α induced NF-κB oscillations are sustained in most cells.(A) 79% of the cells (n = 95) had sustained oscillations in nuclear p65. Ten temporal profiles from TNF-α treated cells are shown, with eight oscillating (left 4 columns) and two non-oscillating (last column) cells. The lower panel is from control time lapse data where cells were followed by time lapse microscopy without TNF-α treatment. The red line at ratio 1 indicates the state where the nuclear and the total mean intensities are the same. (B) The distribution of the period identified by Fourier analysis (see Methods).
Mentions: Figure 1 (C, center plot in D) shows an example of a time lapse experiment after TNF-α stimulation where we quantify the nuclear:total ratio of GFP-p65. The long-term dynamics of p65 in individual single cells ranged from 1∼2 cycles to dramatic oscillations with 6∼7 cycles. A minority of cells had a single cycle with subsequent minor and irregular fluctuations (8 oscillating cells and 2 non-oscillators are shown in Fig. 2A). Most cells showed multiple striking peaks; later peaks had gradually decreasing amplitudes but were clearly discernible. Often the p65 flux out of the nucleus between the periodic cycles was so efficient as to reduce the nuclear level to a basal state. We subjected the time course data from 95 cells to Fourier analysis (Fig. 1D) and found that the majority of the cells (79%) oscillated with a somewhat variable period with a median value of 2.2 hours (n = 75, Fig. 2B). Cells that were not treated with TNF-α had no nuclear translocation of p65 as expected (Fig. 2A bottom panel). Although we classified the time courses into two categories by the presence/absence of a strong periodic component, we cannot rule out the possibility that even the ‘non-oscillating’ cells may have weak oscillations which can nevertheless mediate some signaling effect.

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