1. Cell Biology
  2. Computational and Systems Biology
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Dynamic NF-κB and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation

  1. John M Ankers
  2. Raheela Awais
  3. Nicholas A Jones
  4. James Boyd
  5. Sheila Ryan
  6. Antony D Adamson
  7. Claire V Harper
  8. Lloyd Bridge
  9. David G Spiller
  10. Dean A Jackson
  11. Pawel Paszek
  12. Violaine Sée
  13. Michael RH White  Is a corresponding author
  1. Institute of Integrative Biology, United Kingdom
  2. Faculty of Life Sciences, United Kingdom
  3. University of Swansea, United Kingdom
Research Article
Cite this article as: eLife 2016;5:e10473 doi: 10.7554/eLife.10473
18 figures and 4 tables

Figures

NF-κB dynamics following TNFα treatment in HeLa and SK-N-AS cells: Mapping the NF-κB response over the cell cycle in synchronized HeLa cells.

(A,B,C and D) The dynamics of RelA-dsRedxp following 10 ng/ml TNFα treatment in transiently transfected SK-N-AS (A), or following 30 pg/ml TNFα treatment in SK-N-AS, and 10 ng/ml TNFα treatment in HeLa cells (C), and at 30 pg/ml for HeLa (D) cells (n=30 cells analysed per condition). (E) The localization of endogenous RelA in different cell cycle phases, observed by immunocytochemistry at 2 hr (G1/S transition), 4 hr (mid S-phase), post-release from double thymidine block and with 15 min TNFα treatment. (F and G) The dynamics of RelA-dsRedxp in transiently transfected HeLa cells synchronized by a double thymidine block, following 10 ng/ml TNFα treatment at G1/S (F), or passing through S-phase (G) (n=20 cells analysed per condition). (H) Western blot of Ser536phopho-RelA (p-RelA), IκBα, and cyclophilin-A (cyclo-A) levels in synchronized HeLa cells harvested at 1 hr time intervals over the G1/S transition following 15 min treatment with TNFα. Also shown are asynchronous, non-stimulated (ASY NST) and asynchronous, stimulated (ASY ST) controls, harvested at t=0.

https://doi.org/10.7554/eLife.10473.003
Figure 2 with 3 supplements
Mapping the NF-κB response over the cell cycle through virtual synchronization.

(A) Selected images from time-lapse imaging of RelA-dsRedxp transiently expressing Hela cells treated with 10 ng/ml TNFα. (B) Virtual synchronization of HeLa cells treated with 10 ng/ml TNFα. Cells were imaged through two successive divisions (M) allowing correlation of cell cycle timing of TNFα treatment (parameter 1) to RelA dynamics (parameters 2, 3 and 4) and cell cycle duration (parameters 1 plus 5). (C) Representative cells of RelA-dsRedxp dynamics following TNFα treatment in asynchronous cells, then virtually synchronized into G1 (n=115), G1/S (n=32), S (n=52) and G2 (n=38) phases.

https://doi.org/10.7554/eLife.10473.004
Figure 2—figure supplement 1
Analysis of cell cycle duration and G1/S timing in HeLa and SK-N-AS cells.

(A) Time series for FUCCI expression in single representative HeLa and SK-N-AS cells. White arrows mark cells before and after the fluorescence levels were detectable. (B) Analysis of cells in (A), showing the G1/S crossing point in fluorescence levels from reporters of SCF (SKP2) (Orange) and APC (Green) E3 ubiquitin ligase activity. (C) Analysis of the G1/S crossing point and cell cycle duration in populations of HeLa and SK-N-AS cells transfected with FUCCI vectors (n ≥ 30 cells for all conditions).

https://doi.org/10.7554/eLife.10473.005
Figure 2—figure supplement 2
Statistical analysis of NF-κB translocation in HeLa cells at inferred cell cycle stages following 10 ng/ml TNFα stimulation.

(A) Analysis of dynamics of initial RelA-dsRedxp translocation with respect to cell cycle phase, using virtual synchronization in HeLa cells. Data were analyzed using nonparametric Anova analysis with Dunn correction for multiple comparisons. Red lines indicate mean normalised amplitude of NF-κB nuclear translocation for different cell cycle phases, and the population average (dotted line). (B) Analysis of nuclear RelA occupancy assessed in non-synchronized cells expressing RelA-dsRedxp following treatment with 10 ng/ml TNFα. Statistical analysis showed significant difference between cell cycle phases with respect to distribution of amplitude of the response (Anova analysis with Dunn correction for multiple comparisons).

https://doi.org/10.7554/eLife.10473.006
Figure 2—figure supplement 3
Statistical analysis of NF-κB translocation in SK-N-AS cells at inferred cell cycle stages following 30 pg/ml TNFα stimulation.

(A) Correlation of estimated cell cycle timing with RelA-dsRedxp N:C peak amplitude following 30 pg/ml TNFα treatment (n=138). (B) Analysis of dynamics of initial RelA-dsRedxp translocation with respect to cell cycle phase. Statistical analysis showed a difference between G1 and S with respect to distribution of amplitude of the response (Anova analysis with Dunn correction for multiple comparisons).

https://doi.org/10.7554/eLife.10473.007
Cell cycle length and variability is modified by TNFα addition at G1/S.

Analysis of the timing and variability of mitosis (parameter 1 plus 5 from Figure 2B) following 10 ng/ml TNFα treatment of asynchronous untransfected HeLa cells, compared to subsets of those cells stimulated at late G1- or S-phase. Mean durations were analysed using nonparametric Anova analysis with Dunn correction for multiple comparisons. Variability in the data was analysed using Levene’s test for equality of variance.

https://doi.org/10.7554/eLife.10473.008
Figure 4 with 2 supplements
Physical and functional interaction between NF-κB and E2F-1 systems.

(A) NF-κB-dependent transcription was assessed by luciferase reporter assay (NF-luc), in SK-N-AS cells (n=3, +/- s.d) expressing EGFP-E2F-1, RelA-dsRedxp or both. (B) IκBα and IκBε mRNA levels in SK-N-AS cells (n=3, +/- s.d) following transient expression of EGFP-E2F-1, RelA-DsRedxp or both. (C) E2F-1-dependent transcription as assessed by luciferase reporter assay (CyclinE-luc), in SK-N-AS cells (n=3, +/- s.d) expressing EGFP-E2F-1, RelA-dsRedxp or both. (D) E2F-1 mRNA levels in SK-N-AS cells (n=3, +/- s.d) transiently transfected with RelA-dsRedxp. (E) Representative SK-N-AS cells transiently expressing EGFP-E2F-1 (green), RelA-dsRedxp (red), both fluorescent fusion proteins at different levels, or EGFP-E2F-1, RelA-dsRedxp and IκBα-AmCyan (blue).

https://doi.org/10.7554/eLife.10473.009
Figure 4—figure supplement 1
E2F-1 modulates NF-κB dynamics in the absence of stimulus in SK-N-AS cells.

(A) Time-lapse confocal microscopy of representative SK-N-AS cells transiently transfected with RelA-dsRedxp and EGFP-E2F-1. (B) Trajectories of three representative cells expressing different levels of EGFP-E2F-1. (C) Correlation between RelA-dsRedxp T½ nuclear occupancy (NO) time and EGFP-E2F-1 T½ nuclear degradation time, based on data in (A). (D) Recapitulation of the observed dynamics with an in silico model for physical interaction between RelA (NFkB) and E2F-1 (E2F) (E) Correlation between NF-κB nuclear occupancy time and nuclear E2F-1 degradation time, based on data in (D) (n= 30 cells).

https://doi.org/10.7554/eLife.10473.010
Figure 4—figure supplement 2
E2F-1 modulates NF-κB dynamics in the absence of stimulus in HeLa cells.

(A) Representative HeLa cells transiently transfected with combinations of RelA and E2F-1 fluorescent fusion proteins. (B) Time-lapse confocal microscopy of representative HeLa cells transiently transfected with RelA-dsRedxp and EGFP-E2F-1. (C) Trajectories of three representative cells expressing different levels of EGFP-E2F-1. (D) Correlation between RelA-dsRedxp T½ nuclear occupancy (NO) time and EGFP-E2F-1 T½ nuclear degradation time, based on data in (C) (n=20).

https://doi.org/10.7554/eLife.10473.011
Interaction of E2F-1 with RelA.

(A) Representative cell demonstrating co-localisation of E2F1-EGFP and RelA-dsRedxp upon transient transfection. (B) Co-Immunoprecipitation of E2F-1 with RelA pull down in HeLa cells synchronized in late G1 (HeLa cells used for this experiment due to their greater ease of synchronization). (C) FCCS assay between transiently transfected EGFP-E2F-1 and RelA-dsRedxp (red line) or empty-dsRedxp (blue line) fluorescent fusion proteins in single live SK-N-AS cells (+/- s.e.m based on 10 measurements from 10+ cells per condition). (D) Qualitative FRET assay between transiently transfected ECFP-E2F-1 and RelA-EYFP fluorescent fusion proteins in live SK-N-AS cells. First negative control between IkB-ECFP and EYFP-E2F1, and second negative control between free ECFP and EYFP fluorophores expressed in an SK-N-AS cell (shown are average ECFP and EYFP signals (+/- s.e.m based on 20 cells per condition normalised to pre-bleach intensity. p.b. indicates the time point at which photo-bleaching occurred).

https://doi.org/10.7554/eLife.10473.012
Mathematical modelling predicts an additional key component for NF-κB - cell cycle interactions: E2F-4 identified as a putative candidate.

(A) Model simulations of RelA-dsRedxp dynamics when co-expressed with EGFP-E2F-1 in cells treated with TNFα. (B) Dynamics analysed in representative SK-N-AS cells treated with 10 ng/ml TNFα expressing RelA-dsRedxp and EGFP-E2F-1 (C) Model simulation of experimental conditions in B, incorporating interactions between NF-κB complexes and a putative E2F-1-induced target protein, subsequently proposed as E2F-4. (D) Analysis of average timing to second peak of NF-κB translocation following TNFα treatment in SK-N-AS cells expressing RelA-dsRedxp alone or with EGFP-E2F-1 (n=20 cells per condition, error bars show s.d.) (E) Assessment of the extent of RelA Ser536 phosphorylation (p-RelA), E2F-4 and IκBα stability by western blot compared to cyclophilin A (cyclo A) amounts in SK-N-AS cells either untreated or treated with 10 ng/ml TNFα and expressing combinations of either untagged or fluorescent RelA-dsRedxp and EGFP-E2F-1. (F) Western blot of E2F-1 and E2F-4 in synchronized HeLa cells, where t=0 is late G1-phase.

https://doi.org/10.7554/eLife.10473.013
Figure 7 with 1 supplement
E2F-4 directly interacts with NF-κB and perturbs RelA dynamics in response to TNFα stimulation.

(A) Single cell trajectories from groups of HeLa cells expressing RelA-dsRedxp and different levels of EGFP-E2F-4 showing the dynamics of RelA-dsRedxp after 10 ng/ml TNFα treatment (n=60 cells). (B) HeLa cells synchronized in S-phase, co-immunoprecipitated with anti-RelA antibody and probed for E2F-4. Also shown are IgG negative controls and whole cell lysate unsynchronized positive control (ctrl). (C) Representative SK-N-AS cells transiently transfected with RelA-dsRedxp and EGFP-E2F-4. (D) FRET assay in live SK-N-AS cells expressing ECFP-E2F-4 and RelA-EYFP fluorescent fusion proteins (shown are average ECFP and EYFP signals (+/- s.e.m) based on 20 cells per condition normalised to pre-bleach intensity. p.b. indicates the point of photo-bleaching). (E) FCCS assay in cells transiently expressing EGFP-E2F-4 and RelA-dsRedxp (red line) or dsRedxp (blue line) fluorescent proteins in single live SK-N-AS cells (+/- s.e.m based on 10 measurements in each of 10+ cells per condition).

https://doi.org/10.7554/eLife.10473.014
Figure 7—figure supplement 1
Analysis of RelA-dsRedxp dynamics in HeLa and SK-N-AS cells co-expressing EGFP-E2F-4 following TNFα stimulation.

The effects of different EGFP-E2F-4 expression levels on the amplitude and timing of the first peak of RelA translocation in HeLa and SK-N-AS cells treated with 10 ng/ml and 30 pg/ml TNFα, respectively. These data indicate how ectopically expressed EGFP-E2F-4 can inhibit the translocation of RelA-dsRedxp in response to TNFα.

https://doi.org/10.7554/eLife.10473.015
Figure 8 with 4 supplements
Effect of cell cycle timing on RelA-dsRedXP translocation in dual BAC HeLa cells (C1-1 line) that co-express E2F-1-Venus fusion protein.

(A) Selected images from time-lapse experiment of dual BAC transfected HeLa stable clone 1-1 showing translocation of RelA-dsRedXP and E2F-1-Venus expression at different cell cycle phases. Cells were treated with 10 ng/ml TNFα. (B) Analysis of the dynamics of initial RelA-dsRedxp translocation in cells ordered at specific cell cycle times with respect to the peak of E2F-1 expression (n = 128). Data were analysed using nonparametric Anova analysis with Dunn correction for multiple comparisons. Red lines indicate mean normalised amplitude of NF-κB nuclear translocation for different cell cycle phases, and the population average (dotted red line). Analysis of nuclear RelA occupancy was assessed in virtually synchronised C 1-1 cells, based on time from cell division and relative to peak E2F-1-Venus expression level. RelA-dsRedxp localization was visualized to allow quantification of translocation, following treatment with 10 ng/ml TNFα. The dotted black line shows the spline fitted level of E2F1 at different times and cell cycle stages (see also Figure 8—figure supplement 1 below). Statistical analysis showed a difference between G1 vs S, and G2 vs S with respect to distribution of amplitude of the RelA translocation response. (C) RelA-dsRedxp dynamics following 10 ng/ml TNFα treatment in asynchronous cells (left panel) and cells virtually synchronised into G1, G1/S, S and G2 phases. The data for each cell was normalised to the amplitude (N:C ratio) at t = 0 min.

https://doi.org/10.7554/eLife.10473.016
Figure 8—figure supplement 1
Virtually synchronized HeLa C 1-1 cells.

Normalised E2F-1-Venus expression at the time of TNFα stimulation of C1-1 cells (data also shown in Figure 8B). E2F-1-Venus expression was normalised to its peak expression. The time axis represents the time of TNFα stimulation relative to the peak of E2F-1-Venus for each cell, where time 0 is the peak of E2F-1-Venus expression. Positive times indicate stimulation after the peak of E2F-1-Venus expression and negative values indicate stimulation events before the peak of E2F-1-Venus expression. The black line shows a spline interpolation of the level of E2F-1-Venus expression. Cell cycle phases were estimated based on measured the E2F1 profile and average cell cycle timing.

https://doi.org/10.7554/eLife.10473.017
Figure 8—figure supplement 2
Physiological and functional expression of E2F-1-Venus in stable BAC-transduced HeLa cells.

(A) HeLa cells stably expressing an E2F-1-Venus fluorescent fusion protein from a 5KB endogenous E2F-1 promoter (Green), transiently transfected with a FUCCI reporter for SCF (SKP-2) activity (Orange). Showing the profile of E2F-1 over two consecutive cell cycles (one parent and two daughter cells), with a peak in late G1. E2F-1 levels dropped during S-phase consistent with rapid rise in SCF (SKP-2) activity and a loss of FUCCI fluorescence. (B) Representative cell from the E2F-1-Venus and RelA-DsRedxp stably transfected population of Hela cells through one full cell cycle.

https://doi.org/10.7554/eLife.10473.018
Figure 8—figure supplement 3
Analysis of the expression of E2F-1-Venus and RelA-DsRedxp translocation in single C1-1 HeLa cells stimulated with 10ng/ml TNFα at different cell cycle phases.

Grey line shows the E2F-1-Venus expression level plotted agains the right y-axis. The red, green blue and orange lines show the timecourse of RelA-dsRedxp localization in exemplar cells in the G1, G1/S, S and G2 phases respectively plotted against the left y-axis. The black vertical line represents the point at which cells were treated with TNFα.

https://doi.org/10.7554/eLife.10473.019
Figure 8—figure supplement 4
Expression and interaction of RelA-dsRedxp and E2F-1-Venus.

(A) Western blot of RelA and α-tubulin levels in dual BAC stable C1-1 and WT HeLa showing exogenous expression of RelA-dsRedxp. (B) Western blot of E2F-1 and cyclophilin A levels in dual BAC stable C1-1 and WT HeLa cells expressing the E2F-1-Venus BAC (C) Total fluorescent molecules per cell for E2F1-Venus at peak expression and RelA-dsRedxp in unstimulated cells (data obtained from Fluorescent Correlation Spectroscopy measurements, and calculated using volume estimates from z-stacked WT HeLa in suspension). (D) FCCS mean correlation curves (+/- s.e.m) between E2F-1-Venus and RelA-dsRedxp (red line, n=46) for TNFα treated BAC stable cells. A comparison to transient empty-dsRedxp is shown (blue line, n=15) (E) Kd determination results using a scatter plot and linear regression (Theil-Sen estimator). The slope of the regression gives the Kd value.

https://doi.org/10.7554/eLife.10473.020
Schematic representation of NF-κB – E2F interactions.

(A) Predicted mechanisms for NF-κB interaction with E2F proteins over the G1/S transition (B) Model simulations of single cell behaviour.

https://doi.org/10.7554/eLife.10473.021
Appendix 1—figure 1
Oscillations in the NF-κB system.

(A) Dynamics of RelA-dsRedxp in transiently transfected SK-N-AS cells following 10 ng/μl TNFα stimulation, plotted over 450 and 150 min respectively. (B) Dynamics of RelA-dsRedxp in transiently transfected HeLa cells following 10 ng/μl TNFα stimulation, plotted over 450 and 150 min respectively.

https://doi.org/10.7554/eLife.10473.022
Appendix 1—figure 2
Use of double-Thymidine block to synchronize HeLa cells at G1/S.

Flow cytometric analysis of the distribution of DNA content of non-synchronized HeLa cells and cells harvested at relevant times post-release from Thymidine block.

https://doi.org/10.7554/eLife.10473.023
Appendix 1—figure 3
Effect of Double-Thymidine block on tagged and endogenous RelA levels HeLa cells.

Endogenous RelA and tagged RelA-DsRedxp expression levels in unsynchronised and synchronised WT-HeLa and double BAC stable cells. Synchronized fractions at 0, 2 and 4 hr post release of thymidine block. α-Tubulin used as loading control.

https://doi.org/10.7554/eLife.10473.024
Appendix 1—figure 4
Negative Co-IP. 

(A) Co-Immunoprecipitation of E2F-1 with RelA (pulled down with a RelA antibody) in asynchronous HeLa cells showing no detectable band of E2F-1. (B) Co-Immunoprecipitation of E2F-4 with RelA (pulled down with a RelA antibody) in HeLa cells synchronized in late G1-phase cells showing no detectable band of E2F-4.

https://doi.org/10.7554/eLife.10473.025
Appendix 1—figure 5
A typical simulation protocol of the NF-κB:E2F-1 mathematical model.

Simulation protocol of a live cell imaging experiment involving transfection and TNFα stimulation.

https://doi.org/10.7554/eLife.10473.026
Appendix 1—figure 6
Simulations from the NF-κB:E2F model.

Blue lines represent E2F1= 0, TR= 1. Black lines represent E2F1= NFkB= 0.1 and TR=0. Red lines represent E2F1= NFkB= 0.1 and TR=1.

https://doi.org/10.7554/eLife.10473.031
Appendix 1—figure 7
Cell Cycle length of Clonal HeLa BAC population.

(A) Analysis of cell cycle duration in populations of dual BAC stable cell line (C1-1) with wild type HeLa cells. (B) Analysis of the effects of TNFα treatment in C1-1 and WT HeLa cells.

https://doi.org/10.7554/eLife.10473.032
Appendix 1—figure 8
FCCS control.

FCCS assays between transiently transfected RelA-dsRedxp and IκBα-EGFP (red line) and Empty-DsRedxp and Empty-EGFP (blue line) in single live SK-N-AS cells (+/- s.e.m based on 10 measurements in each of 10+ cells per condition).

https://doi.org/10.7554/eLife.10473.033
Appendix 1—figure 9
FCCS autocorrelation analysis.

Autocorrelation lines for RelA/IkBα, RelA/E2F-1 and RelA/E2F-4 FCCS studies in single live SK-N-AS cells (+/- s.e.m based on 10 measurements in each of 10+ cells per condition).

https://doi.org/10.7554/eLife.10473.034

Tables

Appendix 1—table 1

A Initial conditions for the NF-κB:E2F mathematical model. Concentrations of NF-κB and E2F-1 added to mimic cell transfection are also included.

https://doi.org/10.7554/eLife.10473.027
SpeciesBiological nameInitial Conditions
Equilibration stage(μM)
Initial Conditions
TNFα stimulation
(μM)
NFkBCytoplasmic RelA00.004 (+0.1)
nNFkBNuclear RelA00.015
E2F1Cytoplasmic E2F100 (+0.1)
nE2F1Nuclear E2F100
tIkBaIκBα mRNA01e-005
IkBaCytoplasmic IκBα00.017
nIkBaNuclear IκBα00.004
IKKnNeutral IKK0.10.1
IKKActive IKK00
IKKiInactive IKKi00
tA20A20 mRNA01e-005
A20A2000.001
pIkBaphospho-IκBα00
pIkBaNFkBphospho-IκBα RelA complex00
NFkBE2F1cyto. RelA E2F-1 complex00
nNFkBE2F1nuclear RelA E2F-1 complex00
IkBaNFkBcyto IκBα RelA complex0.10.091
nIkBaNFkBnuclear IκBα RelA complex00.001
tE2F4E2F-1 target E2F-4 mRNA00
E2F4E2F-1 target E2F-400
E2F4NFkBE2F-4 RelA complex00
E2F4IkBaNFkBE2F-4 IκBα RelA complex00
Appendix 1—table 2

NF-κB:E2F-1 model equations Symbol ‘n’ denotes nuclear variables,‘t’ denotes mRNA transcripts, ‘p’ denotes phosphorylated form of IκBα. Symbols denoting cytoplasmic localisation were omitted.

https://doi.org/10.7554/eLife.10473.028
ddtNFκB(t)=ka1a×IκBα(t)×NFκB(t)+kd1a×(IκBα:NFκB)(t)ki1×NFκB(t)+ke1×nNFκB(t)+kt2a×(pIκBα:NFκB)(t)+c5a×(IκBα)(t)+kd2e×(NFκB:E2F1)(t)ka2e×E2F1(t)×NFκB(t)+c8ne×(NFκB:E2F1)(t)ka3e×NFκB(t)×E2F4(t)+kd3e×(E2F4:NFκB)(t)+c4x×(E2F4:NFκB)(t)\eqno(1)
ddtnNFκB(t)=+ka1a×nIκBα(t)×nNFκB(t)+kd1a×(nIκBα:nNFκB)(t)+ki1×kv×NFκB(t)ke1×kv×nNFκB(t)+kd2e×(nNFκB:nE2F1)(t)ka2e×nE2F1(t)×nNFκB(t)+c9ne×(nNFκB:nE2F1)(t)\eqno(2)
ddtE2F1(t)=kie×E2F1(t)kee×kv×nE2F1(t)c6e×E2F1(t)+kd2e×(NF κB:E2F1)(t)ka2e×nE2F1(t)×NF κB(t)+kdis×(NF κB :E2F1)(t)×IκBα(t)\eqno(3)
ddtnE2F1(t)=+kie×kv×E2F1(t)kee×kv×nE2F1(t)c7e×nE2F1(t)+kd2e×(nNF κB:nE2F1)(t)ka2e×nE2F1(t)×nNF κB(t)+kdis×(nNF κB :nE2F1)(t)×nIκBα(t)\eqno(4)
ddttIκBα(t)=+c1a×nNFκBh(t)nNFκBh(t)+khc3a×tIκBα(t)\eqno(5)
ddtIκBα(t)=kd1a×(IκBα:NFκB)(t)ka1a×IκBα(t)×NFκB(t)+c2a×tIκBα(t)c4a×IκBα(t)ki3a×IκBα(t)+ke3a×nIκBα(t)kc1a×IKK(t)×IκBα(t)kdis×(NFκB:E2F1)(t)×IκBα(t)ka3e×(NFκB:E2F4)(t)×IκBα(t)+kd3e×(E2F4:IκBα:NFκB)(t)\eqno(6)
ddtnIκBα(t)=kd1a×(nIκBα:nNFκB)(t)ka1a×NIκBα(t)×nNFκB(t)c4a×nIκBα(t)+ki3a×kv×IκBα(t)ke3a×kv×nIκBα(t)kdis×(nNFκB:nE2F1)(t)×nIκBα(t)\eqno(7)
ddtIKKn(t)=kp×(kbA20kbA20+TRA20×A20(t))×IKKi(t)TR×ka×IKKn(t)\eqno(8)
ddtIKK(t)=TR×ka×IKKn(t)ki×IKK(t)\eqno(9)
ddtIKKi(t)=ki×IKK(t)kp×kbA20kbA20+TRA20×A20(t)×IKKi(t)\eqno(10)
ddttA20(t)=+c1×nNFκBh(t)nNFκBh(t)+khc3×tA20(t)\eqno(11)
ddtA20(t)=c2×tA20(t)c4×A20(t)\eqno(12)
ddtpIκBα(t)=kc1a×IKK(t)×IκBα(t)kt1α×pIκBα(t)\eqno(13)
ddt(pIκBα:NFκB)(t)=κc2α×IKK(t)×(IκBα:NFκB)(t)κt2α×(pIκBα:NFκB)(t)\eqno(14)
ddt(NFκB:E2F1)(t)=ka2e×E2F1(t)×NFκB(t)kd2e×(NFκB:E2F1)(t)kine×(NFκB:E2F1)(t)+kene×(nNFκB:nE2F1)(t)c8ne×(NFκB:E2F1)(t)kdis×(NFκB:E2F1)(t)×IκBα(t)\eqno(15)
ddt(nNFκB:nE2F1)(t)=ka2e×nE2F1(t)nNFκB(t)kd2e×(nNFκB:nE2F1)(t)+kine×kv×(NFκB:E2F1)(t)kene×kv×(nNFκB:nE2F1)(t)c9ne×(nNFκB:nE2F1)(t)kdis×(nNFκB:nE2F1)(t)×nIκBα(t)\eqno(16)
ddt(IκBα:NFκB)(t)=ka1a×IκBα(t)×NFκB(t)kd1a×(IκBα:NFκB)(t)c5a×(IκBα:NFκB)(t)+ke2a×(nIκBα:nNFκB)(t)kc2a×IKK(t)×(IκBα:NFκB)(t)+(kdis)×(NFκB:E2F1)(t)×IκBα(t)ka3e×(IκBα:NFκB)(t)×E2F4(t)+kd3e×(E2F4:IκBα:NFκB)(t)+c4x×(E2F4:IκBα:NFκB)(t)\eqno(17)
ddt(nIκBα:nNFκB)(t)=ka1a×nIκBα(t)×nNFκB(t)kd1a×(nIκBα:nNFκB)(t)ke2a×kv×(nIκBα:nNFκB)(t)+kdis×(nNFκB:nE2F1)(t)×nIκBα(t)\eqno(18)
ddttE2F4(t)=+clx×nE2F1h(t)nE2F1h(t)+khc3x×tE2F4(t)\eqno(19)
ddtE2F4(t)=c2x×tE2F4(t)c4x×E2F4(t)ka3e×NFκB(t)×E2F4(t)+kd3e×(E2F4:NFκB)(t)ka3e×E2F4(t)×(IκBα:NFκB)(t)+kd3e×(E2F4:IκBα:NFkB)(t)\eqno(20)
ddt(E2F4:NFκB)(t)=ka3e×NFκB(t)×E2F4(t)kd3e×(E2F4:NFκB)(t)c4x×(E2F4:NFκB)(t)+c5a×(E2F4:IκBα:NFκB)(t)ka3e×(NFκB:E2F4)(t)×IκBα(t)+kd3e×(E2F4IκBαNFκB)(t)\eqno(21)
ddt(E2F4:IκBα:NFκB)(t)=+ka3e×(IκBα:NFκB)(t)×E2F4(t)kd3e×(E2F4:IκBα:NFκB)(t)c5a×(E2F4:IκBα:NFκB)(t)+ka3e×(NFκB:E2F4)(t)×IκBα(t)kd3e×(E2F4:IκBα:NFκB)(t)c4x×(E2F4:IκBα:NFκB)(t)\eqno(22)
Appendix 1—table 3

Model reactions and associated parameters.

https://doi.org/10.7554/eLife.10473.029
ReactionSymbolValueReferences
Spatial parameters
Total cell volumetv2700 µm3Measured
C:N ratiokv3.3Measured
Conversion to nuclear volumenv×(kv+1)-
Conversion to cytoplasmic volumecv×(1/kv+1)-
Initial concentration
Total NF-κBNF0.08 µMInitialized as cytoplasmic IκBα·NF-κB
Total IKK-0.08 µMInitialized as IKKn
Complex formation & dissociation
IκBα + NF-κB → IκBα·NF-κB
nIκBα + nNF-κB → nIκBα·NF-κB
ka1a0.5 µM-1s-1(Hoffmann et al., 2002)
IκBα·NF-κB → IκBα + NF-κB
nIκBα·nNF-κB → nIκBα + nNF-κB
kd1a0.0005 s-1(Hoffmann et al., 2002)
NF-κB + E2F (1 or 4) → NF-κB·E2F
nNF-κB + nE2F → nNF-κB·nE2F
ka2e0.5 µM-1s-1fitted, same as IκBα + NF-κB
NF-κB·E2F → NF-κB + E2F
nNF-κB·nE2F → nNF-κB + nE2F
kd2e0.0005 s-1fitted, same as IκBα + NF-κB
NF-κB·E2F1 + IκBα→ IκBα·NF-κB + E2F1
nNF-κB·nE2F1 + nIκBα → nIκBα·NF-κB + nE2F1
kdis0.001 s-1fitted
Transport
NF-κB → nNF-κBki10.0026 s-1Measured fitting range: Average 0.0026 ± 0.0018s-1
nNF-κB → NF-κBke10.000052 s-1ki1/50 (Carlotti et al., 2000)
E2F1→ nE2F1kie0.0026 s-1fitted, same as NF-κB
nE2F1 → E2F1kee0.000052 s-1fitted, same as NF-κB
IκBα → nIκBαki3a0.00067 s-1Measured fitting range:
Average 0.00043 ± 0.00024 s-1
nIκBα → IκBαke3a0.000335 s-1ki3a/2 (Carlotti et al., 2000)
nIκBα·nNF-κB → IκBα·NF-κBke2a0.01 s-1Fitted
NF-κB·E2F1 → nNF-κB·nE2F1kine0.0026 s-1fitted, same as NF-κB
nNF-κB·nE2F1 → NF-κB·E2F1kene0.000052 s-1fitted, same as NF-κB
Protein synthesis & degradation
nNF-κB → nNF-κB + tIκBα
Order of hill function, h=2
Half-max constant, k=0.065h(fitted)
c1a1.4×10-7
 µM-1s-1
Fitted (constrained):
1.07×10-7 – 8.2×10-7µM-1s-1 (Femino et al., 1998);
(Cheong et al., 2006)
tIκBα→ tIκBα + IκBαc2a0.5 s-1(Lipniacki et al., 2004)
NF-κB·IκBα→ NF-κBc5a0.000022 s-1(Pando and Verma, 2000;
Mathes et al., 2008)
nNF-κB·nIκBα→ nNF-κB-0 s-1Assumed (O'Dea et al., 2007;
Mathes et al., 2008)
nNF-κB → nNF-κB + tA20
Order of hill function, h=2 Half-max constant, k=0.065h
c11.4×10-7 
µM-1s-1
Assumed to be the same as IκBα
nE2F1 → nE2F1 + tE2F4
Order of hill function, h=2 Half-max constant, k=0.065h
c1x9.8×10-7
µM-1s-1
Fitted
tA20→ tA20 + A20c20.5 s-1-
tE2F-4→ tE2F-4 + E2F4c2x0.5 s-1-
tIκBα→ Sinkc3a0.0003 s-1Fitted (constrained): 0.00077-0.00029 s-1
(Blattner et al., 2000)
tA20→ Sinkc30.00048 s-1Fitted, constrained >tIκBα
turnover (Ashall et al., 2009)
tE2F4→ Sinkc3x0.00048 s-1Fitted
IκBα→ Sinkc4a0.0005 s-1Fitted (constrained): 0.000105 – 0.002 s-1
(Pando and Verma, 2000; O'Dea et al., 2007;
Mathes et al., 2008)
A20 → Sinkc40.0045 s-1Fitted
E2F4 → Sinkc4x0.00016 s-1Fitted
E2F1 → Sinkc6e0.00016 s-1Fitted
nE2F1 → Sinkc7e0.00016 s-1Fitted
NF-κB·E2F1 → Sinkc8ne0.00016 s-1Fitted
nNF-κB·nE2F1 → Sinkc9ne0.00016 s-1Fitted
TNFα stimulation
TNFαTR1/0on/off (Lipniacki et al., 2004)
IKK parameters
IKKn → IKKaka0.004 s-1Fitted, as above
IKKa → IKKiki0.003 s-1Fitted, as above
IKKi → IKKnkp0.0006 s-1Fitted
A20 inhibition rate constantkbA200.0018Fitted, scales kp dependent on
receptor state kbA20×TR
IKKa + IκBα → pIκBαkc1a0.074 s-1Assumed (0.037×2) (Heilker et al., 1999)
IKKa + IκBα·NF-κB → pIκBα·NF-κBkc2a0.37 s-1Assumed (0.037×5×2)
(Heilker et al., 1999;
Zandi and Karin, 1999)
pIκBα → Sinkkt1a0.1 s-1Fitted
pIκBα·NF-κB → NF-κBkt2a0.1 s-1Fitted
Appendix 1—table 4

Simulation protocols used throughout the manuscript. TNFα stimulation is invoked via TR=0/1. E2F refers to levels of 'transfection' (in μM). E2F4 off/on refers to whether its transcription is switched on or off.

https://doi.org/10.7554/eLife.10473.030
FigureModel conditions
3ETR=0, E2F1= (0.05, 0.1, 0.15), E2F4 off
4ATR=1, E2F1 = 0.1, E2F4 off
4CTR=1, E2F1 = 0.1, E2F4 on
4I (G1, G2)TR=1, E2F1= 0, E2F4 on (but unaffected)
4I (G1/S)TR=1, E2F1 = 0.2, E2F4 on
4I (S)TR=1, NFkBIkBa = 0.1, E2F1 = 0, E2F4= 0.1

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