Modelling the influence of delayed <i>BCL3</i> expression on <i>TNFA</i> transcription.

<p>The ODE model shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077015#pone-0077015-g004" target="_blank">Figure 4B</a> was simplified by removing the chromatin remodelling step (A) to simulate how this delay influences <i>TNFA</i> transcription (B-D). At different times following TNFα stimulation, the output from this model (B, solid line) is lower than with the time-delayed model (B, dashed line), though differences can be partly recapitulated simply by reducing expression of BCL-3, when both a single (C) or double (D) pulse of TNFα is used. This model motif can be represented by an Incoherent Feed-forward Loop (E), to test a simplified version of the <i>TNFA</i> transcription/BCL-3 model with time-delayed or continuous induction (with decreasing magnitude, red numbers) of <i>BCL3</i> transcription (F). Simulations for <i>TNFA</i> mRNA (G) and BCL-3 protein (H) are shown (solid lines) and compared to the chromatin delay model (dashed lines).</p

Chromatin remodelling is required for <i>BCL3</i> transcription.

<p>Following TNFα treatment of HT1080 cells clear differences in the induction dynamics of <i>TNFA</i> and <i>BCL3</i> expression were seen (A). ChIP analysis showed RNAP to be bound at the <i>TNFA</i> promoter (30) prior to TNFα treatment (B) but not within the protein coding region (Cds). At this time, RNAP at the <i>BCL3</i> promoter is not detectable and binding is clearly delayed, concomitant with a clear increase in promoter-associated histone H3 acetylation (C). Changes at the <i>BCL3</i> promoter correlated with dynamics of association of NF-κB (p56; D) and chromatin accessibility measured qualitatively (E) and quantitatively (F) by qRT-PCR. Clear changes in chromatin structure at the Xcm1 site shown were seen at 90 min following TNFα treatment and in control cells treated with TSA (400 nm for 12 h). Error bars show standard deviation: *P<0.05; **P<0.01.</p

Nuclear localisation of p65/NF-κB following TNFα treatment.

<p>HT1080 cells were treated with TNFα and the distribution of NF-κB (p65 subunit) visualised by indirect immuno-flourescence (A,B) or live cell imaging of p65-dsRed (C,D) at the times shown. Time-lapse imaging shows that p65 accumulates in nuclei from 20-40 min before returning to the cytoplasm (C,D) and this is confirmed for endogenous p65 in fixed cells (B). Error bars show standard deviation: *P<0.05; **P<0.01.</p

Temporal dynamics of <i>TNFA</i> and <i>BCL3</i> gene transcription in TNFα treated HT1080 cells.

<p>HT1080 cells were treated with TNFα and signalling through NF-κB monitored. Expression of <i>TNFA</i> and <i>BCL3</i> was assessed using qRT-PCR (A; n=3) to measure fold-changes in mRNA (relative to t=0) and shown to be dependent on nuclear translocation of NF-κB using the inhibitor SN50 (B; n=3). Transient over expression of BCL-3 from a constitutive promoter significantly reduced <i>TNFA</i> transcription (C; n=3) and siRNA-induced depletion of BCL-3 (inset) prior to TNFα treatment significantly increased <i>TNFA</i> transcription (D; 3h time points are shown; n=3). Temporal changes in BCL-3 occupancy at a distal (-869) κB site in the <i>TNFA</i> promoter (E; n=3) correlated with increased expression of BCL-3 (F), which though present throughout HT1080 cells was enriched in nuclei (G; 3h time-point shown). Error bars show standard deviation: *P<0.05; **P<0.01.</p

Modelling TNFα induced transcription at the <i>BCL3</i> and <i>TNFA</i> promoters.

<p>A model linking <i>TNFA</i> and <i>BCL3</i> expression shows how the timing of events that regulate <i>BCL3</i> transcription (A; data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077015#pone-0077015-g001" target="_blank">Figures 1</a>and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077015#pone-0077015-g002" target="_blank">2</a>) can be recapitulated using an ODE model (B; Information S2 for parameters) that mimics the data in simulations (C,D). During pulsatile stimulation, the separation of two TNFα treatments only weakly affects the nuclear localisation of NF-κB (E; p65 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077015#pone-0077015-g003" target="_blank">Figure 3B</a>) while dramatically influencing the time-dependent expression of <i>TNFA</i>, as a result of promoter-bound BCL-3 (F). This behaviour is reproduced by the model (G).</p

HeLa FUCCI ANALYSIS

Analysis of cell cycle transition from G1 to S using Fucci vectors in HeLa cells as well as analysis of mean Mitosis length in the cell typ

RelA-DsRedxp E2F1-Venus BAC stables FCS Autocorrolation and cross-corrolation data

RelA-DsRedxp E2F1-Venus BAC stables FCS autocorrolation and cross-corrolation dat

BAC BAC Stable cell phase Meta data_Dryad

Traces of BAC-BAC E2F-1-Venus and Rel-A-dsRedexpress sorted into different cell cycle phases

Peak RelA Nuclear Amplitude for HeLa Cells

Peak RelA Nuclear Amplitude for HeLa Cells following TNF stimulation arranged by virtual synchronization of cell cycle. Single cell traces upon which the peak amplitude data is based are also include

RelA-DsRedxp E2F1-Venus BAC stables FCS molecular fluoresence Data

Molecular fluorescence counts in single cells of RelA-DsRedxp E2F1-Venus BAC stables and correlation data