Modelling autoregulation of histone gene expression.

<p>(A) Model structure. Expansion of the basic models to test the effect of additional H2B genes on the expression of endogenous H2B and H3 genes. (B) and (C). Panels describe the time evaluation of histone RNA (<i>R</i>), histone proteins (<i>H</i>), free histone binding sites on DNA (<i>D</i>), nucleosome packed DNA formed from endogenous histone H2B and H3 (<i>T</i>_<i>e</i>), formed from exogenous H2B and endogenous H3 (<i>T</i>_<i>p</i>) simulated by the mathematical models as a function of the external influence of SLBP (<i>S</i>) and DNA synthesis (<i>V</i>_<i>5</i>). Shown are results for the histone feedback loop (B) and DNA coupled (C) model.</p

Model structures and equations.

<p>Histone feedback loop model and DNA coupled model. (A) Model structure. Dashed lines illustrate the links between free histone proteins and histone RNA synthesis and degradation (histone feedback loop model). The links between DNA replication and histone RNA are illustrated by dotted lines (DNA coupled model). Solid lines are common to both models. (B) and (C). Full set of equations for the histone feedback loop model (B) and the alternative fluxes <i>v</i>_<i>1</i> and <i>v</i>_<i>2</i> for the DNA coupled model (C). Other equations and fluxes are common to both models.</p

Evidence for a feedback control mechanism that regulates histone gene expression.

<p>U2OS cells were transfected with plasmids pEGFP- H2B (H2B-GFP) or pEGFP (GFP) and subjected to antibiotic selection produce stable lines prior to FACS and protein analysis. (A, C) Analysis of H2B protein levels. Proteins were separated by SDS PAGE and analysed by Western blotting. Shown are H2B, H3 and GAPDH protein levels. Histone protein levels were standardised with respect to GAPDH protein, with H2B or H3 protein levels in cells transfected with pEGFP (GFP) defined as 1. Note that endogenous H2B levels (H2B) are significantly reduced in cells expressing H2B-GFP. (B, D) Analysis of H2B RNA levels. H2B RNA levels were analysed by Northern blotting. Shown are H2B and GAPDH RNA levels. The RNA levels were standardised with respect to GAPDH RNA with H2B RNA levels in cells transfected with pEGFP (GFP) defined as 1. Model predictions of the averaged H2B proteins (E, G) and RNAs (F, H) from the histone feedback loop (E, F) and DNA coupled (G, H) model. Endogenous H2B is in black, exogenous H2B in red. The different bars in each plot illustrate the effect of the strength of the promoter controlling exogenous H2B expression.</p

A comparison of histone RNA levels measured experimentally and predicted by the model.

<p>(A) U2OS cells were synchronised and released into S phase as described in Materials and Methods. Total RNA was prepared before release into S phase (0 min time point) and at regular intervals after that, and histone H2B RNA levels were subsequently analysed by Northern blotting. 28S rRNA levels were also measured and H2B RNA levels were standardised using 28S rRNA as a reference. (B) The graph shows the model prediction of histone RNA based on the analysis of DNA replication from two independent experiments (RNA simulation exp1 and 2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165848#pone.0165848.s003" target="_blank">S2 File</a>). Also shown is the quantitation of the RNA analysis by Northern blotting from these two experiments (RNA exp 1 and 2). Note these data were scaled to match the maxima of the model predictions.</p

The histone feedback loop and DNA coupled models differ in their response to the inhibition of DNA synthesis but not in their response to transcription blocks.

<p>Shown variables and colour coding are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165848#pone.0165848.g002" target="_blank">Fig 2</a>. In (A), inhibition of DNA replication was implemented by setting <i>V</i>_<i>5</i> to 0 at 15000 s. In (B), inhibition of transcription was implemented by setting the transcription flux <i>v</i>_<i>1</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165848#pone.0165848.g001" target="_blank">Fig 1</a>) to 0.</p