miR-17 and miR-20 form an incoherent positive feedback loop with the target mRNA <i>Cd69</i>.

<p>A) The strength of activation signals (0.1, 1, 10 μg/ml H57) determines the expression of <i>Cd69</i>, miR-17 and miR-20a (normalised to snRNA-135 and -202, n = 2–3, mean ± SE). B) Perceived signal strength varies among individual DP thymocytes and determines the expression of <i>Cd69</i>, miR-17 and miR-20a (microRNA expression is normalised to snoRNA-135 and snoRNA-202). At a fixed extracellular signal of 1u/ml H57, the fold-change in miR-17 and miR-20 relative to CD69 negative DP and normalised to snoRNA-135 was proportional to the expression of CD69 (n = 3, mean ± SD). C) The microRNA target mRNA <i>Cd69</i> and microRNAs of the miR-17-92 cluster are co-regulated in response to activation signals and form an incoherent feed-forward loop downstream of the TCR. D) Modeling CD69 protein expression with and without microRNA feed-forward regulation. The state of the system is described by five major variables: the number of mRNAs transcribed from the TF gene, the number of TF molecules, the number of miRNAs, the number of mRNAs and the number of target proteins. Of these variables we can estimate the number of mRNA copies and the number of microRNA copies. As detailed in the legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005020#pgen.1005020.s004" target="_blank">S4 Fig.</a>, the number of <i>Cd69</i> mRNA copies was estimated as 0 in resting and 6 in activated cells, miR-17 and miR-20 were estimated as 6–12 copies per cell the resting state and 30–60 copies per cell after activation. Simulations of transcriptional networks were carried out using the Gillespie exact stochastic simulation algorithm, programmed and analysed using R based on a microRNA feed-forward model [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005020#pgen.1005020.ref008" target="_blank">8</a>] to simulate CD69 protein expression in resting T cells (unfilled histogram), in a scenario where activation increases <i>Cd69</i> mRNA but the expression of miR-17 and miR-20a remained the same as in resting T cells (activated without microRNA FFL, filled red histogram), and in a scenario where activation increases both <i>Cd69</i> mRNA and miR-17 and miR-20a expression (activated with microRNA FFL, filled grey histogram). The plot represents 10,000 simulations. The model predicts that thymocyte activation with co-regulation of <i>Cd69</i> mRNA and miR-17/miR-20a reduces the mean (887 versus 1300) and the CV (10.2 versus 14.6) of CD69 expression compared to the regulation of <i>Cd69</i> mRNA alone (P<10^–4). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005020#pgen.1005020.s004" target="_blank">S4 Fig.</a> for details of the underlying circuitry, the parameters used, and a model based on microRNA effects on mRNA degradation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005020#pgen.1005020.ref049" target="_blank">49</a>].