(a) Steady state Xist levels simulated deterministically (see Fig. 2e) to indicate that a sharp threshold is required between a single (1x) and a double (2x) tXA dose. (b) Maintenance of the XaXi state was simulated by initiating an allele either from the Xa (light green) or from the Xi state (dark green). For an example parameter set (kT=113 h-1, t1/2repr =0.7 h) mean and standard deviation of Xist expression across 100 cells from the chromosomes that initiated as Xa (light green) and Xi (dark green), respectively, is shown for different values of kX for the full Xist-Tsix model (left) and the reduced model without transcriptional interference (right). The vertical lines indicate the kX threshold value, above which >1 (Thlow, red) or >99 (Thhigh, grey) out of 100 cells up-regulate Xist from the Xa. (c) Distribution of the Thhigh-to-Thlow ratio (red and grey in (b)) across all parameter sets of the Full model (grey) and the reduced model without transcriptional interference (black). Since tXA reduces kX 2-fold upon Xist up-regualtion a threshold ratio of <2 is required to allow reliable Xist up-regulation with a double dose (2x) of tXA and stable maintenance of the XaXi state with a single dose (1x). This is only possible in the Full model with transcriptional interference.