Macrophage Genome-Wide Control Figure 5. Emergence of regulatory signature from scattered expressions. Genome-wide expression changes (Dx) between time points, 0– 1 h (x-axis) vs. 1–4 h (y-axis) for single ORF (left panels) in A) wildtype, B) MyD88 KO, C) TRIF KO), and D) DKO. Center panels: corresponding plots for group of 200 ORFs sorted by their 0–1 h expression change (x-axis). Each point represents the average of Dx for 200 ORFs. Right panels: Gaussian distribution of Dx for 1–4 h. Superposed profiles (lighter color for increasing upregulated groups and darker color for increasing downregulated groups) represent density distribution (Gaussian) of each group of 200 ORFs sorted from highest to lowest for 0–1 h in A) wildtype, B) MyD88 KO, C) TRIF KO and D) DKO. x-axis represents Dx for 1–4 h and y-axis represents the density of ORFs. doi:10.1371/journal.pone.0004905.g005 these should be due to experimental shift or normalization process. Thus, we eliminated these shifts from MyD88 KO, TRIF KO and DKO contributions. In general, the emergent smooth lines in collective mode can be presented by y = ax+b (Figure 8A–C) where slope a represents net outcome between transcriptional and mRNA decay machineries on average expression change and y-axis interception, b, implies contribution from the equilibrium state of transcriptional and mRNA decay machineries. Note that we ignore analysis around the origin of axes, where the densely distributed average points results in overlapping of their Gaussian distributions between genotypes. For WTc+, we observed same flat profile for DKO in both WT+ and randomly selected wildtype ORFs, which indicates that WT+ in DKO possess no response, i.e., U = 0 (Figure 8A and C, x.0). The flat response in DKO stems from different set of upregulated ORFs from WT+, confirming that DKO response (U) is independent of MyD88 and TRIF. From all genotypes’ plot, we obtain M+T+MT = x, T = 20.04 and M = 0.15x, thus, MT = 0.85x+0.04 (Figure 8A–C, x.0). This result points to i) MyD88 & TRIF-dependent processes (MT) show synergized activation of genes through transcription, i.e., Tr&Dm, since MT possesses dominant positive slope, ii) MyD88-dependent pathway (M) also activates these genes but to a lower extent, i.e., Tr.Dm, since M possesses smaller positive slope, iii) TRIF-dependent pathway (T) shows the equilibrium of transcriptional and mRNA decay machineries i.e., Tr–Dm = constant, since non-zero flat response between positive (transcriptional) and negative (mRNA decay) contribution. For wildtype downregulated ORFs (WTc2), we obtained: M+T+MT+U = x (Note: x,0 for WTc2), T+U = 20.35x, M+U = 0.15x, U = 0.15x; thus, M = 0, T = 20.5x, MT = 1.35x PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4905