Table 1. Biological processes enriched in wildtype upregulated acute-stochastic mode. Biological processes (GO) and pathways (KEGG) # genes p-value inflammatory response response to wounding defense response response to stress response to external stimulus immune system process immune response response to stimulus Cytokine-cytokine receptor interaction doi:10.1371/journal.pone.0004905.t001 13 13 14 16 13 14 11 21 9 5.6.E-06 9.9.E-05 2.9.E-04 3.7.E-03 3.8.E-03 2.8.E-02 3.6.E-02 4.6.E-02 2.5.E-02 and U = 0.15x (Figure 8A–C, x,0). This result points to i) MyD88 & TRIF-dependent (MT) pathways mainly show synergized repression of genes through mRNA decay, i.e., Tr%Dm; ii) TRIF-dependent (T) pathways can activate the transcription of the same processes as MT, i.e., Tr.Dm; iii) M has no regulatory role for wildtype downregulated ORFs and iv) unknown processes (U) can weakly repress the same genes through decay machinery, i.e., Dm.Tr. To determine biological processes regulated by the whole genome, we selected genes satisfying upregulation in TRIF KO and downregulation in MyD88 KO for WTc+ and genes satisfying downregulation in TRIF KO and DKO, and upregulation in MyD88 KO for WTc2, due to the observed emergent linearity (Figure 8D). We obtained 3798 out of 11793 ORFs for WTc+ and 1916 out of 10897 ORFs for WTc2. Next, combining WTc+ and WTc2 ORFs, and using DAVID platform analysis, we retained the biological processes of genes with significant enrichment (Tables 2 and 3, p,0.05). We observed biological processes related to immunity (Cytokine- cytokine receptor interaction, Toll-like receptor signaling pathway, etc.) were upregulated in WTc+, which indicates immune-related genes are not restricted to the acute-stochastic mode alone (Table 2). Predominantly, however, in WTc+, a) pre-transcriptional and transcription-related genes, such as genes related to cell surface receptor mediated signal transduction (including Protein phosphorylation required for signal transduction and Metabolism of cyclic nucleotides Macrophage Genome-Wide Control used for kinase activities), and mRNA transcription regulation and b) genes related to post-translational processes (Proteolysis, Exocytosis, etc.) were upregulated (Table 2). In WTc2, c) genes related to post- transcriptional processes, (Pre-mRNA processing, mRNA splicing, Protein biosynthesis, Ribosome, etc.) were downregulated (Table 3). We therefore hypothesize that wildtype cells prepare for secondary immune activation (possibly through cytokine receptors) by upregulating signaling and transcription processes (Figure 8E). To self-regulate the secondary immune response, post-transcrip- tional processes (mRNA splicing and translation) are repressed. Interestingly, we observe, from Figure 8E, a complete reverse (switching) behavior in MyD88 KO where only T and U are active, e.g. pre-transcriptional processes and transcription become downregulated while post transcriptional processes are upregu- lated. This suggests MyD88 KO cells compensate lack of activation (transcription) by enhancing post-transcriptional pro- cesses through TRIF-dependent (T) pathways [29]. These findings seem to indicate that MyD88 is a key regulator in collective mode. In summary, we observe differential activation of group of ORFs between genotypes (Table S2). From these results, we obtained the individual effects of M, T, MT and U on a genome- wide scale (Figure 8E). This shows genome-wide differential activation machinery of biological processes. Unlike acute-stochastic mode which is dedicated to immune system, collective mode may also participate in diverse regulatory processes; to prepare the cell for activation (signal transduction), prevent over expression of genes (regulation of post-transcriptional processes), and compen- sate lack of activation in KO conditions (enhancement of genes related to post-transcription when transcription process is lacking). Conclusion In this report, we found quantitative genome-wide differential control of transcriptional and mRNA decay machineries through signaling processes superimposing over the general strong co- regulation of expression levels that is largely invariant between genotypes and related to the global expression attractor corre- spondent to the specific cell type [15,20]. Moreover, each genotype, except DKO, possess two modes of responses; acute- stochastic (small number of immune-related genes) and collective modes (rest of ORFs). The collective mode, which consists of myriad cellular processes, is often ignored in most analyses as they are made of ORFs displaying small expression changes in time and hence cannot be captured if high cut-off thresholds (e.g. 3-fold) are used. Also in collective mode, for all genotypes, we notice scalable response and emergent linear behavior arise when ORFs are Figure 7. Global shifts in each genotype distribution. Genome-wide expression changes (Dx) for groups of 200 randomly chosen ORFs between genotypes for 0–1 h: wildtype vs. A) MyD88 KO, B) TRIF KO, C) DKO. doi:10.1371/journal.pone.0004905.g007 PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4905