Functional Modules Identified by the “Domain Influence”

<div><p>(A) Identification of domain influencing groups. The effects of modifier deletions on the signaling of AHR and AHRΔPASB were compared in parallel. It was found that 28 modifiers were required for the function of the PASB domain (i.e., their deletions affected the AHR, but not the AHRΔPASB). The other 25 modifiers were found to be required for the shared TAD region (i.e., their deletions affected the signaling of both AHR and AHRΔPASB).</p> <p>(B) Overlay of the “domain influence” layer (blue boundary) and the network-clustering layer (shadowed) on the AHR–PIN. The PASB influence group corresponds to a central region in the AHR–PIN. The TAD influence group corresponds to two peripheral areas. Occasional outlier nodes are marked with their corresponding module names.</p></div

Functional Modules Identified by Network Clustering

<div><p>(A) Network clustering of AHR–PIN. Protein nodes in the AHR–PIN (D_max = 2) were clustered by a hierarchical clustering algorithm. A tree-depth threshold was set to delimit cluster boundaries (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020065#pbio-0020065-Rives1" target="_blank">Rives and Galitski 2003</a>). Clusters with at least two M-nodes are shown. See text for details.</p> <p>(B) Overlay of the network clusters on the AHR–PIN. The ten network clusters correspond to ten local areas in the AHR–PIN. Each network cluster (local area) is labeled with its significant functional enrichment as calculated using the FunSpec program (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020065#pbio-0020065-Robinson1" target="_blank">Robinson et al. 2002</a>).</p> <p><b>Color scheme.</b> Nodes: modifier deletions that incurred down- and up-regulation of AHR signaling are marked in green and red, respectively. For intervening nodes, essential genes are marked in gray and nonessential genes in white. Links: physical interactions are labeled in black and genetic interactions in red. If both interactions are available for a given link, only the physical interaction is shown. This color scheme is also applied to Figures 4–7.</p></div

Regulatory Network of AHR Signaling

<div><p>(A) The summary map of AHR–PIN. Functional modules were determined by the overlapped annotations from three experimental layers (domain influence, pharmacology clustering, and localization influence) as well as from network clustering. For each functional module, the main “stacking pattern” of experimental layers is noted in italics. Modifiers initially left outside the single large cluster of the AHR–PIN were assigned to corresponding functional modules by sharing the similar stacking pattern where applicable. See the legend of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020065#pbio-0020065-g003" target="_blank">Figure 3</a> for the color scheme of the nodes and links.</p> <p>(B) An expanded model of AHR signaling. The AHR signaling pathway is regulated by at least five functional modules that are involved in the control of receptor folding, nuclear translocation, transcriptional activation, receptor level, and a PASB-related nuclear event. Within each functional module, modifers intially enclosed in the single large cluster of the AHR–PIN are highlighted in bold. Known human homologs of the modifiers are noted at the side with a smaller font (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020065#pbio-0020065-Costanzo1" target="_blank">Costanzo et al. 2001</a>) . ARNT is dimmed because modifiers were identified in this study from an “ARNT-free” chimeric AHR system. See text for details.</p></div

Functional Modules Revealed by Effect on AHR Pharmacology

<div><p>(A) Cluster analysis of the effect of modifier deletion on AHR pharmacology. AHR signaling was examined at various doses, timepoints, and temperatures, and with the two AHR agonists βNF and αNF. The influence of modifier deletion on the dose-response of the AHR was analyzed by a hierarchical clustering algorithm. Rows in the clustering diagram represent modifier deletions. Columns correspond to experimental conditions. Green and red indicate down- and up-regulated AHR signaling, respectively. Color brightness is proportional to fold change. Black indicates <i>wt</i> signaling. Sparse gray boxes represent missing datapoints. (Insert) Diagram of corresponding dose-response curves of the <i>wt</i> strain and the average of cluster C.</p> <p>(B) Overlay of the “pharmacology clustering” layer (shadowed, black boundary) and “domain influence” layer (blue boundary) on the AHR–PIN. The major pharmacology clusters are coincident with five local areas in the AHR–PIN. In addition, clusters A, D, and E correspond to the PASB influence module, and clusters B and C correspond to the TAD influence module. Functional annotations determined by pharmacology clustering are indicated in black, and those derived from domain influencing are indicated in blue. Occasional outlier nodes are marked with their corresponding module designation. See the legend of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020065#pbio-0020065-g003" target="_blank">Figure 3</a> for the color scheme of the nodes and links.</p></div

Functional Modules Identified by the “Localization Influence”

<div><p>(A) The AHR–GFP fusion protein translocates to nucleus in the presence of agonist βNF. Nucleus position in the cell was confirmed by DAPI staining (data not shown). Dimethyl sulfoxide (DMSO) is a vehicle control for βNF.</p> <p>(B) Classification of modifier deletion strains according to AHR–GFP phenotype (with βNF). Group I displays <i>wt</i> phenotype. Group II contains decreased level of receptor protein. Group III contains aggregated misfolded receptor. Group IV displays the AHR that is not capable of translocating to the nucleus.</p> <p>(C) Overlay of “localization influence” layer (shadowed, red boundary) and the “pharmacology clustering” layer (black boundary) on the AHR–PIN. Group I corresponds to modules C and D. Groups II, III, and IV overlap with modules of B, A, and E, respectively. Functional annotations determined by localization influence are indicated in red, and those derived from pharmacology clustering and domain influencing studies are indicated in black. Occasional outlier nodes are noted with their corresponding module designation. See the legend of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020065#pbio-0020065-g003" target="_blank">Figure 3</a> for the color scheme of the nodes and links.</p></div

AHR–PIN versus Random PINs

<div><p>(A–D) AHR–PINs at various D_maxlevels. AHR modifiers are highlighted with bigger green nodes. A total of 48, 46, 34, and three AHR modifiers are interconnected in the AHR–PINs with D_max values of 4, 3, 2, and 1, respectively.</p> <p>(E–H) Distribution of random PINs at various D_maxlevels in histogram. Each distribution graph represents 5,000 randomly generated PINs. The density estimation curve (in red) is plotted on top of the histogram where applicable. The number of M-nodes in the AHR–PIN and the average number of M-nodes in random networks are marked in each distribution graph. See text for details.</p></div