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. 2010 Dec 10;143(6):991-1004.
doi: 10.1016/j.cell.2010.11.021.

Functional Overlap and Regulatory Links Shape Genetic Interactions Between Signaling Pathways

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Free PMC article

Functional Overlap and Regulatory Links Shape Genetic Interactions Between Signaling Pathways

Sake van Wageningen et al. Cell. .
Free PMC article

Abstract

To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy, and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multiprocess control.

Figures

Figure 1
Figure 1. Expression Profiles of Kinase/Phosphatase Single Gene Deletions
(A and B) Activity profiles of all deletion strains, ranked as box-whisker plots for kinases (A) and phosphatases (B), showing fold changes (vertical axis), with significantly changing genes (p < 0.05, FC > 1.7) as red dots and unresponsive genes as black dots. Green triangles indicate the doubling time of each mutant (-log2 relative to WT). Dashed gray lines indicate 1.7-fold change. The solid gray line is the threshold for distinguishing deletions with significant profiles (≥8 genes changing) versus deletions that behave similarly to WT (<8 genes changing). This threshold is based on the maximum number of changes observed in the 200 WT profiles, excluding the WT variable genes (Experimental Procedures). (C) Lanes 1–7 are expression profiles of strains indicated to the right. All genes with significantly changed expression in any single mutant (p < 0.05, FC > 1.7) are depicted, with gene names on top. STE20, STE11, STE7 and FUS3 are the MAPK components of the mating pheromone response pathway. FUS3 is redundant with KSS1 and the profile of the double mutant is therefore shown in lane 4. Profiles of the single mutants are depicted in Figure 4C. SSK2, PBS2 and HOG1 are MAPK components of the HOG pathway. The opposite effects of the HOG pathway on some of the genes affected by the mating pathway agrees with inhibition of the mating pathway by the HOG pathway (Chen and Thorner, 2007). See also Figure S1.
Figure 2
Figure 2. Expression Profiles of Genetically Buffered Pairs
(A–K) Single and double deletion gene expression scatter plots of four genetically buffered pairs. In each scatter plot the normalized, dye-bias corrected and statistically modeled fluorescent intensity value is plotted for each gene. For each mutant this is the average of four measurements. For WT this is the average of 200 cultures grown throughout the project. Genes with significant increase or decrease in mRNA expression (p < 0.05, FC > 1.7) are represented by yellow and blue dots respectively. Gray dots are all other genes. (L) Scatter plot of all genes that have a significant change in mRNA expression in either bck1Δ ptp3Δ (J), slt2Δ ptp3Δ (K) or in both double mutants. The log2 FC is plotted for each of these genes in both double deletions, showing that the same mRNAs are changing in both strains. See also Figure S2.
Figure 3
Figure 3. Kinase-Phosphatase Buffering Is Caused by Phosphatase-Mediated Inhibitory Crosstalk between Kinase Pathways
(A) The bck1Δ ptp3Δ and slt2Δ ptp3 kinase-phosphatase double mutants are sensitive to elevated temperature. Ten-fold dilutions of cultures were spotted onto plate and incubated at 30°C or 37°C. (B) The bck1Δ ptp3Δ and slt2Δ ptp3 kinase-phosphatase double mutants show more sensitivity to zymolyase. Bars and standard deviations are based on the average of three. (C) Active, phosphorylated Hog1 is increased in the bck1Δ ptp3Δ and slt2Δ ptp3 kinase-phosphatase double mutants. Immunoblots for phosphorylated Hog1 (top), all Hog1 (middle) and Tubulin (bottom). Lane 1 is a positive control of WT exposed to 0.4 M NaCl for five minutes prior to harvesting. (D) All genes with significant changes in bck1Δ ptp3Δ or slt2Δ ptp3Δ (p < 0.05, FC > 1.7) are depicted. Lane 7 shows the same genes for the ptp2Δ ptp3Δ expression profile. (E) As in (C). (F) Model of interactions for the buffering observed between PTP3-SLT2 and PTP3-BCK1. Gray lines indicate buffering. Black line indicates redundancy. The two arrows between Slt2 and Ptp2 indicate that this activation may be direct or indirect.
Figure 4
Figure 4. Expression Profiling Reveals Three Different Genetic Buffering Interactions
For each set of three profiles all genes with changes in mRNA expression in any single profile are shown (p < 0.05, FC > 1.7). (A) Complete redundancy whereby the single mutants have less than eight genes changing significantly and the double have more than eight. (B) Quantitative redundancy, whereby one single mutant shows no significant profile (<8 genes p < 0.05, FC > 1.7), the other single mutant has a significant profile and in the double the same genes change to a higher degree. (C) Mixed epistasis. Here at least 8 more genes change significantly in the double versus the two singles, with at least 8 genes behaving in other ways than in complete or quantitative phenotypes. The two bars below the FUS3-KSS1 profiles indicate the two gene sets selected for modeling (Figure 5). (D) Unclassified buffering interactions due to inviability of the double mutant (Table 1). (E) Quantification of the profiles shown in B, plotted for all genes with significant (p < 0.05, FC > 1.7) changes in mRNA expression in any one single or double mutant strain. M is the log2 ratio of normalized fluorescent mRNA expression in the mutant divided by WT. Asterisks indicate strains showing aneuploidy in the double mutant whereby all genes on aneuploid chromosomes were excluded from analyses. (F) Complete redundancy can result from two proteins able to directly substitute for all of each other’s activity. (G) Expression profiles of the ark1Δ prk1Δ double mutant and the target sla1Δ. All genes are depicted with significant changes (p < 0.05, FC > 1.7) in mRNA expression in any profile. (H) Quantitative redundancy resulting from the ability of two proteins to directly substitute for each others activity qualitatively, but not quantitatively. (I) Immunoblot as described in Figure 3C.
Figure 5
Figure 5. Mechanisms of Mixed Epistasis: Partial Overlap in Function Coupled to Unidirectional Repression
(A) A minimal mixed epistasis pattern consisting of two gene sets selected from the FUS3-KSS1 profiles (Figure 4C). The names “mating” and “filamentous growth” are based on the enrichment for Ste12 and Tec1 transcription factor binding sites respectively, upstream of each gene, as indicated in the vertical bars. (B) Experimentally-derived/literature-based model for regulation of the mating and filamentous growth gene sets under basal, unactivated conditions in WT cells. The model omits details such as activation of Ste12 and Tec1 transcription factor complexes through phosphorylation of the Dig1, Dig2 repressors (Chen and Thorner, 2007). The black line between Kss1 and Fus3 indicates redundancy. (C) Model for fus3Δ. (D, F, and H) Boolean solution models for a minimal mixed epistasis pattern. (E, G, and I) The accompanying state transitions for one of the eight simulated initial states (Experimental Procedures). R1 and R2 indicates the activities of the two responder gene sets, depicted for the mutants relative to WT, similarly to the expression profiles, with blue indicating decrease, black no change and yellow increase in expression. K1 and K2 indicate the absolute activities of the regulator nodes with red for True and white for False. The numbers at the bottom indicate the first five time steps of simulation. See also Figure S4, Table S2, and Table S3.
Figure 6
Figure 6. Multiprocess Control through Signaling Components with Mixed Epistasis
Yellow circular nodes represent the single and double mutant profiles for the pairs with mixed epistasis (Table 1). Single mutants with no significant changes are not shown. Square nodes (numbered 1–50) indicate gene sets that show differential expression patterns across this set of mutants, obtained by QT clustering all genes with a significant change (p < 0.05, FC > 1.7) in any one profile. Yellow edges between mutants and gene sets indicate that a gene set is upregulated in the mutant, blue indicates downregulation. Diamonds indicate significant (p < 0.05) enrichment of a particular GO category in the gene set. Only the top three categories are shown. Three-quarters of the gene sets are significantly enriched for at least one GO category. Triangles depict enrichment for transcription factor binding sites in the gene set, indicating which transcription factor may be mediating the response. See also Figure S5.

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