Research Article

Systematic analysis of complex genetic interactions

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Science  20 Apr 2018:
Vol. 360, Issue 6386, eaao1729
DOI: 10.1126/science.aao1729
  • Systematic analysis of trigenic interactions.

    We surveyed for trigenic interactions and found that they are ~100 times as prevalent as digenic interactions, often modify a digenic interaction, and connect functionally related genes as well as genes in more diverse bioprocesses (multicolored nodes). PPI, protein-protein interaction.

  • Fig. 1 Triple-mutant synthetic genetic array (SGA) analysis.

    (A) Criteria for selecting query strains for sampling trigenic interaction landscape of singleton genes in yeast. The gene pairs were grouped into three general categories based on a range of features: (1) Digenic interaction strength. Gene pairs were directly connected by zero to very weak (digenic interaction score: 0 to –0.08, n = 74 strains), weak (–0.08 to –0.1, n = 32), or moderate (<–0.1, n = 45) negative digenic interactions. (2) Number of digenic interactions. Gene pairs had a low (10 to 45 interactions, n = 50), intermediate (46 to 70, n = 53), or high (>71, n = 48) average digenic interaction degree (denoted by the number of black edges of each node). (3) Digenic interaction profile similarity. Gene pairs had low (score: –0.02 to 0.03, n = 46; represented by genes A and B, which show a relatively low overlap of genetic interactions with genes K to R), intermediate (0.03 to 0.1, n = 59; represented by genes C and D, which display an intermediate overlap of genetic interactions), or high (>0.1, n = 46, represented by genes E and F, which display a relatively high level of overlap of genetic interactions) functional similarity, as measured by digenic interaction profile similarity and coannotation to the same GO term(s). Query mutant genes were either nonessential deletion mutant alleles (Δ) or conditional temperature-sensitive (ts) alleles of essential genes. (B) Diagram illustrating the triple-mutant SGA experimental strategy. To quantify a trigenic interaction, three types of screens are conducted in parallel. To estimate triple-mutant fitness, a double-mutant query strain carrying two desired mutated genes of interest (red and blue filled circles) is crossed into a diagnostic array of single mutants (black filled circle). Meiosis is induced in heterozygous triple mutants, and haploid triple-mutant progeny is selected in sequential replica pinning steps. In parallel, single-mutant control query strains are used to generate double mutants for fitness analysis. (C) Triple-mutant SGA quantitative scoring strategy. The top equation shows the quantification of a digenic interaction, where εij is the digenic interaction score, ƒij is the observed double-mutant fitness, and the expected double-mutant fitness is expressed as the product of single-mutant fitness estimates ƒiƒj. In the bottom equation, the trigenic interaction score (τijk) is derived from the digenic interaction score, where ƒijk is the observed triple-mutant fitness and ƒiƒjƒk is the triple-mutant fitness expectation expressed as the product of three single-mutant fitness estimates. The influence of digenic interactions is subtracted from the expectation, and each digenic interaction is scaled by the fitness of the third mutation.

  • Fig. 2 Functional characterization of trigenic interactions.

    (A) Frequency of negative genetic interactions within biological processes. For our analysis, we used the fraction of screened query-array combinations exhibiting negative interactions belonging to functional gene sets annotated by SAFE (spatial analysis of functional enrichment) on the global genetic interaction network (55). The “within process” category received a count for any combination in which both genes for digenic interactions or all three genes for trigenic interactions were annotated to the same term. The size of the circle assigned to each “within process” element reflects the fold increase over the background fraction of interactions (digenic = 0.023, trigenic = 0.016). Significance was assessed with a hypergeometric test; P < 0.05. Blue circles represent significant enrichment; gray circles denote no significant change. (B) Enrichment of negative digenic and trigenic interactions across four functional standards. The dashed line indicates no enrichment. The functional standards are merged protein-protein interaction (PPI) (5660), coannotation (based on SAFE terms) (7), coexpression (61), and colocalization (62). Significance was assessed with a hypergeometric test; * represents 10−4P < 0.01, ** represents P < 10−4.

  • Fig. 3 The MDY2-MTC1 double mutant: a hub on the trigenic interaction network.

    Representative digenic interactions are highlighted for MDY2 and MTC1 single-mutant query genes, and representative trigenic interactions are shown for the MDY2-MTC1 double-mutant query. The network was visualized using Cytoscape (63). Genes were chosen from representative protein complexes (8) in which ≥50% of members on the diagnostic array display genetic interactions. Negative genetic interactions (ε or τ < –0.08, P < 0.05) are depicted. All of the digenic and trigenic interactions displayed have been confirmed by tetrad analysis. Nodes are color coded on the basis of their biological roles and are labeled with gene names. Genes are grouped according to specific protein complexes.

  • Fig. 4 Enrichment of genetic interactions within bioprocesses defined by a global network of digenic interaction profile similarities.

    (A) The global digenic interaction profile similarity network (7) was annotated using SAFE (55), identifying network regions enriched for similar GO biological process terms as outlined by dashed lines. rRNA, ribosomal RNA; ncRNA, noncoding RNA; MVB, multivesicular body. (B) MDY2 digenic interactions showing bioprocess enrichments. (C) MTC1 digenic interactions showing bioprocess enrichments. (D) MDY2-MTC1 trigenic interactions showing bioprocess enrichments.

  • Fig. 5 Trigenic interactions reflect the physiology of the MDY2-MTC1 double-mutant query strain.

    (A) Endocytic membrane trafficking is impaired in the mdy2Δ-mtc1Δ double-mutant query strain. (Top) Example of tetrad analysis confirmations for the mdy2Δ-mtc1Δ-sla1Δ triple-mutant strain. (Bottom left) Endocytic uptake dynamics were examined with the Sla1–green fluorescent protein (GFP) reporter. Representative kymographs are displayed for the wild type and the mdy2Δ-mtc1Δ double mutant. (Bottom right) Lifetime of Sla1-GFP endocytic vesicle formation was quantified across ~100 different patches in two independent experiments. Error bars represent SD. (B) Peroxisome biogenesis was monitored in the wild type (wt) and in mdy2Δ, mtc1Δ, and mdy2Δ-mtc1Δ mutants using Pex14p-GFP reporter. (C) Growth response to HU and MMS for the wild type and mdy2Δ, mtc1Δ, and mdy2Δ-mtc1Δ mutants. YPD, yeast extract, peptone, and dextrose.

  • Fig. 6 Trigenic interactions are more functionally distant than digenic interactions.

    (A) Distribution of genetic interaction profile similarities of genes showing digenic and trigenic interactions. P values are based on a Wilcoxon rank sum test; **P < 10−30. (B) Frequency of negative genetic interactions between biological processes using SAFE annotations for digenic and trigenic interactions (55). The size of the circle assigned to each “between process” element reflects the fold increase over the background fraction of interactions (digenic = 0.023, trigenic = 0.016); P < 0.05 based on a hypergeometric test. The “between process” category received a count for any combinations that were not counted in the “within process” category shown in Fig. 2A. Filled blue circles represent significant enrichment, the open blue circle represents significant underenrichment, and gray circles denote no significant change. Trigenic versus digenic fold change (the ratio of trigenic interaction enrichment to digenic interaction enrichment) is represented by filled squares (black is maximal fold change; white is no fold change). In cases for which the “between process” enrichment was observed but is not significant (P < 0.05), the square is outlined with a dashed line. (C) Number of SAFE bioprocess clusters enriched for digenic or trigenic interactions.

  • Fig. 7 Relation of digenic and trigenic interaction networks.

    (A) Trigenic interaction degree distribution correlated with three quantitative features of genes on the digenic interaction network: (i) Interaction profile similarity of the two genes in the double-mutant query gene pair (bin thresholds: –0.02, 0.03, 0.1, +∞), which generates three bins for average digenic interaction profile similarity (r): –0.02 < r < 0.03; 0.03 ≤ r < 0.1; 0.1 ≤ r. (ii) Negative digenic interaction strength associated with the double-mutant query gene pair (bin thresholds: 0, –0.08, –0.1, –∞), which generates three bins for digenic interaction score (ε): ε < –0.1; –0.1 ≤ ε < –0.08; –0.08 ≤ ε < 0. (iii) Average digenic interaction degree, which represents the average number of negative genetic interactions associated with each of the genes of the double-mutant query gene pair (bin thresholds: 10, 45, 70, +∞), which generates three bins for average digenic interaction degree: 10 ≤ degree < 45; 45 ≤ degree <70; 70 ≤ degree. The bin with the highest average negative trigenic interaction degree at the intermediate interaction score cutoff (τ < –0.08) of 63.5 is shown in dark blue. (B) Essentiality determines trigenic interaction degree. Number of single mutants: 254 nonessential genes, 47 essential genes. Number of double mutants: 111 nonessential gene pairs, 40 essential or mixed essentiality gene pairs. Mean genetic interaction is represented; error bars indicate SEM; P values are based on a t test. Negative genetic interactions (ε or τ < –0.08, P < 0.05) are depicted. (C) Cumulative distribution of negative digenic and trigenic interaction score magnitudes. Pairwise significance was assessed with a Wilcoxon rank sum test. (D) Estimates of the number of digenic and trigenic interactions at the intermediate score cutoff (ε or τ < –0.08, P < 0.05). Bootstrapping was used to generate the estimate by sampling 10,000 times with replacement. Dashed lines indicate the 95% CIs; solid lines denote the estimated extent of the trigenic interaction landscape. This conservative estimate of the total number of trigenic interactions in the yeast genome covers ~26% of the interaction space. For the total genome-wide estimate, see fig. S15B and table S3.

  • Systematic analysis of complex genetic interactions

    Elena Kuzmin, Benjamin VanderSluis, Wen Wang, Guihong Tan, Raamesh Deshpande, Yiqun Chen, Matej Usaj, Attila Balint, Mojca Mattiazzi Usaj, Jolanda van Leeuwen, Elizabeth N. Koch, Carles Pons, Andrius J. Dagilis, Michael Pryszlak, Jason Zi, Yang Wang, Julia Hanchard, Margot Riggi, Kaicong Xu, Hamed Heydari, Bryan-Joseph, San Luis, Ermira Shuteriqi, Hongwei Zhu, Nydia Van Dyk, Sara Sharifpoor, Michael Costanzo, Robbie Loewith, Amy Caudy, Daniel Bolnick, Grant W. Brown, Brenda J. Andrews, Charles Boone, Chad L. Myers

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S16
    • Tables S1 to S5
    • Captions for Data S1 to S7
    • References
    Data S1
    Raw genetic interaction dataset.
    Data S2
    Digenic and adjusted trigenic interaction dataset.
    Data S3
    Query strains and plasmids list.
    Data S4
    Fitness standard for single and double mutant query strains.
    Data S5
    Diagnostic array strain list.
    Data S6
    MDY2-MTC1 genetic interaction list.
    Data S7
    Query bins for trigenic interaction space extrapolation.

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