Hierarchical modularity and the evolution of genetic interactomes across species

Abstract

To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional crosstalk between biological processes, suggesting general design principles of genetic interactomes.

Figure 1. Overview of the genes included in this study

(A) Species distribution of the genes in the fission yeast E-MAP. Species distribution data obtained from Pombase. For a complete list of the genes in each category, see Table S1. (B) Coverage of the non-essential genes with respect to different biological processes. Shown are genes present on the library array from Bioneer (http://pombe.bioneer.co.kr/) only (blue), as queries only (red) and present as both arrays and queries (orange). For each process, the total number of non-essential genes present in the E-MAP is given as the figure in brackets. For a full assignment of genes to different biological processes, see Table S1.

Figure 2. Hierarchical clustering of genetic interaction profiles

Genes are grouped based on the similarity of their genetic interaction profiles. Modules discussed in the text are magnified and labeled and uncharacterised genes within these modules are highlighted in bold red. Genes are labeled using their S. pombe common name, followed by the common names of their S. cerevisiae orthologs if present (with paralogs separated by underscores). Only genes with at least one similarity score ≥ 0.1 are included in this representation (a complete dataset is provided in Dataset S3.

Figure 3. Functional Characterization of SPAC1610.01 and SPCC2H8.05c

(A) Temperature sensitive phenotype of SPAC1610.01Δ. Serial dilutions of wild type (WT), SPAC1610.01Δ and SPAC1610.01Δ prp43-DAmP mutants grown at 30°C, 37°C and 16°C. (B) Intron accumulation in SPAC1610.01Δ measured by qRT-PCR expressed as mean fold change over WT. Error bars indicate standard errors derived from at least two replicate experiments. See Figure S3A for a semi-quantitative PCR experiment. (C) SPCC2H8.05cΔ results in sensitivity to the DNA damaging agent, MMS (Methyl methanesulfonate). (D) MMS-induced S-phase delay in SPCC2H8.05cΔ. (E) Quantification of the S-phase distribution from (D). 3 hours after 0.03% MMS exposure, significantly fewer (p<0.01) SPCC2H8.05cΔ cells are in S-phase compared to WT. Means and standard errors (shown as vertical lines) were derived from five independent experiments.

Figure 4. Conserved Functional Modules

(A) Groups of genes with highly correlated genetic interaction profiles in both S. pombe and S. cerevisae are shown. S. cerevisiae gene names were used for labeling, as many of the S. pombe orthologs lack common names. Modules are manually grouped and colored according to the biological process they are involved in. Modules from the insets are boxed and correspond to the Set3 complex (B), the Rpd3C(S) (C) and the DSC complex (D). A full list of the modules identified, and their S. pombe counterparts, is given in Table S2. Blue edges correspond to pairs of genes that have high E-MAP similarity scores, green edges represent pairs of factors that are physically associated from previous studies whereas dashed red edges represent paralogs within one species. For the immunoprecipitation assay in (D), Dsc2 binding proteins were immunopurified from detergent lysates of wild-type and dsc2Δ cells using anti-Dsc2 affinity purified polyclonal antibody. Equal amounts of total (lanes 1 and 2) and unbound fractions (lanes 3 and 4) along with 10× bound fractions (lanes 5 and 6) were immunoblotted using the indicated HRP-conjugated antibodies. See Figure S6 for an additional experiment confirming that Dsc2 binds to Ubx3, but not Ubx2.

Figure 5. Conserved Network Features

Pairs of genes which interact genetically in S. pombe (A) and S. cerevisiae (B) are more likely to display the same knockout phenotype and share membership of the same biological process and protein complex. Data are expressed as fold change over all gene pairs in the E-MAP. S. pombe and S. cerevisiae process annotations are presented in Table S1. (C) Members of known protein complexes have more genetic interactions. Bar height represents the median normalized genetic interaction degree for different categories of genes. (D) Sequence orphans have fewer genetic interactions (E) Non-essential S. pombe genes, whose S.cerevisiae ortholog is essential, have more genetic interactions than genes whose ortholog is non-essential. The same applies for S. cerevisiae. For (C), (D) and (E), the normalized genetic interaction degree for a gene is the number of significant genetic interactions for that gene, divided by the total number of measured interactions involving that gene. Error bars are calculated using 1000-fold bootstrap resampling. P-values are calculated using a two-sided Mann-Whitney U test.

Figure 6. Hierarchical Conservation of Genetic Interactions

(A) Calculated percentage of conserved genetic interactions for different categories of gene pairs. Estimates were derived by comparing the observed cross-species conservation of genetic interactions to the within-species reproducibility of genetic interactions in the same category. See Supplemental Methods for full details. (B) A scatter plot of S. pombe and S. cerevisiae genetic interaction scores for pairs of genes belonging to different categories. r values are calculated using Pearson’s correlation coefficient. (C) A model for the evolution of genetic interactions with different colors representing the level of conservation. Genetic interactions between gene pairs whose products are co-complexed are highly conserved (orange), those between genes participating in the same biological process are less conserved (green), while interactions between genes involved in distinct biological processes are poorly conserved (blue).

Figure 7. Conservation of Functional Cross-talk Between Biological Processes

(A) Genetic cross-talk between distinct biological processes in S. pombe. The size of each circle represents the fraction of significant interactions between two processes compared to the fraction of significant interactions between all annotated genes. Purple circles represent significant enrichment of interactions between processes. Significance is assessed using a two-tailed binomial test, and the Bonferonni method is used to correct for multiple testing. Enrichment values and p-values are given in Table S3. For the S. cerevisiae enrichment map and data see Figure S7 and Table S3. (B) Enrichments observed in S. pombe are highly correlated with the ones in S. cerevisiae. Pairs of processes that are highly connected in both species are colored purple, highlighted inside the yellow box and are drawn as a network diagram in (C). Within process enrichments (i.e. the diagonal in Figure 7A) are not shown. (C) Conserved links between biological processes. Links represent pairs of processes that are linked by at least 1.4 times the background rate of genetic interactions in both species.