Conservation and rewiring of functional modules revealed by an epistasis map in fission yeast

Abstract

An epistasis map (E-MAP) was constructed in the fission yeast, Schizosaccharomyces pombe, by systematically measuring the phenotypes associated with pairs of mutations. This high-density, quantitative genetic interaction map focused on various aspects of chromosome function, including transcription regulation and DNA repair/replication. The E-MAP uncovered a previously unidentified component of the RNA interference (RNAi) machinery (rsh1) and linked the RNAi pathway to several other biological processes. Comparison of the S. pombe E-MAP to an analogous genetic map from the budding yeast revealed that, whereas negative interactions were conserved between genes involved in similar biological processes, positive interactions and overall genetic profiles between pairs of genes coding for physically associated proteins were even more conserved. Hence, conservation occurs at the level of the functional module (protein complex), but the genetic cross talk between modules can differ substantially.

Fig. 1

Data set overview. (A) Functional classification of the genes contained within the S. pombe E-MAP. The map contains 550 genes that were classified into 11 functional categories (table S4). (B) Distribution of interaction scores for pairs of genes corresponding to physically interacting proteins (green) and noninteracting proteins (black). (C) Distribution of Pearson correlation coefficients of the genetic interaction profiles for the same set of genes used in (B). For a complete list of PPIs used in this analysis, see table S2.

Fig. 2

The S. pombe chromosome function E-MAP. A section of the E-MAP with specific regions of interest annotated. Further highlighted are the factors involved in DNA repair/recombination (1), as well as two complexes contained within the chromatin remodeling/modification region: the SWR-C chromatin remodeling complex (2) and the Set1, Lid2, and CPF complexes (3). The names of the budding yeast orthologs are shown in parentheses (table S3). The final data set consists of 118,575 measurements and contains 5772 negative (S score ≤ -2.5) and 1812 positive (S score ≥ 2) interactions.

Fig. 3

Characterization of genes involved in the RNAi pathway. (A) Genetic profiles for genes involved in RNAi with individual protein complexes or processes annotated. (B) Schematic of the centromeric region of chromosome 1 with the position of the ura4⁺ reporter gene within the otr1 region. (C) Loss of Rsh1p abolishes heterochromatic silencing of the ura4⁺ reporter gene inserted at the outer repeat region of centromere 1 (otr1∷ura4⁺). NS, nonselective; FOA, counterselective; -URA, uracil-deficient media. (D) Levels of dh transcripts analyzed by reverse transcription polymerase chain reaction (RT-PCR) using RNA prepared from indicated strains. (E) Loss of siRNAs derived from dg/dh repeats in rsh1Δ detected by Northern blotting. nt, nucleotides. (F)Rsh1 localizes to outer (otr) centromeric repeats. An epitope-tagged version of Rsh1 (mycRsh1) was used to perform chromatin immunoprecipitation (ChIP). wce, whole-cell extract. (G) Rsh1 is required for localization of Ago1. Localization of mycAgo1 at otr1∷ura4⁺ in wild-type and rsh1Δ cells was assayed using ChIP. leu1 is an internal loading control for ChIP experiments. (H) Effect of rsh1Δ on heterochromatin assembly at centromeric repeats. Levels of histone H3 lysine 9 dimethylation (H3K9me2) and Swi6/HP1 at otr1∷ura4⁺ were assayed using ChIPs. (I and J) Loss of Mediator and RNAPII subunits affects centromeric silencing. The levels of transcripts corresponding to dh centromeric repeats were analyzed by RT-PCR. leu1 and act1 are used as internal loading controls.

Fig. 4

Modular conservation of genetic interaction patterns. A set of 239 one-to-one orthologs (table S3) was used for the analysis. (A) Conservation of positive and negative genetic interactions based on comparison with S. cerevisiae. Conservation rates are higher for the subset of negative interactions between genes with the same functional annotation and the subset of positive interactions corresponding to known PPIs in S. cerevisiae. Pairs of genes whose proteins are physically associated or functionally related did not contribute significantly to the general trends (second bar), because removal of these pairs (third bar) resulted in similar conservation rates. P values were determined using a two-sided Student's t test (7). (B) Scatter plot of Pearson correlation coefficients of genetic interaction profiles. Sc, S. cerevisiae; Sp, S. pombe. (C) Distribution of the cross-species Pearson correlation coefficient of genetic profiles.

Fig. 5

Rewiring of the conserved functional modules. (A) Comparison of genetic interaction profiles of the SWR-C in S. cerevisiae and S. pombe. Analogous sets of genetic interactions from the two organisms are shown (database S2). (B) Genetic cross talk between functional modules. Modules are represented as circles or boxes (in yellow if the interactions within the module are primarily positive). Negative and positive interactions between modules are represented as blue and yellow lines, respectively. The diagram was generated using the method described in (41).