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The Synthetic Genetic Interaction Spectrum of Essential Genes

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The Synthetic Genetic Interaction Spectrum of Essential Genes LETTERS The synthetic genetic interaction spectrum of essential genes Armaity P Davierwala1, Jennifer Haynes2, Zhijian Li1,2, Rene´e L Brost1, Mark D Robinson1, Lisa Yu3, Sanie Mnaimneh1, Huiming Ding1, Hongwei Zhu1, Yiqun Chen1, Xin Cheng1, Grant W Brown3, Charles Boone1,2,4, Brenda J Andrews1,2,4 & Timothy R Hughes1,2,4 The nature of synthetic genetic interactions involving essential mapping these genetic interactions2,3. Essential gene function is also genes (those required for viability) has not been previously buffered by both nonessential and other essential genes because hypomorphic (partially functional) alleles of essential genes often http://www.nature.com/naturegenetics examined in a broad and unbiased manner. We crossed yeast strains carrying promoter-replacement alleles for more than have synthetic lethal interactions with deletion alleles of nonessential half of all essential yeast genes1 to a panel of 30 different genes and hypomorphic alleles of other essential genes2,3,7.Genetic mutants with defects in diverse cellular processes. The resulting interactions among essential genes have not been examined system- genetic network is biased toward interactions between atically and objectively because of the inherent difficulty in creating functionally related genes, enabling identification of a and working with hypomorphic alleles. previously uncharacterized essential gene (PGA1) required for specific functions of the endoplasmic reticulum. But there are also many interactions between genes with dissimilar functions, Query gene (crossed to array) Gene Ontology labels (array strains) suggesting that individual essential genes are required for buffering many cellular processes. The most notable feature of the essential synthetic genetic network is that it has an interaction density five times that of nonessential synthetic 2005 Nature Publishing Group Group 2005 Nature Publishing genetic networks2,3, indicating that most yeast genetic © -linked glycosylation -linked interactions involve at least one essential gene. SLT2 ERG11 SEC18 CDC12 SEC7 SEC15 SEC1 EXO84 KRE1 BNI1 ACT1 ARP2 CDC42 RFC5 RAD50 MRC1 CSM3 RPA49 DBP10 LRP1 NOP1 NIP7 CDC40 RPS17A SET2 LOS1 ARL3 BIM1 PRT1 SRB6 Lipid biosynthesis Lipid metabolism GPI anchor metabolism Cytokinesis Morphogenesis Exocytosis of cell polarity Establishment docking/exocytosis Vesicle Glycoprotein biosynthesis Bud site selection N Actin polymerization Ubiquitin-dependent proteolysis DNA repair DNA-dependent DNA replication DNA replication Response to DNA damage elongation DNA strand G1/S transition rRNA transcription mRNA splicing mRNA polyadenylation of spliceosome Formation mRNA splicing II transcription Regulation of Pol G1-specific transcription ER to Golgi transport elongation Lagging strand Amino acid biosynthesis Nucleotide catabolism The complete analysis of gene-deletion mutations for each of the B6,000 known or predicted genes in the budding yeast Saccharomyces cerevisiae identified B5,000 viable deletion mutants and B1,000 essential genes4. Many (if not most) genes are nonessential because multiple pathways of eukaryotic cells buffer one another to create systems that are robust to environmental and genetic perturbation5,6. Large-scale identification of ‘synthetic lethal’ phenotypes among nonessential genes, in which the combination of mutations in two genes causes cell death or reduced fitness, provides a means for Array strains Array Figure 1 Matrix display of SGA data. Left, synthetic genetic interactions between a query and array strain are represented as red rectangles; black indicates no interaction. Right, Gene Ontology classifications of genes mutated in the array strains are indicated; black rectangles indicate that the gene mutated in the array strain has the given annotation. We determined row and column order by hierarchical clustering analysis followed by rearrangement of higher-level nodes to achieve a diagonal appearance. Full spreadsheets are posted on our project website. 1Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6, Canada. 2Departments of Medical Genetics and Microbiology and 3Biochemistry, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada. 4These authors contributed equally to this work. Correspondence should be addressed to T.R.H. ([email protected]). Received 3 May; accepted 27 July; published online 11 September 2005; doi:10.1038/ng1640 NATURE GENETICS VOLUME 37 [ NUMBER 10 [ OCTOBER 2005 1147 LETTERS To examine systematically the basic principles of genetic networks Query strain (crossed to array) with essential genes, we used synthetic genetic array (SGA) technol- ogy2 and carried out large-scale analysis of synthetic genetic interac- ∆ tions among temperature-sensitive conditional alleles and conditional sec1-1 sec15-1 rfc5-1 bni1- arp2-14 expression alleles (using the tetracycline (tet) promoter) of essential SEC2 yeast genes1. In the tet-promoter strains, the expression of the gene of SEC8 DBF4 interest is controlled by a promoter that can be shut off by adding CDC42 doxycycline (an analog of tetracycline) to the growth medium. In SGA SEC10 analysis, a mutation in a particular query gene is first crossed to an CDC5 input array of single mutants and then a series of robotic pinning steps SEC3 EXO84 is used to generate an output array of double mutants, which is scored SEC1 2 for fitness defects relative to either of the single mutants .Wecarried SEC5 out 30 SGA screens using an array of tet-promoter mutants in 575 ABD1 Both alleles essential genes1. The set of query genes represents diverse biological CDC34 tet-promoter only CDC16 ts only processes and the query mutations include 14 temperature-sensitive CDC2 Neither and 2 promoter mutation alleles of essential genes and 14 deletion CDC27 alleles of nonessential genes (Fig. 1 and Supplementary Table 1 CDC9 online). We examined both the tet-promoter strains and the tempera- DFR1 KIN28 ture-sensitive strains at a semipermissive condition: we scored inter- CDC11 actions at the semipermissive temperature for temperature-sensitive strains in array Gene mutated CDC19 (i.e., query) strains, and because of a delay of several hours in the onset RNA1 of growth cessation in tet-promoter strains, virtually all of the tet- SEC15 CDC25 http://www.nature.com/naturegenetics promoter (i.e., array) strains formed at least a small colony. We SEC14 evaluated growth phenotypes both with and without doxycycline in CDC6 the medium, because tet-promoter strains may show abnormal MCM10 phenotypes even with the promoter ‘on’1. We identified instances of CDC3 RFC5 smaller colony size for a double mutant versus either single mutant grown under the same conditions and confirmed them by random Figure 2 Allele specificity of essential gene interactions. Genetic spore analysis (RSA). Although there were variations between strains interactions obtained by crossing five query strains into an array containing with respect to growth rate as single mutants (in the case of tet- strains with both tet-promoter and temperature-sensitive (ts) alleles of 42 promoter strains, this is presumably a result of the varying response of essential genes are shown. cells to depletion of different transcripts1), we obtained interactions at roughly comparable frequencies from strains with both mild and severe growth phenotypes (Supplementary Methods online). This compared with distribution of number of interactions for randomly analysis resulted in a genetic network of 567 interactions, 386 of which reshuffled row and column labels; Fig. 1). In some screens, these 2005 Nature Publishing Group Group 2005 Nature Publishing © occur between two essential genes, encompassing 286 essential genes relationships were easily rationalized. For instance, most interactions (Fig. 1). Only two interactions (between RFC5 and RFC2 and with RFC5, which encodes a DNA replication–repair–chromatid between SEC15 and MYO2) were found among the 4,922 known cohesion factor9–11, occurred with components of the DNA replication yeast genetic interactions cataloged by the GRID database8.Allthe (P ¼ 6.4 Â 10À11) or chromosome segregation machinery (chromo- data, including RSA images, are available from our project website (see some cycle, P ¼ 4.2 Â 10À10; Fig. 3). Synthetic interactions with LRP1, URL in Methods). which encodes an accessory component of the exosome that processes To determine whether the interactions we obtained were general or and degrades many types of RNA12,13, were enriched for genes allele specific, we created a second array of strains carrying both tet- involved in RNA processing (P ¼ 9.1 Â 10À7) and ribosome biogen- promoter and temperature-sensitive alleles of 42 essential genes for esis (P ¼ 1.0 Â 10À5; Fig. 3). Synthetic interactions with SLT2, a gene which temperature-sensitive alleles could be obtained. We then involved in cell-wall organization and biogenesis14, were biased toward crossed this array to five query strains that interacted with these lipid biosynthesis (P ¼ 7.1 Â 10À10), exocytosis (P ¼ 2.1 Â 10À5), genes (Fig. 2). We detected a total of 40 interactions by at least one of ergosterol biosynthesis (P ¼ 8.2 Â 10À5)andproteinaminoacid the two allele types (tet-promoter or temperature-sensitive alleles), of glycosylation (P ¼ 0.0013; Fig. 3). We selected the query genes to which 22 were common to both allele types, showing that there is a represent diverse cellular functions, and consequently, their interaction strong tendency for genetic interactions
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