Systematic analysis of bypass suppression of essential genes

Abstract Essential genes tend to be highly conserved across eukaryotes, but, in some cases, their critical roles can be bypassed through genetic rewiring. From a systematic analysis of 728 different essential yeast genes, we discovered that 124 (17%) were dispensable essential genes. Through whole‐genome sequencing and detailed genetic analysis, we investigated the genetic interactions and genome alterations underlying bypass suppression. Dispensable essential genes often had paralogs, were enriched for genes encoding membrane‐associated proteins, and were depleted for members of protein complexes. Functionally related genes frequently drove the bypass suppression interactions. These gene properties were predictive of essential gene dispensability and of specific suppressors among hundreds of genes on aneuploid chromosomes. Our findings identify yeast's core essential gene set and reveal that the properties of dispensable essential genes are conserved from yeast to human cells, correlating with human genes that display cell line‐specific essentiality in the Cancer Dependency Map (DepMap) project.


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Page Appendix Figure S1. Suppressor isolation, identification and confirmation 2 Appendix Figure S2. Mechanisms of suppression 3 Appendix Figure S3. Suppression by aneuploidies 4 Appendix Figure S4. Partial aneuploidies and suppressor prediction 5 Appendix Figure S5. Evolutionary properties of dispensable essential genes 6 Appendix Figure S6. Properties of dispensable essential human genes 7 Appendix Figure S7. Dispensable essential gene prediction 9 Appendix Figure S8. Bypass  Strategy for isolating bypass suppressors of essential gene deletion mutant alleles. 1179 temperature sensitive (TS) alleles were amplified from available TS strains, thereby including regions of homology to a plasmid carrying the counter-selectable marker and a haploid-selection cassette (HSC). The PCR product and linearized plasmid were cotransformed into a diploid yeast strain that was heterozygous for a deletion allele of the corresponding essential gene. The resulting diploid strains carrying an assembled plasmid were sporulated, and haploid progeny carrying the deletion allele of the essential gene and the TS allele on plasmid were selected using the haploid selection cassette present on the plasmid. For each strain, ~25 million cells were subsequently incubated at a restrictive temperature for 4 days, and suppressor colonies were isolated and tested for growth on media containing 5-FOA, which selects for loss of the plasmid. Suppressor isolation was repeated 4-6 times for each strain. In total, 380 bypass suppressor strains were isolated, for 124 essential gene deletion mutants. Suppressor identification and validation. 1) All 380 suppressor strains were sequenced whole-genome to identify the suppressor mutations. To complement the sequencing data, 89 haploid suppressor strains that had a relatively mild fitness defect were screened by Synthetic Genetic Array (SGA) analysis to identify the genomic location of the suppressor mutation as a stretch of slow-growing colonies corresponding to mutants that have a genomic location in close proximity to the suppressor mutation (Jorgensen et al., 2002). SGA data of a mutant that is suppressed by a nonsense mutation in is shown as an example. 2) Suppression interactions were validated using several assays. First, the query strain carrying the suppressor mutation was crossed to a wild-type strain, a strain deleted for a gene linked to the suppressor, and a strain carrying a deletion or conditional allele of the suppressor gene, to determine proper segregation, linkage, and loss-of-function (LOF) suppression, respectively. Next, the query strain carrying the suppressor mutation was transformed with a plasmid carrying the wild-type allele of either the suppressor gene or an empty vector control, and growth was scored. 3) Finally, for 17 suppression interactions, diploid strains were constructed that were heterozygous for both the query and the suppressor mutant alleles. Random sporulation analysis was used to verify the suppression phenotype. In the example that is shown, haploid progeny from a heterozygous MATa/ diploid strain were replica plated onto media selecting for the allele, the tao3 also carried a one of the validation assays. Dataset EV2 contains a summary of the SGA, sequencing, tetrad dissection, complementation, and random sporulation results.
Paralog is found as suppressor The fraction of all suppressor or passenger missense mutations that occur in an essential gene, that are predicted to be deleterious (SIFT score < 0.05), or that map to a disordered region of a protein or to a protein-protein interaction (PPI) interface. * = p < 0.05; ** = p < 0.005; *** = p < 0.0005. List of dispensable essential query genes that have a paralog in the yeast genome. The last column indicates whether suppression occurs via mutation of the paralog. Fraction of strains with the same suppressor gene for suppressor strains derived from query strains carrying either the same or a different TS allele for the same query gene. Significance (panels A and C) was determined using Fisher's exact test. Fitness distribution of (C) euploid and aneuploid suppressor strains, and (D) aneuploid suppressor strains in which the aneuploidy is either involved in the suppressor phenotype or is a random event not involved in suppression. Statistical significance was calculated using Mann-Whitney's U test. Distribution of aneuploidy sizes for aneuploid suppressor strains in which the aneuploidy is either involved in the suppressor phenotype or is a random event not involved in suppression. Significance was determined using Mann-Whitney's U test.
Histograms showing the fraction of suppressor strains for a given query gene that carry an aneuploidy (F) or that are suppressed by SNP (G). Only queries with 2 or more suppressor strains are shown.  Heatmaps showing genomic copy number based on 30,000 bp regions. Maps are shown for all suppressor isolates of query genes for which at least one of the suppressor isolates carried a partial aneuploidy. Where known, suppressor genes are indicated. LTR = long terminal repeat. Normalized RDN1 coverage is plotted for each of the 380 suppressor strains. Strains with a substantial increase in RDN1 coverage are highlighted. Strategy for identifying causal suppressor genes on aneuploid chromosomes. For each query gene for which suppressor strains carrying aneuploidies were identified, the corresponding parental strain (without the aneuploidy, and with the TS allele on plasmid still present) was crossed into a collection of strains each carrying a multicopy plasmid expressing one of the genes located on the identified aneuploid chromosome. Synthetic genetic array (SGA) analysis was used to isolate haploid progeny carrying the deletion allele of the essential query gene, the corresponding TS allele on plasmid, and the multicopy plasmid. The resulting strains were tested for growth in the absence of the plasmid carrying the TS allele, to determine whether overexpression of the gene located on the multicopy plasmid could rescue the lethality caused by deletion of the essential query gene.

B
Fraction of dispensable and indispensable essential query genes in S288c that are nonessential in Saccharomyces cerevisiae strain Sigma1278b. The fraction of dispensable or indispensable essential query genes that are not essential in 0, 1, 2, 3, or 4 of the species Candida albicans, Schizosaccharomyces pombe, Caenorhabditis elegans, and Homo sapiens cell lines KBM7/HAP1. Different orthology mapping tools were used to identify orthology relationships. The fraction of dispensable essential query genes that are not essential in 0, 1, 2, 3, or 4 of the species indicated in (B), split by the number of suppressors that were identified for a query gene (C) or by the average fitness of the obtained suppressor strains for a query gene (D).
All p values were calculated using a 1-sided Fisher's exact test. Distribution of the fraction of human cell lines in which 1-to-1 orthologs of nonessential, dispensable essential and indispensable essential yeast genes are essential. Statistical significance was determined using a Mann-Whitney's U test. Fraction of nonessential, dispensable essential, and indispensable essential query genes in Saccharomyces cerevisiae that are nonessential, dispensable essential (= context-depedent or selective essential), or indispensable essential (= core essential) in human cell lines. * = p < 0.05 (Fisher's exact test). Enrichment of dispensable essential human genes for various gene-and protein-level properties. Fisher's exact or Mann-Whitney's U tests were performed to determine statistical significance of the results. Fold enrichment for the absence of orthologs of human dispensable essential genes, compared to human indispensable essential genes, in 11 species. P values were calculated using Fisher's exact test. For all analyses, multiple different thresholds for calling a gene essential in any given cell line or dispensable essential across cell lines were used.