Genome-wide consequences of deleting any single gene

Abstract

Loss or duplication of chromosome segments can lead to further genomic changes associated with cancer. However, it is not known whether only a select subset of genes is responsible for driving further changes. To determine whether perturbation of any given gene in a genome suffices to drive subsequent genetic changes, we analyzed the yeast knockout collection for secondary mutations of functional consequence. Unlike wild-type, most gene knockout strains were found to have one additional mutant gene affecting nutrient responses and/or heat-stress-induced cell death. Moreover, independent knockouts of the same gene often evolved mutations in the same secondary gene. Genome sequencing identified acquired mutations in several human tumor suppressor homologs. Thus, mutation of any single gene may cause a genomic imbalance, with consequences sufficient to drive adaptive genetic changes. This complicates genetic analyses but is a logical consequence of losing a functional unit originally acquired under pressure during evolution.

Figure 1. Heat-ramp stress test detects phenotypic variation within knockout strains

(A) Potential heterogeneity within individual knockout strains in the yeast knockout (YKO) collection consisting of ~5,000 unique single-gene deletion strains in which non-essential gene were replaced with a bar-coded kanamycin resistance cassette. (B) Original knockout strains (BY MATa) and their single-cell-derived substrains were heat-stressed using a programmable thermocycler. (C) Yeast viability was assessed as colony-forming units (cfu per 5 µl starting concentration) following heat-ramp treatment. Data are presented as mean ±SD for 3 independent experiments (all data generated are presented). See also Figure S1.

Figure 2. Widespread heterogeneity within knockout strains

(A) Six morphologically indistinguishable colonies from each of 250 randomly selected knockout strains (BY MATa) were archived as 1,500 substrains. (B) Example results of heat-ramp stress tests on colony-derived substrains from the 250 randomly selected YKOs before and after treatment in 3 independent experiments; first test performed prior to freezing substrains. Colony counts differed significantly between substrains from BY MATa YKOs Δfrt1, Δizh2, Δgyp5, Δrpl1A, and Δypl191c (ANOVA, p<10⁻⁵). Variant Δypl191c substrain #6 is an example false negative as it was below the cut-off threshold (set to avoid false positives in this screen). (C) Summary of observed results from all experiments performed. Inferred proportion was estimated using a mathematical model. See Supplemental Experimental Procedures. (D) Wild type substrains were analyzed as for knockout substrains in panel B. (E) Example PCR genotyping results of variant substrains from panel B. See also Figure S2, Tables S1 and S2, and Supplemental Experimental Procedures.

Figure 3. Variant growth phenotypes of knockout strains in low amino acids

(A) Variant overgrowth phenotypes among substrains from example YKO strains in the 749 group (BY MATa, see text) plated simultaneously on control (SCD_CSH) and low amino acid media (SCD_ME). The first test was performed before freezing substrain stocks. (B) Percent (observed) of original YKO strains with at least one substrain among six tested with obvious variation for indicated phenotypes. Observed results from Figure 2C are plotted for direct comparison (black bar). (C) Summary of results from heat-ramp stress and overgrowth assays for 3,809 substrains of the 749 original YKOs. Rationale for the number of substrains tested is found in Supplemental Experimental Procedures; groupings do not overlap and were grouped without regard to substrain variation. See also Tables S3 and S4.

Figure 4. Prevalence of partially/fully fixed secondary mutations in knockout populations

(A) Diagram of a tetratype tetrad in which the knockout (Δ) and second mutant gene (red star) segregate independently. (B) Example results for tetrads (tetratypes) produced from substrains with second gene mutations affecting overgrowth and heat-stress phenotypes that segregate independently of the knockout locus conferring kanamycin resistance (Kan). (C) Number of original knockouts (BY MATa) with/without variant substrains that have a second mutation responsible for heat ramp-sensitive (HR) and low amino acid overgrowth (LAA) phenotypes based on tetrad analysis. Final frequency estimates: 17.1% of YKOs with invariant substrains plus the percent of YKOs with variant substrains from Figure 2 [(44%×17.1%)+56%] and Figure 3 [(28.3%×17.1%)+71.7%]. See also Table S5.

Figure 5. Recurrent secondary mutant genes indicate knockout-driven selection

(A) Flow chart for the 40 pairs of independent knockout strains tested by complementation. (B) Frequency of knockout strain pairs with the same deleted gene in which both strains (BY MATa and BY MATα) have substrains bearing the same phenotype constellations known to be caused by a secondary mutation (from Figure 4). (C) Summary of complementation tests (analysis of diploids after mating substrains with the indicated BY MATa and BY MATα YKOs). Of the 40 YKO pairs with secondary mutations, both partners of 26 pairs share the same secondary phenotypes (black gene names for Parent 2), 15 of which contain secondary mutations in the same gene or complementation group (blue bars), while the remaining 11 define two different complementation groups (open circles). Complementation groups shared by >1 BY MATa strain are color-coded (brightly colored solid circles); secondary mutations in unique complementation groups among all BY MATa strains, or that are shared only by their corresponding MATalpha strain (gray solid circles); overgrowth-suppressor phenotypes not testable by complementation between unique MATa strains (black filled circles). See also Figure S4, Table S5, Table S6, and Supplemental Experimental Procedures.

Figure 6. Knockout-driven evolution by the same and different paths

(A) Example DNA sequence chromatograms of independently acquired secondary mutations in WHI2 in each of two independently constructed knockout strains of STE20. (B) Demonstration that the secondary gene/locus and corresponding phenotypes segregate independently from the knockout locus in knockout substrains. (C) DNA sequence results for strains with WHI2 mutations. (D) Model depicting influences of gene deletion versus environmental conditions on genome evolution. (E) Identification of secondary mutations by whole genome sequencing. All mutations unique to the two phenotypically similar spore-derived substrains are shown. Additional intergenic mutations and a mutation in mitochondria-encoded ATP6, which is not expected to segregate 2:2 in tetrads, are unlikely to be of consequence. Presumed passenger mutations identified by genome sequencing are summarized in Table S6.

Figure 7. Model of genome evolution driven by gene mutation

(A) Most yeast knockout strains are quasispecies harboring prevalent additional mutations. (B) Though there are potentially several evolutionary paths to compensate for the loss of any one gene, independently constructed knockouts of the same gene tend to evolve similar phenotypes, often by acquiring secondary mutations in the same gene (parallel evolution), indicating a selection process driven by the specific knockout. (C) Deletion of different genes can drive the selection of mutations in the same genes.