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. 2007 Mar 6;104(10):3925-30.
doi: 10.1073/pnas.0610642104. Epub 2007 Feb 28.

Systematic genome instability screens in yeast and their potential relevance to cancer

Karen W Y Yuen  1 , Cheryl D Warren, Ou Chen, Teresa Kwok, Phil Hieter, Forrest A Spencer
Affiliations

Systematic genome instability screens in yeast and their potential relevance to cancer

Karen W Y Yuen et al. Proc Natl Acad Sci U S A. .

Abstract

To systematically identify genes that maintain genome structure, yeast knockout mutants were examined by using three assays that followed marker inheritance in different chromosomal contexts. These screens identified 130 null mutant strains exhibiting chromosome instability (CIN) phenotypes. Differences in both phenotype severity and assay specificity were observed. The results demonstrate the advantages of using complementary assays to comprehensively identify genome maintenance determinants. Genome structure was important in determining the spectrum of gene and pathway mutations causing a chromosome instability phenotype. Protein similarity identified homologues in other species, including human genes with relevance to cancer. This extensive genome instability catalog can be combined with emerging genetic interaction data from yeast to support the identification of candidate targets for therapeutic elimination of chromosomally unstable cancer cells by selective cell killing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Three marker loss screens. (a) Haploid yeast knockout mutants (ykoΔ) containing a chromosome fragment (CF, blue line whose centromere is depicted as a circle) and ade2-101 were generated. Red colony color is caused by accumulation of pigment because of a block in adenine production caused by the ade2-101 (ochre) mutation. This block is relieved in the presence of the SUP11 gene (blue rectangle) located on the telocentric arm of the CF, encoding an ochre-suppressing tRNATyr. Cells that contain the CF are therefore unpigmented, whereas cells that lose the CF develop red color (–29). Colonies exhibiting unstable inheritance of this CF develop red sectors. (b) Homozygous diploid yeast knockout mutants were tested for bimater phenotype. For example, loss of the MATa allele (depicted in gray, Left) causes the development of an α-type mating cell, which is detected by its ability to mate with a MATa tester strain containing complementing auxotrophy to support selection of mated diploids. Mutant strains exhibiting unstable inheritance of the MAT locus will lose either allele in individual cell and exhibit a bimater phenotype in a population. The mutants in the squares show elevated formation of mated cells when exposed to either MATα or MATa testers (18). (c) MATα haploid yeast knockout mutants were tested for elevated frequency of ALF cells. Loss of the MATα locus in haploids results in dedifferentiation to a-mating type. The presence of these cells is detected by selection for mated products after exposure to a MATα tester strain.
Fig. 2.
Fig. 2.
One hundred thirty nonessential yeast CIN genes. The small diagram (Upper Left) depicts the distribution of the 293 mutants identified initially across the three screens. The numbers in parentheses denote single-assay knockouts confirmed in independent transformants, which are included in the detailed diagram to the right. The detailed diagram (Right) summarizes the 130 high confidence genes described in SI Table 3. (For the other 163 genes, see SI Table 4). All gene names are connected to one or more of the three major nodes, indicating the screen phenotypes for which each knockout was positive (CTF, BiM, or ALF). Gene names in black typeface are those validated in all three deletion arrays: i.e., tag sequencing indicated the presence of the correct mutation in uncontaminated form. For these mutants, present and absent phenotypes are meaningful. Gene names in blue italic typeface failed tag validation in at least one of the deletion collections, and therefore phenotype information is missing from at least one screen. These partially characterized genes are placed to indicate observed tag-sequence validated phenotypes (SI Table 3 contains details). The node colors indicate biological process. Genes associated with more than one biological process are represented by the one highest in the color key for simplicity.
Fig. 3.
Fig. 3.
A-like fakers result from whole chromosome loss, gross chromosomal rearrangement, and gene conversion. (A) Electrophoretic karyotypes of ALF mated products were examined for chromosome III status as described (42), and examples are shown. Individual colonies selected after mating were characterized by using pulsed-field gel electrophoresis (top, ethidium bromide stained gel) and in-gel hybridization with a radiolabeled probe (bottom, autoradiogram) that hybridizes chromosomal bands containing the mating type locus and/or silent mating type loci located distally on each arm. Chromosomes III from the mating tester and deletion mutant were of distinct size (top and bottom chr III bands, respectively). In some strains, a less intense signal for rearrangement chromosomes reflects poor mitotic transmission, or hybridization only to HMRa which has imperfect homology to the radiolabeled probe. (B) Discordant CTF sectoring phenotypes are observed in knockout mutations with similar ALF frequencies.
Fig. 4.
Fig. 4.
Common synthetic lethal interactions among yeast CIN genes that have human homologs mutated in cancer. Eight yeast CIN genes with top hit human homologs mutated in cancer (e-value <10−10) are also found in the public interaction database BioGrid (56). These 8 yeast CIN genes are placed peripherally and are shown in black. There were 61 interactors with at least two synthetic lethal connections to the yeast cancer homologs. The arrows point from a query to a target gene hit in the synthetic lethal screens. The interactors include high-confidence CIN genes (blue), low-confidence CIN genes (purple), and genes not detected by the CIN screens (gray). The three genes that have six common synthetic lethal interactions with the cancer gene homologs are indicated by their blue connections. Node color indicates biological process as in Fig. 2.
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