Heterozygous Screen in Saccharomyces cerevisiae Identifies Dosage-Sensitive Genes That Affect Chromosome Stability

Abstract

Current techniques for identifying mutations that convey a small increased cancer risk or those that modify cancer risk in carriers of highly penetrant mutations are limited by the statistical power of epidemiologic studies, which require screening of large populations and candidate genes. To identify dosage-sensitive genes that mediate genomic stability, we performed a genomewide screen in Saccharomyces cerevisiae for heterozygous mutations that increase chromosome instability in a checkpoint-deficient diploid strain. We used two genome stability assays sensitive enough to detect the impact of heterozygous mutations and identified 172 heterozygous gene disruptions that affected chromosome fragment (CF) loss, 45% of which also conferred modest but statistically significant instability of endogenous chromosomes. Analysis of heterozygous deletion of 65 of these genes demonstrated that the majority increased genomic instability in both checkpoint-deficient and wild-type backgrounds. Strains heterozygous for COMA kinetochore complex genes were particularly unstable. Over 50% of the genes identified in this screen have putative human homologs, including CHEK2, ERCC4, and TOPBP1, which are already associated with inherited cancer susceptibility. These findings encourage the incorporation of this orthologous gene list into cancer epidemiology studies and suggest further analysis of heterozygous phenotypes in yeast as models of human disease resulting from haplo-insufficiency. C ANCERS are commonly characterized as having an abnormal number of chromosomes, termed aneuploidy, which arises due to genomic instability. Aneuploidy has been proposed as a mutagenic mechanism and a driving force of tumor progression and not just a phenotype of the disease (Weaver and Cleveland 2006). Many of the genes whose disruption results in aneuploidy were first identified in budding yeast on the basis of analysis of haploid strains containing null mutations. Aneuploidy can be caused by inherited or somatic mutations in genes that function in the maintenance of genomic stability, such as those involved in centrosome formation,chromosomemetabolism,andcellcyclecheckpoints. Among these, cell cycle checkpoint pathways play an especially important role in maintaining genomic integrity when cells are challenged with genotoxic agents or other stressors (Zhou and Elledge 2000).Mutations in checkpoint genes including, BRCA1, PTEN, ATM, and CHEK2, lead to increases in breast cancer susceptibility (Li et al. 1997;Nathanson and Weber 2001;Meijers-Heijboer et al. 2002;King et al. 2003). Women who inherit BRCA1 mutations have an increased lifetime breast cancer risk of 50-80% (12% risk in the general population) and an ovarian cancer risk of 15-65% (1.5% risk in the general population). However, the age of cancer onset and type and number of cancers can vary, even among women carrying the same mutation. One mechanism for this variation is the presence of unlinked genetic loci, which modify this risk (Rebbeck 2002). For instance, ovarian cancer ...

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity

“…However, unlike strong loss of function mutations gene dosage effects are likely to be relatively subtle and requiring reliable and sensitive quantitative assays. Two previous studies ( Choy et al 2013 ; Strome et al 2008 ) identified sets of genes that cause increased CIN when hemizygous. We refer to these genes as dcCIN genes.…”

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

“…Genome-wide genetic screens of this species have examined deletion mutants for a growth defect upon exposure to different DNA-damaging agents (62)(63)(64)(65) or for synthetic genetic interaction with other mutant genes important for the cellular response to DNA damage (66)(67)(68)(69). Other screens identified mutants with increased genomic instability by investigating the mutation rates within a specific gene (70) or the rates of gross chromosomal rearrangements (51,(71)(72)(73)(74)(75). While several studies have used S. cerevisiae to study genome stability, drastic genome changes are not well tolerated by this organism, thus correlating with a high fitness cost (76).…”

Branching Processes in Biology

“…However, Wu et al (2008) reported that mutation rates showed variability from experiment to experiment that exceeded expectations. This can be seen by comparison of the experimentally observed data in Fig.…”