Chromosome-Specific and Global Effects of Aneuploidy In
Total Page：16
File Type：pdf, Size：1020Kb
HIGHLIGHTED ARTICLE | INVESTIGATION Chromosome-Specific and Global Effects of Aneuploidy in Saccharomyces cerevisiae Stacie E. Dodgson,*,† Sharon Kim,*,† Michael Costanzo,‡,§ Anastasia Baryshnikova,** Darcy L. Morse,† Chris A. Kaiser,† Charles Boone,‡,§ and Angelika Amon*,†,1 *Koch Institute for Integrative Cancer Research, Howard Hughes Medical Institute, and †Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, ‡The Donnelly Centre and §Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S3E1, Canada, and **Lewis-Sigler Institute for Integrative Genomics, Princeton University, New Jersey 08544-1014 ORCID ID: 0000-0002-3129-8853 (S.E.D.) ABSTRACT Aneuploidy, an unbalanced karyotype in which one or more chromosomes are present in excess or reduced copy number, causes an array of known phenotypes including proteotoxicity, genomic instability, and slowed proliferation. However, the molecular consequences of aneuploidy are poorly understood and an unbiased investigation into aneuploid cell biology is lacking. We performed high-throughput screens for genes the deletion of which has a synthetic fitness cost in aneuploid Saccharomyces cerevisiae cells con- taining single extra chromosomes. This analysis identified genes that, when deleted, decrease the fitness of specific disomic strains as well as those that impair the proliferation of a broad range of aneuploidies. In one case, a chromosome-specific synthetic growth defect could be explained fully by the specific duplication of a single gene on the aneuploid chromosome, highlighting the ability of individual dosage imbalances to cause chromosome-specific phenotypes in aneuploid cells. Deletion of other genes, particularly those involved in protein transport, however, confers synthetic sickness on a broad array of aneuploid strains. Indeed, aneuploid cells, regardless of karyotype, exhibit protein secretion and cell-wall integrity defects. Thus, we were able to use this screen to identify novel cellular consequences of aneuploidy, dependent on both specific chromosome imbalances and caused by many different aneuploid karyotypes. Interestingly, the vast majority of cancer cells are highly aneuploid, so this approach could be of further use in identifying both karyotype-specific and nonspecific stresses exhibited by cancer cells as potential targets for the development of novel cancer therapeutics. KEYWORDS aneuploidy; synthetic lethality; dosage imbalance; protein transport NEUPLOIDY, defined as an imbalanced karyotype in one aneuploid chromosome (Weaver and Cleveland 2006; Awhich the copy number of one or more chromosomes Nagaoka et al. 2012; Chen et al. 2015). deviates from base ploidy, has myriad phenotypic conse- Across both cellular and organismal aneuploid model systems, quences onboth the cellular and organismal levels. In humans, the expression of genes present on imbalanced chromosomes aneuploidy is the leading cause of spontaneous abortions, and causes a set of fitness defects including slowed proliferation—in aneuploid organisms display severe developmental defects particular, delays in the G1 phase of the cell cycle (Torres et al. exemplified by the growth delays and mental retardation 2007; Williams et al. 2008; Thorburn et al. 2013). Aneuploidy characteristic of trisomy 21, or Down syndrome. Paradoxically, also induces a characteristic stress-associated transcriptional the vast majority of cancer cells are also aneuploid; recent program called the “environmental stress response,” causes mul- estimates indicate that .90% of solid tumors harbor at least tiple forms of genomic instability, and broadly disrupts protein homeostasis (Torres et al. 2007; Sheltzer et al. 2011; Stingele Copyright © 2016 by the Genetics Society of America doi: 10.1534/genetics.115.185660 et al. 2012; Oromendia et al. 2012; Dephoure et al. 2014). In Manuscript received December 3, 2015; accepted for publication January 25, 2016; principle, these phenotypes shared among many different aneu- published Early Online January 29, 2016. Supporting information is available online at www.genetics.org/lookup/suppl/ ploidies could be due to copy-number imbalances of specific doi:10.1534/genetics.115.185660/-/DC1. genes the misexpression of which has particular cellular con- 1Corresponding author: Massachusetts Institute of Technology, 77 Massachusetts Ave., E17-233A, Cambridge, MA 02139. E-mail: [email protected] sequences or due to the aggregate effect of imbalances in the Genetics, Vol. 202, 1395–1409 April 2016 1395 levels of many genes. Recent work suggests that the prolif- disomes as query strains. Data were analyzed using SGAtools, eration defects of aneuploid yeast cells cannot be explained a normalization and scoring methodology developed for small- by changes in the copy number of specific dosage-sensitive scale screens (Wagih et al. 2013). Validations were performed genes (Bonney et al. 2015). In contrast, specific drug sensi- by crossing deletions generated de novo into disomic strains tivities of aneuploid yeast strains in some cases are attribut- lacking the screen-specific markers, followed by tetrad dissec- able to gene-specific effects (Chen et al. 2012, 2015). Although tion and fitness measurements of the resultant disomic deletion some effects of genomic imbalance have been characterized, mutants. an unbiased investigation into the molecular consequences of Doubling time analysis aneuploidy is lacking. The development of a high-throughput synthetic lethal Cells were grown overnight at room temperature in yeast screening technology, synthetic genetic array (SGA) analysis, extract/peptone medium containing 2% glucose (YPD) and has enabled the unbiased, genome-wide interrogation of novel diluted to OD600 = 0.1 the next morning. The growth rate of aspects of yeast biology (Tong 2001; Baryshnikova et al. 2010; these cultures at 25° was measured in triplicate using a BioTek Costanzo et al. 2010). This method utilizes the Saccharomyces plate reader to take measurements every 15 min for 24 hr.Data cerevisiae deletion collection as a basis for screens to identify were accumulated using Gen5 BioTek software. The period of genes the deletion of which causes synthetic lethality or exponential growth was used to calculate doubling time using synthetic sickness when combined with a genetic manipu- GraphPad Prism software. Data shown are the average of two lation of interest (Giaever et al. 2002). In this work, we used to four biological replicates performed on different days. stable haploid yeast strains that carry an additional copy Assessment of disomic drug sensitivities of single yeast chromosomes, henceforth known as disomes, as query strains in these screens to further investigate the Overnight cultures in YPD were diluted to OD600 = 1.0, and biology of aneuploid cells. 1.9 ml of 10-fold serial dilutions were spotted on YPD plates Using this technique, we identified a number of candidate that contained 250 mg/ml Calcofluor white, 10 mg/ml Congo genes the deletion of which negatively impacts the fitness of red, 50 mg/ml Congo red, 16 mg/ml fluconazole, 32 mg/ml aneuploid cells. We identified a subset of these candidate gene fluconazole, or 200 mg/ml Brefeldin A. Plates were incubated deletions that either affect the fitness of specific disomes or at 30° for 2 days before images were taken. impair proliferation in a large number of different disomic yeast Lucifer yellow uptake experiments strains. We then used this analysis to identify pathways the functionofwhichiscompromisedindisomicyeastcells.Notably, Uptake of Lucifer yellow (LY) was assayed essentially as de- we have discovered previously unknown phenotypes of aneu- scribed (Duncan et al. 2001). Briefly, 1 ml o f c el ls at O D 600 = ploid cells, namely defects in the secretory pathway and in the 0.1–0.2 was resuspended in 100 ml YPD containing 4 mg/ml integrity of the cell wall. Importantly, the utility of this method to Lucifer Yellow carbohydrazide (Sigma-Aldrich). Cells were uncover commonalities of aneuploid cells as well as chromosome- incubated at room temperature for 1 hr before 1 ml ice-cold specific phenotypes could ultimately be utilized to selectively 50 mM potassium phosphate buffer containing 10 mM NaN target aneuploid cells in the context of cancer therapy. and 10 mM NaF was added. Cells were washed three times andresuspendedin20ml of the same buffer before imaging Materials and Methods usingaZeissAxioplan2microscopewithaHamamatsu OCRA-ER digital camera. Image analysis was performed using Yeast strains and plasmids Volocity software. Disomes used in this study are derivatives of those published Zymolyase sensitivity assay in Torres et al. (2007) or were generated using the same method. Disomes used for screening were crossed to the Sensitivity to zymolyase was assayed as described (Castrejon Boone lab starting strain for SGA technology, Y7092, with et al. 2006). Briefly, doubling times were measured as de- the genotype can1delta::STE2pr-Sp_his5 lyp1delta his3delta1 scribed above with the addition of 10, 25, or 50 mg/ml of leu2delta0 ura3delta0 met15delta0. De novo gene deletions zymolyase (20T, MP Biomedicals). The ratio of doubling were generated using published methods (Longtine et al. times with zymolyase to those without was calculated and 1998) in a wild-type W303 yeast strain. Disomes carrying plotted relative to wild type. candidate gene deletions were constructed by crosses. Kar- Detection of immature Ccw14p protein intermediates yotypes of key disomic strains were verified by comparative