Chromosome-Specific and Global Effects of Aneuploidy in Saccharomyces Cerevisiae

Genetics: Early Online, published on February 2, 2016 as 10.1534/genetics.115.185660 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‡, Angelika Amon*,† * Koch Institute for Integrative Cancer Research, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA † Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA ‡ The Donnelly Centre, University of Toronto, Toronto, ON M5S3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S3E1, Canada § Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544-1014 1 Copyright 2016. Aneuploidy Synthetic Interactions Keywords: aneuploidy, synthetic lethality, dosage imbalance, protein transport Corresponding author: Angelika Amon, 77 Massachusetts Avenue 76-561, Cambridge, MA 02139; 617-258-6559; [email protected] 2 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 whose deletion has a synthetic fitness cost in aneuploid Saccharomyces cerevisiae cells containing 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 as well as 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. 3 INTRODUCTION Aneuploidy, defined as an imbalanced karyotype in which the copy number of one or more chromosomes deviates from base ploidy, has myriad phenotypic consequences on both the cellular and organismal levels. In humans, aneuploidy is the leading cause of spontaneous abortions, and aneuploid organisms display severe developmental defects exemplified by the growth delays and mental retardation characteristic of trisomy 21, or Down syndrome. Paradoxically, the vast majority of cancer cells are also aneuploid; recent estimates indicate that greater than 90% of solid tumors harbor at least one aneuploid chromosome (Weaver and Cleveland 2006; Nagaoka et al. 2012; Chen et al. 2015). Across both cellular and organismal aneuploid model systems, the expression of genes present on imbalanced chromosomes causes a set of fitness defects including slowed proliferation – in particular, delays in the G1 phase of the cell cycle (Torres et al. 2007; Williams et al. 2008; Thorburn et al. 2013). Aneuploidy also induces a characteristic stress-associated transcriptional program called the environmental stress response (ESR), causes multiple forms of genomic instability and broadly disrupts protein homeostasis (Torres et al. 2007; Sheltzer et al. 2011; Stingele et al. 2012; Oromendia et al. 2012; Dephoure et al. 2014). In principle, these phenotypes shared among many different aneuploidies could be due to copy number imbalances of specific genes whose misexpression has particular cellular consequences or to the aggregate effect of imbalances in the levels of many genes. Recent work suggests that the proliferation defects of aneuploid yeast cells cannot be explained by changes in the copy number of specific dosage-sensitive genes (Bonney et al. 2015). In contrast, specific drug sensitivities of aneuploid yeast strains are in some cases attributable to gene-specific effects (Chen et al. 2012; 2015). Although some effects of genomic imbalance have been characterized, an unbiased investigation into the molecular consequences of aneuploidy is lacking. 4 The development of a high-throughput synthetic lethal screening technology called synthetic genetic array (SGA) analysis has enabled the unbiased, genome-wide interrogation of novel aspects of yeast biology (Tong 2001; Costanzo et al. 2010; Baryshnikova et al. 2010). This method utilizes the Saccharomyces cerevisiae deletion collection as a basis for screens to identify genes whose deletion causes synthetic lethality or synthetic sickness when combined with a genetic manipulation of interest (Giaever et al. 2002). In this work, we used stable haploid yeast strains that carry an additional copy of single yeast chromosomes, henceforth known as disomes, as query strains in these screens to further investigate the biology of aneuploid cells. Using this technique, we identified a number of candidate genes whose deletion negatively impacts the fitness of aneuploid cells. We identified a subset of these candidate gene deletions that either affect the fitness of specific disomes or that impair proliferation in a large number of different disomic yeast strains. We then used this analysis to identify pathways whose function is compromised in disomic yeast cells. Notably, we have discovered previously unknown phenotypes of aneuploid cells, namely defects in the secretory pathway and in the integrity of the cell wall. Importantly, the utility of this method to uncover commonalities of aneuploid cells as well as chromosome-specific phenotypes could ultimately be utilized to selectively target aneuploid cells in the context of cancer therapy. MATERIALS & METHODS Yeast strains and plasmids Disomes used in this study are derivatives of those published in Torres et al. 2007 or were generated using the same method. Disomes used for screening were crossed to the Boone lab starting strain for SGA technology, Y7092, with the genotype can1delta::STE2pr-Sp_his5 lyp1delta his3delta1 leu2delta0 ura3delta0 met15delta0. De novo gene deletions were generated using published methods (Longtine et al. 5 1998) in a wild-type W303 yeast strain. Disomes carrying candidate gene deletions were constructed by crosses. Karyotypes of key disomic strains were verified by comparative genomic hybridization as described (Torres et al. 2007) and analyzed with Java TreeView. All strains are listed in Table S1. Synthetic lethal screens The SGA screens were performed robotically in triplicate, as previously described (Tong and Boone 2012) using MATα disomes as query strains. Data were analyzed using SGAtools, a normalization and scoring methodology developed for small-scale screens (Wagih et al. 2013). Validations were performed by crossing deletions generated de novo into disomic strains lacking the screen-specific markers, followed by tetrad dissection and fitness measurements of the resultant disomic deletion mutants. Doubling time analysis Cells were grown overnight at room temperature in yeast extract/peptone medium containing 2% glucose (YPD) and diluted to OD600=0.1 the next morning. The growth rate of these cultures at 25° was measured in triplicate using a BioTek plate reader to take measurements every 15 minutes for 24 hours. Data were accumulated using Gen5 BioTek software. The period of exponential growth was used to calculate doubling time using GraphPad Prism software. Data shown are the average of 2-4 biological replicates performed on different days. Assessment of disomic drug sensitivities Overnight cultures in YPD were diluted to OD600=1.0, and 1.9 µl of 10-fold serial dilutions were spotted on YPD plates that contained either 250 µg/ml Calcofluor white, 10 µg/ml Congo red, 50 µg/ml Congo red, 16 µg/ml fluconazole, 32 µg/ml fluconazole or 200 µg/ml Brefeldin A. Plates were incubated at 30° for 2 days before images were taken. Lucifer yellow uptake experiments 6 Uptake of Lucifer Yellow was assayed essentially as described (Duncan et al. 2001). Briefly, 1 ml of cells at OD600=0.1-0.2 was resuspended in 100 µl YPD containing 4 mg/ml Lucifer Yellow carbohydrazide (Sigma-Aldrich). Cells were incubated at room temperature for 1 hour before 1 ml ice-cold 50 mM potassium phosphate buffer containing 10 mM NaN and 10 mM NaF was added. Cells were washed three times and resuspended in 20 µl of the same buffer before imaging using a Zeiss Axioplan 2 microscope with a Hamamatsu OCRA-ER digital camera. Image analysis was performed using Volocity software. Zymolyase sensitivity assay Sensitivity to zymolyase was assayed as described (Castrejon et al. 2006). Briefly, doubling times were measured as described above, with the addition of 10, 25 or 50 µg/ml of zymolyase (20T, MP Biomedicals). The ratio of doubling times with zymolyase to those without were calculated and

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