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INVESTIGATION HIGHLIGHTED ARTICLE

The Sister Chromatid Cohesion Pathway Suppresses

Multiple Chromosome Gain and

Chromosome Ampli

cation

Shay Covo,*,1,2Christopher M. Puccia,†,3Juan Lucas Argueso,Dmitry A. Gordenin,* and Michael A. Resnick*

*National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, and†Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523

ABSTRACTGain or loss of chromosomes resulting in aneuploidy can be important factors in cancer and adaptive evolution. Although chromosome gain is a frequent event in eukaryotes, there is limited information on its genetic control. Here we measured the rates of chromosome gain in wild-type yeast and sister chromatid cohesion (SCC) compromised strains. SCC tethers the newly replicated chromatids until anaphase via the cohesin complex. Chromosome gain was measured by selecting and characterizing copper-resistant colonies that emerged due to increased copies of the metallothionein geneCUP1. Although all defective SCC diploid strains exhibited increased rates of chromosome gain, there were 15-fold differences between them. Of all mutants examined, a hypomorphic mutation at the cohesin complex caused the highest rate of chromosome gain while disruption ofWPL1, an important regulator of SCC and chromosome condensation, resulted in the smallest increase in chromosome gain. In addition to defects in SCC, yeast cell type contributed significantly to chromosome gain, with the greatest rates observed for homozygous mating-type diploids, followed by heterozygous mating type, and smallest in haploids. In fact,wpl1-deficient haploids did not show any difference in chromosome gain rates compared to wild-type haploids. Genomic analysis of copper-resistant colonies revealed that the“driver”chromosome for which selection was applied could be amplified to overfive copies per diploid cell. In addition, an increase in the expected driver chromosome was often accompanied by a gain of a small number of other chromosomes. We suggest that while chromosome gain due to SCC malfunction can have negative effects through gene imbalance, it could also facilitate opportunities for adaptive changes. In multi-cellular organisms, both factors could lead to somatic diseases including cancer.

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HERE is growing evidence that aneuploidy is an impor-tant factor in adaptive evolution. Although aneuploidy generally has adverse consequences (Torres et al. 2007, 2008; Williams et al. 2008; Oromendia et al. 2012), it may provide selective advantage under various stresses (Pavelkaet al.2010; Sheltzer and Amon 2011). The selec-tive advantage of aneuploidy cells has medical implications as shown for tumors (Weaver et al. 2007; Chandhok and Pellman 2009) and drug resistance in pathogenic fungi

(Selmeckiet al.2006, 2009; Semighiniet al.2011). More-over, advantageous aneuploidy can be a common step in evolution. Recently it was suggested that in yeast, aneu-ploidy may be a“quickfix”to tolerate stress during adaptive evolution since an aneuploidy state was shown to be tran-sient while more “refined”mutations take over the culture (Yonaet al.2012). Moreover, changes in aneuploidy can be an “on–off switch”for colony morphological changes (Tan et al. 2013).

For these reasons it is important to understand how different mutants and different physiological conditions affect aneuploidy. Most of the quantitative analysis in yeast of mutants that influence aneuploidy was done using chromosome loss assays. Relatively fewer studies address chromosome gain (Hartwell and Smith 1985; Spenceret al. 1990; Stirlinget al.2011, 2012). However, measuring chro-mosome loss does not provide a complete view of chromatid malsegregation or aneuploidy tolerance. For example, Copyright © 2014 by the Genetics Society of America

doi: 10.1534/genetics.113.159202

Manuscript received September 9, 2013; accepted for publication November 11, 2013; published Early Online December 2, 2013.

Supporting information is available online athttp://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.113.159202/-/DC1.

1Present address: Department of Plant Pathology and Microbiology, Hebrew University, P. O. Box 12 Rehovot 76100, Israel.

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inability to repair double-strand breaks (DSBs) may cause loss of a chromosome that is not due to a defect in chromatid transmission as evident by the number of DNA repair strains that exhibit increased chromosome loss (Yuen et al.2007) and especially proteins involved in recombination (Nakai et al.2011; Song and Petes 2012). Unlike chromosome gain, loss of chromosomes cannot be measured in haploid cells that contain natural 1n complement of chromosomes, ob-scuring the ability to study ploidy-dependent effects on chromatid segregation. Ploidy may have an effect on chro-mosome transmission as evident by the diploid-dependent lethality of some temperature-sensitive spindle pole body mutants (Storchova et al. 2006). In addition, at least for the budding yeastSaccharomyces cerevisiae, most of the col-onies that were selected for chromosome loss, based on loss of genetic markers following centromere deactivation, actu-ally reduplicated the homologous chromosome and were not aneuploid (Reid et al. 2008). Therefore, measurements of stable aneuploidy cannot be made by these types of assays.

Sister chromatid cohesion (SCC) is a process that tethers the newly replicated chromatids until anaphase and pro-vides fidelity of chromosome transmission (Guacci et al. 1997; Onn et al. 2008; Xiong and Gerton 2010). Defects in SCC are associated with several developmental defects (Bose and Gerton 2010) and cancer (for example, Solomon et al.2011 and summarized in Pfau and Amon 2012). SCC is primarily accomplished by the four-subunit cohesin complex containing Smc1, Smc3, yMcd1/hRad21, and yScc3/hSA1 or hSA2. Cohesin is deposited across chromosomes by the SCC2/4 cohesin loader. Cohesin becomes cohesive during DNA replication through acetylation by Eco1 (Ivanovet al. 2002; Rolef Ben-Shaharet al.2008; Unalet al.2008; Zhang et al.2008; Heidinger-Pauliet al.2009). Activation of cohe-sin is linked to DNA replication via proteins like Ctf4 and Ctf8 (Lengronne et al.2006; Skibbens 2009) that facilitate the acetylation of cohesin. Ctf4 contributes to SCC also in an Eco1-independent manner (Borges et al.2013). Cohesin is specifically enriched around the centromeres (Glynn et al. 2004), which in yeast is due in part to the protein Mcm21 (Ortizet al.1999; Poddaret al.1999; Eckertet al.2007; Ng et al. 2009). The centromere enrichment of cohesin facili-tates sister chromatid biorientation before mitosis (Nget al. 2009; Stephenset al.2013), assuring proper chromatid seg-regation and the prevention of aneuploidy. This function may be independent of SCC, occurring through intra-DNA molecule cohesion (Stephens et al.2011). Aneuploidy due to defects in SCC can occur even if SCC is established prop-erly. Failure to maintain SCC or failure to disrupt SCC before mitosis should lead to aneuploidy. Wpl1 (the yeast homolog of the oncoprotein hWAPL) (Oikawaet al.2004) is consid-ered to be an important regulator of the SCC process. Re-cently, Wpl1 was proposed to have a role in preventing establishment of SCC at G2 by counteracting acetylation of Smc3 (Guacci and Koshland 2012; Borges et al. 2013; Lopez-Serra et al. 2013). On the other hand Wpl1 partici-pates in maintenance of SCC once it is properly established

(Rolef Ben-Shahar et al.2008; Rowlandet al.2009; Sutani et al. 2009) and it controls chromosome condensation (Lopez-Serra et al. 2013). The effect of deletion of WPL1 on genome stability is not fully understood although evi-dence suggest it leads to increased loss of heterozygosity (Yuenet al.2007).

In addition to SCC, cohesin has a role in the proper function of the kinetochore and chromatid biorientation as mentioned above (Ng et al. 2009; Stephens et al. 2011, 2013). Cohesin is also important for gene expression and DNA repair (Sjogren and Nasmyth 2001; Kimet al.2002a, b; Unal et al. 2004; Bauerschmidt et al. 2010; Wu et al. 2012).

As we and others have shown, cohesin facilitates DSB repair between sister chromatids and suppresses recombi-nation between homologous chromosomes (Sjogren and Nasmyth 2001; Covo et al. 2010; Heidinger-Pauli et al. 2010). Cohesin is recruited to DSBs (Stromet al.2004; Unal et al.2004) and stalled replication forks (Tittel-Elmeret al. 2012). SCC is activated in response to DNA damage (Strom et al. 2007; Unal et al. 2007, 2008; Heidinger-Pauli et al. 2008, 2009). Defects in SCC-mediated recombination might lead to aneuploidy, since inefficient resolution of homolo-gous recombination intermediates can cause whole chromo-some gain (Acilanet al.2007; Hoet al.2010).

Here, we show that different mutations in the SCC pathway can result in highly different increases in the rate of chromosome gain. Yet, the focus of this work is the use of our chromosome gain assay to answer questions that were not addressed previously in classical chromosome loss assays. We were able to show increase in chromatid malsegregation in diploidvs.haploid strains. In addition, we showed a clear effect of DNA damage on chromosome gain. Finally, we were able to show, for thefirst time, that defects in cohesin can cause multiple whole chromosome gains, in-cluding chromosome amplification (fast acquisition of mul-tiple copies of one chromosome) that allows cells to survive toxic exposure. Based on thesefindings, we propose that the genome plasticity of diploid cells defective in SCC may fa-cilitate adaptive evolution of pathogenic fungi and provide a selective advantage to cancer cells.

Materials and Methods

Strains used in this study are provided in Table 1.

Strain construction

Gene inactivation was done by knockout of specific open reading frames using the KanMX cassette from theS. cerevisiae deletion collection. The primers that were used forWPL1 knockout were as follows:

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59TCCGGTTGAAGAGGTTCCA 39and 59TTTCGAAGGA GAGCCTGAAT 39 for MAD1; 59 GGACAGTGAGGGTA CATTTCAAGA 39 and 59 CAGCAACATCCGCAGATTTT 39. mcd1-1 strains were created by popin/popout of pVG257 (Guacci et al.1997) and the mutation was verified by se-quencing. For the chromosome gain assay, we used as a starting point strains that were previously developed (Narayanan et al. 2006). These strains were modified by replacing the inverted Alu repeats with the LYS2 gene. Heterozygous mating-type diploids were created by trans-forming MATa haploid cells with a vector containing HO under a native promoter (YEpHO). Nonmating isolates were selected and diploid status was confirmed by low UV mutability ofCAN1.MATa/MATaderivatives were then created by transforming diploid strains with pGAL-HOT, (HO under Gal10 promoter). Transformants were incu-bated in galactose media for 6 hr and mating colonies were selected after assuring that the pGAL-HOT plasmid was cured. To create CUP1/cup1D diploid strains, we inserted KanMax cassette using plasmid pFA6 targeted to the CUP1locus of our diploid strain background (CS2335 for example) using primers 59GCAGCATGACTTCTTGG TTTCTTCAGACTTGTTACCGCAGGGGCATTTGTCGTCGCT GTTACACCCCCGTACGCTGCAGGTCGACGGATCCCC39and 59ATGTTCAGCGAATTAATTAACTTCCAAAATGAAGGTCATG AGTGCCAATGCCAATGTGGTAGCTG-ATCGATGAATTCGAG CTCGTTTTCGA39. For knockout verification primers, 59CA TTTCCCAGAGCAGCATGAC 39and 59GTTCAGCGAATTAA TTAACTTCC 39were used.

General conditions for rate determination of chromosome gain are described below. Experiments were started by patching at least six single colonies from each genotype to YPDA-rich medium followed by incubation overnight in 30°, includingmcd1-1temperature-sensitive strains (mcd1-1strains were grown and maintained at 23°prior to the experiments). Overnight patches were then spread on selective media (CuSO4) and diluted samples were spread on synthetic com-plete media. Putative chromosome gain was indicated by re-sistance to copper. To restrict the effect of themcd1-1mutation to the growth phase and not to the selection phase, plates were incubated at 23°. Plates were incubated for 2–4 days.

Chromosome copy number analysis

Copy number was estimated using array comparative genome hybridization (CGH). Genomic DNA preparation, labeling, hybridization, and data analysis procedures were as described earlier (Zhanget al.2013).

Determination of copper-resistant rate as a measure of chromosome gain

Undiluted cultures of yeast patches were spread in synthetic complete media containing 0.9 mM CuSO4. In parallel, diluted samples were spread in synthetic complete media to determine the amount of cells in each patch. After 3–4 days the number of copper-resistant colonies was determined. For each genotype in thefirst few experiments, the copper-resistant colonies were replica plated to another CuSO4 -containing plate. The great majority of the resistant colonies Table 1 Strains used in this work

Strain Ploidy Genotype

CS1131 Haploid (WT) MATa,bar1-D,his7-2,trp1D,ura3D,leu2-3,112,ade2D,sfa1D,lys2D,cup1-1D, yhr054cD,cup1-2D.LYS2 ec,CUP1-1ec,ADE2 ec,SFA1 ec(Narayananet al.2006)

CS1143 Haploid As 1131 butmcd1-1

CS1152 Haploid As 1131 butwpl1::G418

CS1249 Haploid As 1131 butmcm21::G418

CS1252 Haploid As 1131 butctf4::G418

CS1276 Haploid As 1131 butcin2::G418

CS1275 Haploid As 1131 butmad1::G418

CS2324 Diploid (WT) MATa/MATa

CS2322 Diploid As 2324 butmcd1-1/mcd1-1

CS2318 Diploid As 2324 butwpl1::G418/wpl1::G418

CS2364 Diploid As 2324 butmcm21::G418/mcm21::G418

CS2360 Diploid As 2324 butctf4::G418/ctf4::G418

CS2405 Diploid As 2324 butcin2::G418/cin2::G418

CS2403 Diploid As 2324 butmad1::G418/mad1::G418

CS2335 Diploid As 2324 butMATa/MATa

CS2344 Diploid As 2324 butMATa/MATa

CS2393 Diploid As 2324 butMATa/MATa,CUP1/cup1D

CS2346 Diploid As 2324 butMATa/MATamcd1-1/mcd1-1

CS2336 Diploid As 2324 butMATa/MATawpl1::G418/wpl1::G418

CS2417 Diploid As 2324 butMATa/MATawpl1::G418/wpl1::G418rad51::NAT/rad51::URA3

CS2420 Diploid As 2324 butMATa/MATarad51::G418/rad51::URA3

CS2342 Diploid As 2324 butMATa/MATawpl1::G418/wpl1::G418

CS2373 Diploid As 2324 butdnl4::G418/dnl4::G418

CS2400 Diploid As 2324 butrad52::G418/dnl4::HYGB

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were able to grow again on CuSO4 plates after replica plating.

Results

A genetic system to study chromosome gain

Defects in SCC are expected to affect chromosome gain as well as loss. Chromosome gain has been addressed primarily using systems based on haploid cells. We chose to determine chromosome gain in diploid cells to address the role of homologous chromosome interactions, segregation defects, and aneuploidy tolerance. In yeast, theCUP1gene codes for copper metallothionein, which protects against excessive copper (Ecker et al. 1986) and increased copies of CUP1 result in resistance to high copper levels (Fogel and Welch 1982; Resnicket al.1990). We used a previously developed strain (Narayananet al.2006), where a single copy ofCUP1 has been placed near the telomeric CAN1gene on chromo-some V and the natural copy in chromochromo-some VIII is deleted (Figure 1). Resistance to copper provided selection for gain inCUP1 copy number and potentially gain of chromosome V. After modifying the strain (See Materials and Methods) the resulting cells were sensitive to 0.4 mM CuSO4 and allowed robust selection for chromosome V gain when yeast cells were plated to 0.9 mM CuSO4(or higher).

The vast majority of resistant colonies in our experiments were due to gain of at least one copy of chromosome V. Among 33 copper-resistant colonies examined (haploid and diploid strains and various genetic backgrounds), all exhibited whole chromosome V gain as determined by CGH. There were no chromosome V gains in the absence of copper selection (supporting information,Table S1) among mcd1-1 diploid cells. No other aberrations on chromosome V, such as local increased copy number of the locus sur-rounding CUP1 were observed, although other chromo-somes were also gained (discussed below). Importantly, we did not find any false positives when 0.9 mM CuSO4 was used for selection. Since we cannot exclude some killing of cells with chromosome V gain, the rates of chromosome

gain can be considered as minimal estimates. The median chromosome gain rates for all genetic backgrounds includ-ing 95% confidence of intervals as derived from the rate of copper resistance are presented inTable S2.

SSC defects and DNA damage facilitate chromosome gain

There was an increase in the rate of chromosome gain for SCC defective cells in comparison to wild type (WT). The increase differed considerably among the mutants. Interest-ingly, homozygote deletion ofWPL1, which has several roles in regulation of sister chromatid cohesion, had the least impact on chromosome gain (17-fold over WT, Figure 2). Homozygous deletion ofCTF4that links SCC establishment to DNA replication andMCM21that facilitates SCC around the centromeres increased chromosome gain 180- and 100-fold over WT, respectively. The greatest effect was found for a cohesin temperature-sensitive mutant mcd1-1 grown at the semipermissive temperature of 30°(265-fold increase in chromosome gain over WT) (Figure 2A).

Chromosome behavior can be affected by differences in physiology between various types of cells or different stresses. Particularly relevant is exposure of cells to DNA damage, which can activate dormant cohesin molecules (Strom et al. 2004, 2007; Unal et al. 2004, 2007, 2008). Since cohesin mutants show defects in homologous recom-bination (Covo et al. 2010; Sjogren and Strom 2010) and since defects in resolution of recombination intermediate can lead to chromosome gain (Ho et al. 2010; Rodrigue et al. 2012) the effects of DNA damage and the role of homologous recombination on chromosome gain in WT and SCC defective strains were studied. We examined chro-mosome gain following growth of diploidMATa/MATacells on plates containing a low level of the recombinogen methyl methanesulfonate (MMS; 1 mM). As shown in Figure 2B and Table S2, the high levels of spontaneous chromosome gain in the MATa/MATa wpl1D/wpl1D and MATa/MATa mcd1-1/mcd1-1 mutants were greatly increased by MMS, based on a comparison of rates between treated and untreated Figure 1A genetic system to study chro-mosome gain based on copy number in-crease ofCUP1. CUP1-1andCUP1-2were deleted from their native locus on chromo-some VIII. CUP1-1 was inserted into the telomere proximal site on the short arm of chromosome V next to the native locus of CAN1. The strains used here are derived from previously published strains (Narayananet al. 2006), but were modified by replacinglys2:: Alu with theLYS2alleles. Selection for chro-mosome gain and rate determination were done mainly on 0.9 mM CuSO4 and for

several experiments with 0.7 mM CuSO4.

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cells. Importantly, the MMS exposure and SCC defects resulted in synergistic increases in rates of chromosome gain. These results are consistent with the view that ineffi -cient sister chromatid recombination in SCC-defective strains might lead to chromosome gain. Such recombina-tion-associated aneuploidy is expected to depend on the function of RAD51, a gene that is central to homologous recombination. However, the rate of spontaneous chromo-some gain in MATa/MATa wpl1D/wpl1D rad51D/rad51D diploid cells was significantly higher than in the wpl1D/ wpl1D single mutant (Figure 2B, Table S2). Homozygous deletion of RAD51alone also increased chromosome gain, although to a lesser extent than in the rad51D/rad51D wpl1D/wpl1Ddouble mutant (Figure 2, A and B andTable

S2). Just as for MMS, therad51Dhomozygous deletion syner-gized with deletion of WPL1. Attempts to measure chromo-some gain in the rad51D/rad51D wpl1D/wpl1D double mutant failed due to severe DNA damage sensitivity. These results indicate that recombination intermediates such as joint molecules are not the major source of aneuploidy in SCC de-fective strains. However, it is possible that DSBs, nicks, gaps, or unresolved replication structures (Sofueva et al.2011) might lead to chromosome gain, especially in cells defective in SCC.

Mating-type controls of chromosome gain

We also examined the effect of cell type on chromosome gain. In yeast, changes in mating type greatly affect the transcriptional program (Galitski et al.1999). Also, mating Figure 2 SCC defects, DNA damage, and homozy-gous mating type differentially increase chromo-some gain rates. (A) Chromochromo-some gain was measured by copper resistance inMATa/MATa dip-loid cells (fold increase over WT cells). Shown is the median of at least six repeats. The error was calcu-lated as 95% confident of intervals and is presented inTable S2. Number of repeats forfluctuation tests are as follows: WT andwpl1D, 12; mcd1-1and ctf4D, 6; andmcm21D, 7. (B) Effect of DNA dam-age on chromosome gain inMATa/MATastrains. Left side, effect of 1 mM MMS (presented in paren-theses are the numbers of events/107cell divisions

that were added in comparison with no MMS treat-ment). The number of repeats forfluctuation tests are: WT andmcd1-1, 6 andwpl1D, 9. Right side, effect ofrad51D(in parenthesis is the increase in events over that measured in RAD51 wild-type cells). The number of repeats forfluctuation tests are:wpl1D/rad51D, 18 andrad51D, 6. (C) Effect of mating type on chromosome gain rates where val-ues were determined for heterozygous and homo-zygous mating type for WT and SCC defective strains (the rate forMATa/MATamcd1-1/mcd1-1 was not measured). The number of repeats forfl uc-tuation tests are: WTa/a, 12;a/a, 14;a/a, 3;wpl1D a/a, 12;a/a, 9;a/a, 6; andmcd1-1, 6 (homozygous and hetrozygote matings). (D) Attempts to identify the pathway responsible for the mating-type effect on chromosome gain. Nonhomologous end joining is suppressed in MATa/MATa vs. MATa/MATa

strains. Interhomolog recombination is known to be less efficient in MATa/MATavs. MATa/MATa strains. A connection between aneuploidy and in-efficient recombination was previously shown (Ho et al.2010). Colony formation under CuSO4

expo-sure is highly dependent on the copy number of CUP1as shown with theCUP1/cup1heterozygote (MATa/MATa), indicating that chromosome gain of CUP1is the driving event. The number of repeats forfluctuation tests are as follows: WT, 14; dnl4D, 13;rad52, 14; andCUP1/cup1, 6. (E) SCC defects and mating type increase the chance to survive high doses of CuSO4. Cultures were plated directly on

1.5 mM CuSO4(instead of 0.9 mM) and colonies

were counted after 3 days. The number of repeats forfluctuation tests are: WT a/a, 6;a/a, 6;mcd1-1

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type influences several aspects of chromosome biology. Non-homologous end joining is active only in haploid or diploid cells carrying oneMATallele,MATaorMATa, while homolo-gous recombination is more active in heterozyhomolo-gous MATa/ MATacells (Kegelet al.2001; Valencia-Burtonet al.2006; Fung et al. 2009). Therefore, we measured chromosome gain in aMATa/MATastrain. Surprisingly, for WT and SCC defective strains, the rate of spontaneous chromosome gain was increased at least 10-fold in diploids and up to 100-fold in MATa/MATa compared to MATa/MATa strains, as de-scribed in Figure 2C and Table S2. The high levels in the MATa/MATastrains did not depend on the end-joining gene DNL4 (end joining is suppressed in MATa/MATa cells) (Kegelet al.2001) or on homologous recombination as de-termined using rad52D/rad52D mutants (Figure 2D and Table S2).The rates in rad52D/rad52D mutants were slightly, but statistically significantly, higher than for RAD+ MATa/MATacells, in agreement with the effect of therad51 deletion (Figure 2, B and D andTable S2).

The ability to form colonies on the CuSO4-containing medium was clearly dependent on the copy number of CUP1since the rate of formation of copper-resistant colonies was greatly reduced, from 13031027to 0.531027, when there was only one CUP1 gene (i.e., MATa/MATa CUP1/ CUP1 vs. MATa/MATa CUP1/cup1D; Figure 2D). These results indicate that the system is highly responsive to an additional copy of the CUP1 gene, leading us to conclude that CUP1 copy number gain is the driver of copper resis-tance in the homozygousMATdiploid strains.

Resistance to high chronic exposure of CuSO4is much

more frequent in cohesin-deficient cells than in WT

Increased copy number ofCUP1is expected to provide pro-tection against higher levels of CuSO4. We, therefore, inves-tigated the ability of several yeast strains to form colonies when grown on 1.5 mM of CuSO4.

The rate of colony formation of WTMATa/MATacells on 1.5 mM CuSO4 was extremely low, ,1029 events per cell division, as described in Figure 2E and Table S2. Similar to the results with lower CuSO4levels, the rate of colony forma-tion was much higher when cells were homozygous for mating type. Remarkably, there was more than a 10,000-fold rate in-crease in MATa/MATamcd1-1/mcd1-1 cells that were resis-tant to 1.5 mM CuSO4compared to WTMATa/MATaMCD1/ MCD1. The rate reached nearly 1025/cell/generation. A similar rate was observed with the MATa/MATa mcd1-1/mcd1-1 strain, possibly indicating that there is a limit to the number of additional chromosomes that a cell can tolerate (Figure 2E). We also addressed the ability of cells to tolerate even higher levels of CuSO4. Unlike for 1.5 mM CuSO4, no colony-forming units were found among 109 MATa/MATa mcd1-1/mcd1-1diploid cells spread on 2 mM CuSO4plates. However, highly resistant cells appeared several days after inoculation of 1.5 mM CuSO4-resistant colonies to liquid medium containing 2 mM CuSO4 (growth conditions are described in the legend to Figure 3).

Multiple aneuploidy and chromosome amplification in diploid cohesin mutants chronically exposed to a high level of CuSO4

Since increased copper resistance as well as the stepwise adaptation to high CuSO4exposure was likely due to a change in chromosome number, we analyzed by array CGH all the chromosomes from diploid mcd1-1/mcd1-1 copper-resistant cells grown in 0.9 and 2 mM CuSO4, as described in Table

S1. Typically, array-CGH analyses would compare the chro-mosome content of a resistant culture that was grown in CuSO4 liquid media against a copper-sensitive culture that grew in CuSO4-free medium at permissive temperature (the reference culture). The array CGH was also performed by comparing a reference strain tomcd1-1cells grown at the semipermissive temperature (without CuSO4) and then propagated in liquid cultures without CuSO4. In the ab-sence of CuSO4, there was almost no aneuploidy (Table

S1), indicating that partial inactivation of cohesin does not lead to frequent unselected stable aneuploidy events.

In all cultures of copper-resistant colonies, there was gain of chromosome V, as described inTable S1. Interestingly, there was aneuploidy for other chromosomes as well. (Occasionally, the array-CGH signal indicates a copy number change,1 n, for example, 0.75, which is likely due to heterogeneity in the population). Among 19 cultures of diploid MATa/MATa mcd1-1/mcd1-1 cells, there were 14 isolates showing a gain of chromosome II in addition to chromosome V (Figure 3A). This coincidence of gain was also observed for 11 of 14 other copper-resistant haploid or diploid isolates of different geno-types (SCC proficient or defective). Altogether, chromosome II was gained along with chromosome V in 25 of 33 (75%) of all the resistant cultures examined. The gain of chromosome II in response to copper is statistically significant. TheP-value of Fisher’s exact test corrected by Bonferroni considering each of 16 chromosomes as an independent hypothesis is 0.0016.

Frequently, chromosomal gain for chromosomes other than V and II was also detected, as described in Table S1 and Figure 3. Based on results for all genotypes and condi-tions, chromosomes VII and XI were often increased (13/33 and 14/33, respectively), yet due to the relatively small size of the sample theP-value of Fisher’s exact test is not signif-icant when corrected to multiple hypotheses.

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Finally, the copy number of chromosome V (the target of selection for copper resistance) was determined. Gain of more than one copy was frequently observed among the various WT, mcd1-1, and wpl1D isolates (20/33) resistant to 0.9 and 1.5 mM CuSO4 (Figure 3D, Table S1). Interestingly, in the diploidMATa/MATamcd1-1/mcd1-1cells those were adapted to grow in 2 mM CuSO4liquid culture there was a gain of even more copies of chromosome V. Among 11 highly resistant iso-lates, there was only 1 that acquired just a single extra chro-mosome. In the remaining 10 isolates, half of them gained 2 extra chromosomes and half gained at least 3 extra chromo-somes, as described in Figure 3C. The karyotype of such cells resistant to 2 mM CuSO4dramatically deviates from that of the normal diploid cells (an example is presented in Figure S1). The multiple changes in karyotype are reminiscent of the var-iations in chromosome numbers that can be seen in cancer cells. The severe imbalanced genome was observed also in wpl1-deficient diploids exposed to CuSO4, as can be seen in Table S1isolates Csra84–87.

Diploid cells are more prone to failure in controlling chromosome gain caused by genetic defects

Using the copper-resistance system, we found that the rate of chromosome gain in haploids (MATa) is lower than in

diploid cells (MATa/MATa). While this trend is true for all strains, the effect is modest in WT strains. In SCC mutants, the differences are more striking. For example, the rate for mcd1-1 in haploidsvs.diploids is 170 as compared to 2660 events/107 cell divisions (Figure 4A,Table S2). Hence, in comparison to the haploid cells, there was a further 4-, 5-, 11-, and 17-fold increase in the rates of gain, respectively, in the diploids as compared to the haploid for the mcd1-1, mcm21D, ctf4D, and wpl1D cells. The overall diploid effect for chromosome gain was nearly two orders of magnitude greater forMATa/MATavs. MATastrains (compare rates in Figure 4 and Figure 2C and alsoTable S2).

As shown in Figure 4A, this diploid effect extends to other components of chromosome transmission. Loss of the microtubule protein geneCIN2and the spindle assembly checkpoint protein gene MAD1in haploid cells resulted in 15- and 16-fold increases, respectively, in the rates of chro-mosome gain. The rates (events/107 cell divisions) in dip-loids were increased another 44-fold (658/15) and 89-fold (1433/16). Thus, reduction in the fidelity of chromosome transmission results in chromosome gain that is greatly in-creased in diploid cells.

The apparent diploid-dependent chromosome gain may stem from differential tolerance of chromosome gain, Figure 3 Copper-adapted yeast cul-tures show multiple chromosome aneuploidy and chromosome

ampli-fication. (A)mcd1-1/mcd1-1(MATa/ MATa) diploid cells were grown overnight on YPDA plates at 30°, then spread on either 0.9 or 1.5 mM CuSO4 plates, and incubated

at the permissive temperature of 23° over 3 days. Surviving colonies were inoculated to 10 ml YPDA and grown at 23°; survivors of 1.5 mM were grown in 2 mM CuSO4

and those of 0.9 mM were grown at 0.9 mM. Genomic DNA was iso-lated, labeled, and hybridized to ge-nomic DNA of cells from amcd1-1/ mcd1-1 MATa/MATadiploid colony that was streaked and grown at 23°

for the entire course without any CuSO4exposure (the latter is a

refer-ence culture). Comparative genome hybridization analysis (CGH) was done between experimental and ref-erence cultures using custom-made Agilent arrays (see Materials and Methods). For each experimental cul-ture chromosome loss (red) or gain (blue) events in comparison to the reference culture are presented. The different shades of blue correspond to the number of chromosomes gained; the actual number of chromosome copies appears within the table. (B) Incidence of chromosome loss (gray rectangle) and chromosome gain (black triangle) for each chromosome as determined by CGH analysis for all diploid cultures tested (Table S1). (C) The number of chromosome loss and chromosome gain events as a function of number of genes on the chromosome; data are pooled from all diploid cells analyzed (for the complete list seeTable S1). A nonlinear regression trend line is shown including R2values both for chromosome loss and gain. The most frequent events are circled and identied. (D) For each indicated

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especially since there would be a lower gene dosage effect for chromosome gain in diploids as compared to haploids. To test this idea, an indicator of chromosome gain tolerance (tolerance index) was estimated by determining the frequency of copper-resistant cells within a culture. The tolerance index for haploid and diploidwpl1Dand WT (only data forwpl1Dis presented) strains was evaluated under three scenarios. First it was determined directly in 3–6 copper-resistant colonies freshly harvested from CuSO4-containing media. The chromo-some gain tolerance index was close to 1 (i.e., most of the cells in the colony were resistant) for haploid- and diploid-resistant colonies (Figure S2A). Second, the tolerance index was determined for cultures propagated from several CuSO4 -resistant colonies in the absence of CuSO4. As seen inFigure

S2A, no major difference was observed between haploid and diploid cells. Finally, copper-resistant colonies were diluted and spread to media lacking CuSO4. This was followed by suspending several (8–16) colonies that arose and spotting them to CuSO4and CuSO4-free media (Figure S2B). No ma-jor difference between haploid and diploid CuSO4tolerance was observed (similar amounts of cells grew on media with and without CuSO4). We conclude that chromosome gain is well tolerated both in diploids and haploids for at least 25 generations (the number of generations from a single cell to a colony).

Interestingly, the rates of chromosome gain inwpl1Dand WT haploids were comparable (Figure 4B). The lack of im-pact by the wpl1D mutation is surprising since wpl1D cells have higher rates of chromosome gain and loss in diploid cells than WT cells as described above (Figure 3 and S. Covo, D. A. Gordenin and M. A. Resnick, unpublished results)

Discussion

Cohesin cohesion and chromosome gain

In this study, we have examined the role of various genes involved with SCC, ploidy, and mating type in preventing aneuploidy due to chromosome gain. Specifically, we focused on the broader effect of defects in SCC on the karyotype of cells under stress. The chromosome gain rates

obtained here for the different mutants in SCC are not statistically different from the rates we calculated for chromosome loss with the same mutants (S. Covo, un-published results). Defects in the establishment of sister chromatid cohesion lead to chromosome gain as shown in ctf4Δcells (Figure 2 and Figure 4). Defects in cohesion per se result in premature chromatid separation before the bi-polar attachment is established, which may lead to a random segregation of the two chromatids, increasing the chance that both chromatids will migrate to the same daughter cell. Thus, one daughter cell gains a chromosome and the other loses one (Figure 1).mcm21Δstrains exhibit proficient SCC across the chromosome but show very similar rates of chro-mosome gain, suggesting that the cohesin activity around the centromeres is at least as important in preventing chro-mosome gain as chrochro-mosome-wide SCC. This is in agree-ment with the important role of cohesin and MCM21 in imposing steric constraints on kinetochore orientation to ensure biorientation. Inmcm21-deficient cells, the chroma-tids do not migrate randomly, rather the kinetochore has high probability to be attached to the wrong pole. We sug-gest that defects in cohesin itself cause much higher rates of chromosome gain because both the centromeric and the SCC functions are compromised (Figure 2A and Figure 4), and, therefore, the probability of both premature separation and aberrant attachment of the kinetochore is raised. In addition, as shown in Figure 2, deficiency of RAD51 or RAD52 increases the risk of chromosome gain and, there-fore, the defects of mcd1-1 in homologous recombination may also contribute to the high rate of chromosome gain. Yet, the contribution of homologous recombination function in a SCC mutant is hard to tease apart, until a mutant in cohesin that is only defective in DNA repair is isolated, be-cause as seen in Figure 2, defects in recombination and defects in SCC synergize. Surprisingly, deletion of WPL1 affects chromosome gain to a much lesser extent in diploid cells and has no effect in haploid cells (Figure 2 and Figure 4).

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to the chromosomes (i.e., sister chromatid separation).ctf4D cells exhibit increased sister chromatid separation (for example, see Borgeset al.2013), which is translated to a high rate of chromosome gain (Figure 2 and Figure 4). Deletion of WPL1has been shown to increase somewhat sister chromatid separation as well as decrease cohesin attachment to chroma-tin (Rowland et al. 2009; Sutaniet al.2009; Maradeo and Skibbens 2010).When compared directly, sister chromatid separation was reduced in wpl1D relative to ctf4D (Borges et al.2013); based on that fact alone, the effect on chromo-some transmission should be less profound in wpl1D than ctf4D. Yet,WPL1is supposed to contribute to thefidelity of chromatid transmission also by preventing SCC establishment at G2. The fact that wpl1D/wpl1D cells show only modest increase in chromosome gain rates suggests that deactivation of the antiestablishment function of Wpl1p has relatively low or no impact on thefidelity of chromosome transmission.

DNA damage synergistically interacts with SCC defects to increase chromosome gain but not through

unresolved recombination intermediates

Recently it was shown that unresolved recombination intermediates can cause chromosome gain, specifically due to inability to resolve joint molecules that were generated by Rad51p activity (Acilanet al.2007; Hoet al.2010). Defects in SCC may increase the prevalence of such intermediates because of the role of SCC in recombination. We hypothe-sized that the increased recombination between homolo-gous chromosomes in SCC mutants may result in delayed recombination intermediates. Indeed, we found that the recombinogenic agent MMS greatly increased chromosome gain in SCC mutants (Figure 2B). However, Rad51p-dependent recombination intermediates are not required for chromo-some gain as demonstrated for wpl1D/wpl1D rad51D/ rad51D diploids andMATa/MATarad52D/rad52Dmutants (Figure 2). While the role of SCC in homologous recombi-nation is not clear yet, we suggest based on these observa-tions that SCC is not an important determinant in the efficiency of resolution of recombination intermediates. The nature of the MMS-derived lesions that exacerbate chromosome gain in SCC defective strains remains to be determined. One possibility is that DNA damage-induced separase, which removes established cohesin molecules (McAleenanet al.2013), may cause premature chromatid separation especially in SCC defective strains. Alternatively, while unrepaired chromosomes activate the checkpoint re-sponse, this activation is transient and is eventually shut down. The segregation of unrepaired chromosomes (gaps, breaks) may be less accurate.

Mating type and ploidy along with defects in chromosome transmission increase the risk of chromosome gain

Interestingly, the rate of chromosome gain is greatly affected by mating type (Figure 2 C). The reason for the difference remains to be established, although many genes are under

mating-type control in yeast (Galitski et al.1999). Several genes associated with mitosis, such as components of the aurora kinase and the Ndc80 complex are underexpressed in MATa/MATa cells (Galitski et al. 1999). Whatever the reasons, the mating-type effects on chromosome stability have important implications. For example, in scenarios where diploid pathogenic yeast become homozygous for mating type, there might be opportunities for greater resis-tance to drug challenges. It was previously shown that an-euploidy in pathogenic Candida albicans can lead to drug resistance (Selmeckiet al.2006).

SCC malfunction increases genome plasticity and allows adaption to stress

To our knowledge, this study provides thefirst description of how defects in SCC can be beneficial via genome instability to the toxic effects of an environmental chemical. Compared to WT, the SCC showed increased survival rates to CuSO4 due to genomic changes. Previously, copper resistance was attributed to various modes of copy number increase in the CUP1gene, includingCUP1amplification, but in these cases the amplification ofCUP1was derived bycis-DNA elements such as inverted repeats (Fogel and Welch 1982; Narayanan et al. 2006). In contrast, here the only mode ofCUP1 copy increase was through whole chromosome gain without any adjacent DNA sequence that destabilizes the CUP1 locus (Table S1). This is despite the collateral effects of unbal-anced expression of many genes (Torres et al. 2007, 2008). Importantly, chromosomes could undergo consider-able amplification, increasing by up to four chromosomes in a diploid cell (i.e., a total of six copies).

In agreement with the reduction in chromosome trans-missionfidelity, other chromosomes were gained as well, the most frequent being chromosome II (75% of the chromo-some V gain events). We consider several possible over-lapping hypotheses that provide likely explanations for the frequent gain of chromosome II. In thefirst, chromosome II is gained because it harbors genes that facilitate tolerance of copper (such as BSD2, SCO1, and SCO2). In the second, chromosome II is gained to counterbalance a specific imbal-ance created by gain of chromosome V either at the proteo-mic level or in the geometry of the spindle pole body. We obtained preliminary results using a system similar to that presented here but in which selection for gain of chromo-some copy number is based on selection in only 0.15 mM CuSO4 in combination with formaldehyde (J. L. Argueso, unpublished results). In this system co-gain of chromosome II and V is far less frequent, indicating that copper exposure drives co-gain of chromosome II and V. An alternative mech-anism to how copper may shape the pattern of aneuploidy is by reducing the fidelity of specific chromosomes (in this case, chromosome II).

Ploidy effect on chromosome gain

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a much higher rate than their haploid counterparts. How-ever, the natural diploid yeast (MATa/MATa) shows only a modest diploid-dependent increase in chromosome gain. The diploid-dependent chromosome gain appears to be a general phenomenon that can be revealed under stress to chromosome transmission since it is observed in mutants defective in SCC, spindle body checkpoint (mad1D), or tu-bulin filament formation (cin2D) (Figure 4A). There are several possibilities to explain the diploid-dependent chro-mosome gain. Diploids might tolerate aneuploidy better than haploids because of milder gene dosage effects, al-though we could not find support for this notion. There was no diploid-dependent tolerance of aneuploidy based on our observation that both haploid and diploid cultures contained comparable fractions of copper-resistant cells even when the cultures were grown on media lacking CuSO4 (Figure S2). Alternatively, there are diploid-depen-dent effects relating to thefidelity of chromosome transmis-sion itself rather than aneuploidy tolerance. As a support to this notion, cells deficient in kinetochore functions, such as dam1-10andspc110-1, show temperature sensitivity that is diploid dependent (Storchova et al. 2006). Scaling up the ploidy may cause defects in chromosome transmission as shown for tetraploid vs. diploids (Mayer and Aguilera 1990; Storchovaet al.2006). Another parallel between hap-loid vs.diploids and a further scale up to tetraploids is the combined effect of ploidy change and mutation in SCC (Storchova et al.2006). Most ascomycota fungi are found as haploids in nature although they can go through a diploid cycle. S. cerevisiae, which is also anascomycete, is actually stable as a diploid (Gersteinet al.2006; Nishantet al.2010). Our results suggest that heterozygous mating type counter-acts the effect of ploidy increase, probably through changes in gene expression.

Overall, we observed that even mild defects in SCC increase significantly the chance of beneficial gain of multiple chromosomes under selective pressure. The stabil-ity of aberrant karyotypes, which might be a “quick and temporary fix”(Yonaet al.2012) needs to be determined. Regardless, given the impact of SCC on chromosome gain, we propose that hypomorphic or even temporary defects in the SCC pathway may have a significant role in adaptive evolution and disease, such as cancer.

Acknowledgments

We thank Kerry Bloom for discussion of the results and useful advice. We greatly appreciate the critical evaluation of the manuscript by Jessica Williams and Thuy-Ai Nguyen. This work was supported by the Intramural Research Pro-gram of the National Institute of Environmental Sciences (National Institutes of Health, Department of Health and Human Services) under project 1Z01ES065073 (to M.A.R.), American Cancer Society grant ACS IRG no. 57-001-53, and a Webb-Waring Biomedical Research Award from the Boettcher Foundation (to J.L.A.).

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GENETICS

Supporting Information

http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.159202/-/DC1

The Sister Chromatid Cohesion Pathway Suppresses

Multiple Chromosome Gain and

Chromosome Ampli

cation

Shay Covo, Christopher M. Puccia, Juan Lucas Argueso, Dmitry A. Gordenin, and Michael A. Resnick

(14)
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Figure S2 Comparable aneuploidy tolerance in haploid and diploid cells. The proportion of copper resistant cells under 3 growth scenarios was determined to assess aneuploidy tolerance. A. Immediate. Determine the amount of copper resistant cells within colonies that arose on CuSO4-containing plates by suspending the cells within three individual colonies. Outgrown.

Three to six copper resistant colonies were outgrown on media lacking CuSO4 and cells were collected and plated to CuSO4

containing and CuSO4 free plates. B. Copper resistant colonies were diluted and spread over CuSO4 free media. Eight to sixteen

descendent colonies were then suspended in water followed by 10-fold serial dilutions. The cells from each well were then plated using a pronging device to CuSO4-containing and CuSO4-free media. An example of the results is presented. While for

most colonies there was no significant different in growth between CuSO4 containing or CuSO4 free media, for two diploid

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Table S1 Karyotype of copper resistant colonies as determined by CGH. CGH analysis of 33 of copper-resistant and 8 copper

sensitive cultures, categorized by genotype, mating type and copper exposure. For haploid cells green indicates chromosome

gain in comparison to the reference genome (see details in the text). For diploid cells different shades of blue mark

chromosome gain (Dark blue corresponds with high copy number of chromosomes). Red marks chromosome loss.

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Table S2 Rates of resistance to copper

Genotype Ploidy MAT CuSO4 (mM) (Events /107 Cell

divisions)

95% CI Figure

WT 2 a/α 0.9 3 (3-4) 2A

mcd1-1 2 a/α 0.9 2661 (663-6534) 2A

wpl1Δ 2 a/α 0.9 49 (46-71) 2A

ctf4Δ 2 a/α 0.9 541 (52-541) 2A

mcm21Δ 2 a/α 0.9 319 (153-319) 2A

WT 2 a/α 0.9, MMS 8 (4-8) 2B

mcd1-1 2 a/α 0.9, MMS 10354 (10354-18355) 2B

wpl1Δ 2 a/α 0.9, MMS 350 (171-579) 2B

rad51Δ 2 a/α 0.9 12 (7-20) 2B

wpl1Δrad51Δ 2 a/α 0.9 565 (135-962) 2B

WT 2 a/a 0.9 130 (90-130) 2C

WT 2 α/α 0.9 61 (50-65) 2C

mcd1-1 2 a/a 0.9 16560 (13650-36500) 2C

wpl1Δ 2 a/a 0.9 1591 (587-5302) 2C

wpl1Δ 2 α/α 0.9 289 (60-2295) 2C

dnl4Δ 2 a/a 0.9 175 (100-200) 2D

rad52Δ 2 a/a 0.9 235 (146-390) 2D

CUP1/cup1Δ 2 a/a 0.9 0.5 (*-1) 2D

WT 2 a 1.5 0.5 (0.2-0.9) 2E

WT 2 a/α 1.5 * 2E

mcd1-1 2 a 1.5 71 (50-97) 2E

mcd1-1 2 a/α 1.5 77 (18-502) 2E

WT 1 a 0.9 1 (0.5-1) 4A&B

mcd1-1 1 a 0.9 169 (55-254) 4A&B

wpl1Δ 1 a 0.9 1 (0.7-1) 4A&B

ctf4Δ 1 a 0.9 27 (17-61) 4A&B

mcm21Δ 1 a 0.9 27 (17-43) 4A&B

cin2Δ 1 a 0.9 15 (4-29) 4A

cin2Δ 2 a/α 0.9 658 (161-1897) 4A

mad1Δ 1 a 0.9 16 (13-38) 4A

mad1Δ 2 a/α 0.9 1433 (347-2220) 4A

Cells from different genotypes were grown on YPDA or YPDA+ MMS plates then spread on 0.9 mM or 1.5 mM CuSO4 containing

Figure

Table 1 Strains used in this work
Figure 1 A genetic system to study chro-underestimate the actual rate of chromosome gain but it is unlikely to suffer from false positive calls, as determined by CGH (ecting the difference between the different genotypes was maintained
Figure 2 SCC defects, DNA damage, and homozy-that were added in comparison with no MMS treat-ment)
Figure 3 Copper-adapted yeast cul-reference culture are presented. The different shades of blue correspond to the number of chromosomes gained; the actual number of chromosomecopies appears within the table
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References

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