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Vol. 15, Issue 3, 1297-1312, March 2004
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* Braun Laboratories 147-75, California Institute of Technology, Pasadena, California 91125;
Laboratoire Bordelais de Recherche en Informatique, Universite Bordeaux I, Talence 33405, France
Submitted October 16, 2003;
Revised November 30, 2003;
Accepted November 30, 2003
Monitoring Editor: David Botstein
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Muller et al., 1980). These daughter cells have the potential for a full life span, whereas the mother cell increases in age with each cell division; thus, yeast aging is asymmetric. The probability that an individual yeast cell will produce daughters declines exponentially as a function of its age in cell divisions or generations (Jazwinski et al., 1998). As yeast cells age, their size increases (Mortimer and Johnston, 1959; Nestelbacher, 2000), their cell cycle slows down (Mortimer and Johnston, 1959), their shape is altered (Pichova et al., 1997), their nucleolus tends to be larger and/or more fragmented, and they become sterile (Guarente, 1997). In wild-type cells, extrachromosomal ribosomal DNA circles (ERCs) also accumulate (Sinclair and Guarente, 1997). Cells in the second half of their life span also show a switch to a state inducing increase loss of heterozygosity (LOH) (McMurray and Gottschling, 2003). Although several lines of evidence suggest that accumulation of ERCs is one determinant of life span, aging can occur in their absence (Guarente, 1997; Sinclair, 2002; Mays Hoopes et al., 2002). What causes the asymmetric inheritance of these aging phenotypes and what causes the final arrest of the mother cell is therefore not completely understood.
The median replicative life span of most S. cerevisiae wild-type cells is 
25 generations; the maximum is 40 generations (Jazwinski, 1993). It has recently been shown that several DNA replication mutants show drastically reduced replicative life span (Mays Hoopes et al., 2002). This led to the suggestion that replication mutants represent an exaggerated case of spontaneous replication errors that occur in wild-type cells in every generation and that cessation of cell division in both mutant and wild-type is the consequence, in part, of chromosome damage occurring during DNA replication. Such a model would be consistent with the accumulation of ERCs in old cells (Guarente and Kenyon, 2000; Sinclair, 2002) and with increased frequency of LOH (McMurray and Gottschling, 2003). One of these DNA replication mutants is dna2-1 (Kuo et al., 1983). Its median life span is eight generations, and maximum 15 generations, and this mutant ages in the absence of ERCs (Mays Hoopes et al., 2002). The DNA2 gene in yeast is essential for growth, and Dna2p possesses a helicase and an endonuclease activity (Budd et al., 1995). The dna2-1 mutant is inviable at 37°C; full 2C DNA content is synthesized but the newly synthesized DNA is highly fragmented, indicating that DNA replication is defective (Budd and Campbell, 1995). This defect can be suppressed by overexpression of RAD27. RAD27, the homolog of the mammalian FEN-1, is a structure-specific nuclease implicated in Okazaki fragment maturation during DNA replication. These genetic interactions suggested that at least one specific role for Dna2p is also in the maturation of the 5' ends of Okazaki fragments (Budd and Campbell, 1997), and in vitro reconstitution studies indicate that Dna2 is required for efficient RNA removal and ligation when FEN-1 activity is impaired (Bae and Seo, 2000; Ayyagari et al., 2003; Kao, Campbell, and Bambara, unpublished data). With respect to aging, it is interesting that the human BLM gene, encoding a RecQ helicase defective in Bloom syndrome, can suppress the replication defect of dna2 mutants (Imamura and Campbell, 2003). dna2 mutants show hyperrecombination in the rDNA repeats, which correlates with increased replication fork pausing at the rDNA replication fork barrier and increased double-strand breaks at the replication fork barrier (RFB). Deletion of FOB1, the fork barrier protein, eliminates pausing, reduces the double-strand breaks (DSBs), and also the hyperrecombination (Weitao et al., 2003b). SGS1, DNA2, SRS2, RRM3, and FOB1 interact to maintain rDNA stability as measured by two-dimensional gel analysis of replication fork pausing in the rDNA and synthetic lethality studies (Weitao et al., 2003a,b).
DNA2 is involved in DSB and postreplication repair pathways, as well as in DNA replication (Budd and Campbell, 2000). Indeed, dna2 mutants are sensitive to X-rays, methyl methanesulfonate (MMS), and bleomycin (Choe et al., 2002). The X-ray sensitivity of the dna2-1 strain, like its replication defect, is suppressed by overexpression of Rad27p (Budd and Campbell, 2000).
Yet another aspect of Dna2p function is the fact that the protein is localized to telomeres, unlike other replication proteins (Choe et al., 2002). This localization is regulated in a cell cycle- and DNA damage-dependent manner. Only during S phase or after treatment of cells with DNA-damaging agents is Dna2p released from telomeres and found associated with the rest of the nuclear chromatin (Choe et al., 2002). Overexpression of DNA2 leads to derepression of marker genes inserted at telomeres and a transient increase in single-stranded telomeric G-tails, Dna2 is required both for telomerase-dependent and telomerase-independent telomere biogenesis, and dna2-2 est2
is synthetically lethal (Singer et al., 1998; Parenteau and Wellinger, 1999; Choe et al., 2002).
Our current study was designed to further define the causes of aging in wild-type yeast and of the premature aging of the dna2-1 strain. Our strategy was to determine the genome-wide transcriptional response to progression through the life span by using microarray analysis. To obtain sufficient quantities of sufficiently old cells for reproducible production of RNA and cDNAs for microarray hybridizations, we developed a method for isolating old cells based on size selection, similar to two methods described previously (Egilmez et al., 1990; Laun et al., 2001). The numerous previously published analyses of yeast gene expression patterns under varying conditions were important in interpreting our data.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) were used for all experiments. Liquid YPD media (2% glucose, 1% yeast extract, 2% peptone) was used for growing yeast cells at 24°C, the permissive growth temperature for the dna2-1 strain.
Isolation of Young and Old Cells by Elutriation
For the isolation of young and old cells, the WT4741 strain and the isogenic strain containing the dna2-1 mutation, 4741dna2-1, were diluted appropriately and grown at 24°C in two liters YPD containing 35 µg/ml chloramphenicol. Wild-type cells, WT4741, were first grown for 10 generations (A600 = 10). Then 3 x 108 10-generation-old cells were isolated by elutriation and then inoculated into two liters fresh YPD medium and grown again for 8 generations (A600 = 1.5). Both young (1 to 3 generations) and old cells (16-18 generations) were isolated by a second elutriation, yielding 2 x 109 young cells and 4.8 x 108 old cells. Cells were quick frozen in liquid nitrogen and stored at -80°C. Note that RNA extractions were not performed on the 1- to 3-generation-old and 10-generation-old wild-type cells harvested at A600 = 10. The latter culture of wild-type cells was grown to such a high density to avoid doing three elutriations for each culture, thus minimizing the preparation time. The young wild-type cells and the 18-generation-old wild-type cells used for RNA extraction were harvested well below the diauxie limit based on the density of the culture at the time of collection (<3.2) (DeRisi et al., 1997).
For isolation of young and old dna2-1 mutant cells, 5 x 108 4741dna2-1 cells growing exponentially were inoculated into two liters of YPD containing 35 µg/ml chloramphenicol and grown for 8 generations at 24°C (A600 = 1.5-2). Young and old 4741dna2-1 cells were separated by elutriation. We collected 1.2 x 109 1- to 3-generation-old cells, and the yield of 8-generation-old cells was 3.6 x 108. Again, care was taken to harvest well before the diauxic shift. Samples of elutriated young and old cells were quick frozen in liquid nitrogen and stored at -80°C.
A complete description of the elutriation technique can be found in (Diamond, 1991). Briefly, pellets of cells harvested from the exponentially growing cultures were washed in phosphate-buffered saline (PBS; 136.89 mM NaCl, 2.68 mM KCl, 5.37 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4), and the cells resuspended in 20 ml of PBS and bovine serum albumin (2 mg/ml). Cell clumps were separated by sonic irradiation (30 s), and the cell suspension was filtered with mesh (3-64/32 NITEX; Tetko, Briarcliff Manor, N.Y.). The elutriation chamber was then loaded with 20 ml of cell suspension corresponding to 3 x 1011 cells (2 liters of culture, A600 = 10, first elutriation) for WT4741 and 7 x 1010 cells for 4741 dna2-1 (2 liters of culture, A600 = 2). The young wild-type and dna2-1 cells were isolated by fixing the flow rate of the water injected into the centrifuge at 45 ml/min and the speed of the rotor of the centrifuge at 1400 rpm. The old wild-type and dna2-1 cells were elutriated by fixing the flow rate at 85 ml/min and the speed of the rotor, respectively, at 900 rpm and 1550 rpm (J-6M centrifuge with JE-10 x rotor; Beckman Coulter, Fullerton, CA). The preparation of the cells before loading into the elutriator took 1 h. Once loaded, the young cells were isolated within 30 min at 24°C and then placed at 4°C. It took 3 h to isolate the fractions of old cells. As for the young cells, we performed the elutriation of the old cells at 24°C, and the cells were then placed at 4°C. Flow cytometric analysis revealed that the cell cycle distribution was the same for elutriated and nonelutriated cells.
Calcofluor Staining
We stained the elutriated young and old cells with fluorescent brightener (Calcofluor White M2R Tinopal UNPA-GX #F3543) to count the bud scars and establish the age of the cells isolated by elutriation. About 106 cells were washed once in 150 µl of 1 M sorbitol and resuspended in 150 µl of fluorescent brightener (10 mg/ml) for 5 min at 4°C. After being washed four times with fresh 1 M sorbitol, the cells were resuspended in 20 µl of 1 M sorbitol and then observed under a fluorescence axioscope (Carl Zeiss, Jena, Germany).
Indirect Immunofluorescence
Nucleoli were monitored by indirect immunofluorescence by using monoclonal antibodies directed against the abundant nucleolar protein Nop1 (Aris and Blobel, 1988; Mays Hoopes et al., 2002). After 1-h incubation with potassium phosphate buffer (KPO4), magnesium chloride (MgCl2), and 37% formaldehyde, cells were placed in 2 M sorbitol buffer and treated with Zymolyase (0.3 mg/ml Zymolyase 20T, 0.1% 
-mercaptoethanol) at 30°C for 45 min for the WT4741 strain and 15 min for the 4741dna2-1 strain. The cells were then fixed on eight-well, Teflon-coated slides pretreated with 0.1% polylysine 6040805; ICN Biomedicals, Costa Mesa, CA). After 15 min in blocking solution (PBS, 0.5% bovine serum albumin, 0.5% chicken albumin, 0.5% Tween 20), the cells were incubated with the primary antibodies diluted at 1:2000 in the block solution for 2 h at room temperature. The cells were then washed four times in blocking buffer and incubated for 2 h with the secondary antibody, rabbit anti-mouse diluted at 1:5000 in blocking solution. After incubation, the cells were washed four times in blocking solution and immediately costained with 4',6'-diamidino-2-phenylindole (DAPI). DAPI was used at 50 ng/ml as described previously (Mays Hoopes et al., 2002). Cells were then observed under a fluorescence axioscope (Carl Zeiss) coupled with a Orca 2 camera (Hamamatsu, Bridgewater, NJ).
Flow Cytometry
For flow cytometry analysis, samples were fixed in 70% ethanol. After an overnight digestion with RNase I (8 mg/ml in PBS) at 37°C and a 4-h digestion with proteinase K (1 mg/ml) at 50°C, cells were resuspended in propidium iodide in PBS (50 µg/ml), sonicated (output 3-20% duty cycle, 15 s) and filtered with mesh (3-64/32 NITEX; Tetko). The control strain (WT4741) was grown asynchronously at 24°C (with 52% of control strain cells having 1C and 48% having 2C content) and until saturation to identify the 1C peak (100% having 1C content).
DNA Microarray Experiments and Analysis
Old and young wild-type cells were isolated as described under "Isolation of Young and Old Cells by Elutriation." The elutriated cells from six such experiments (a total of 12 liters), yielding 4.8 x 108 cells each, were pooled. Samples of these cells were used for multiple RNA extractions (see below). Old and young dna2-1 cells were isolated as described under "Isolation of Young and Old Cells by Elutriation." The elutriated cells from eight such experiments (a total of 16 liters), yielding 3.6 x 108 cells each, were pooled. Samples of these cells were used for multiple RNA extractions (see below).
RNA extractions were performed with hot/phenol chloroform followed by an Rneasy column (74104; QIAGEN, Valencia, CA). cDNA was synthesized by using oligo(dT) primer (18418-012; Invitrogen, Carlsbad, CA) and Powerscript reverse transcriptase (8460-1; BD Biosciences Clontech, Palo Alto, CA) by using 60 µg of total RNA for each reaction. The cDNA was labeled during reverse transcription with either Cy5-labeled dCTP (red, PA55021; Amersham Biosciences, Piscataway, NJ), or Cy3-labeled dCTP (green, PA53021; Amersham, Biosciences), according to the directions of the supplier (BD Biosciences Clontech). Independent RNA preparations were used for each cDNA preparation.
RNA levels in young and old cells were determined in duplicate for each strain by a ratiometric method by using microarray hybridization analysis. We performed two control experiments (one for wild-type and one for dna2-1), which consisted of a determination of RNA levels in young cells of the same strain, alternatively labeled with Cy3 and Cy5. We also performed four hybridization experiments that consisted of a comparison between RNA levels in young cells and old cells of the same strain. For each strain, wild-type and dna2-1, two hybridization experiments were performed. In the first experiment, Cy3 was used to prepare the cDNA from young cells and Cy5 for old cells. In the second experiment, the dyes were switched to remove any bias that may have been introduced by gene-specific differences in incorporation of the two dyes. For instance, equal amounts of the Cy3 and Cy5-labeled dyes, attached to their respective cDNAs, were used for each array (at least 20 pmol). In addition, as just mentioned, independent RNA isolations were used for each cDNA preparation to control for possible differences due to extraction procedures. Hybridizations were performed on DNA microarrays from Corning Microarrays Technology (Fountain Valley, CA). The CMT Yeast-S228c Gene Array version 1.31 contains >6160 polymerase chain reaction (PCR) products (1 kb) representing open reading frames (ORFs) from the fully sequenced S. cerevisiae genome, plus 108 control genes from Bacillus subtilis. For hybridization, the microarrays were incubated in 50 µl of hybridization solution (1% sonicated salmon sperm, 24.75% formamide, 4.95x SSC, 0.099% SDS, and 0.99 mM dithiothreitol) for 10-18 h at 42°C. After washing (1 min in 2x SSC, 0.1% SDS at 42°C; 5 min in a new solution of 2x SSC, 0.1% SDS at 42°C; 10 min in 0.1x SSC, 0.1% SDS; 15 s, 2 min, 2 min, and 1 min in 0.1x SSC; 15 s in 0.01x SSC), separate images were acquired for each dye, and fluorescence intensity ratios were obtained for all genes (Genepix-Pro3.0 Axon scanner 4000A).
We performed the analysis using MArray, a software that allows the user to analyze single or paired microarray data sets (Wang et al., 2002). MArray defines the quality of microarray experiments and assesses the reproducibility of replicate experiments. The analysis consisted of three steps: filtering, normalization, and interpretation. During the filtering, all empty or negatively flagged spots (the flag coefficient is given by the Genepix file) were removed (see supplementary data http://woldlab.caltech.edu/~lesur/old_and_young_cells/MArray_output.pdf). We eliminated the genes that did not show a consistent expression profile in the duplicates (for example, genes that were more highly expressed in the young wild-type cells in the first experiment but were then more highly expressed in the old wild-type cells in the repeated experiment). After all filtering, 5733 wild-type genes and 6141 dna2-1 genes were left. The minimum signal intensity of each spot was set to zero. We used two control experiments to quantify the noise due to the technique by itself, especially dye bias, and to set the thresholds for identifying the genes significantly differentially expressed in old or young cells in each strain. This sets the intensity limits for interpreting the experimental arrays. We then normalized the intensity young/old cells by using intensity-dependent normalization of the ratios as described by Yang et al. (2002). The data are presented as the background subtracted, normalized young/old ratios for each array. For each strain, reported expression ratios are the average of the two expression ratios young cells/old cells after normalization of the two hybridizations in which the Cy3 and the Cy5 fluorescent dyes were reversed (see tables in supplementary data). To demonstrate the reproducibility of the hybridizations, we calculated the correlation coefficients for each set of reversed experiments.
The entire experiment was also performed again comparing young cells isolated without elutriation with old cells isolated by elutriation. The genes activated fell in the same pathways identified in the data reported. The results are not included because the young cells were not isolated by elutriation as they were in the data set presented here, making averaging of the data impossible. However, it is noteworthy that the results overlap significantly and lead to identical interpretations.
The Yeast Proteome Database (http://www.incyte.com/proteome/mainmenu.jps), the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces), and the annotations of the MIPS Comprehensive Yeast Genome Database (http://mips.gsf.de/proj/yeast/CYGD/db/index.html) were used for interpretation of the expression profiles. In addition, comparison of our results with databases generated recently describing transcriptional responses in yeast to various environmental stresses, conditions that extend life span, and to various mutations that lead to chromosome damage or repair was valuable (Jelinsky and Samson, 1999; Gasch et al., 2000, 2001; Jelinsky et al., 2000; Rep et al., 2000; Kaeberlein et al., 2002; Lin et al., 2001; Lin et al., 2002).
Real-Time Quantitative Reverse Transcription RT-PCR
Confirmation of differentially expressed transcripts was performed using the iCycler IQ real time PCR detection system (Bio-Rad, Hercules, CA) on cDNA obtained from old and young cells isolated with the elutriation system. Total RNA was obtained from each sample and treated with deoxyribonuclease (catalog no. 1906; Ambion, Austin, TX) to remove DNA contamination. cDNA was synthesized using 5 µg of RNA and the iScript cDNA synthesis kit from Bio-Rad (catalog no. 170-8891).
Four microliters of a serial 10-fold dilution (10, 100, 1,000, and 10,000) of cDNA obtained by reverse transcription was amplified in a 25-µl reaction mix containing 1x SYBR Green Supermix (catalog no. 170-8880; Bio-Rad) and 25 pM of each primer. Each sample was run in triplicate. After a 3-min Taq activation step at 95°C, reactions were subjected to 50 cycles of 10-s denaturation at 95°C and 10-s extension at 54°C. Primers were purchased from IDT Integrated Technologies (Coraville, IA). Primer pairs were chosen to minimize primer dimerization and to generate an amplicon between 100 and 150 base pairs. Optical data were collected during the annealing step of each cycle. After PCR, reaction products were melted for 1 min at 95°C and then the temperature was set to 54°C and increased to 94°C in 0.5°C increments. Optical data were collected over the duration of the temperature increase. This was done to ensure that only one PCR product was amplified per reaction.
Relative expression of the RT-PCR products was determined using the mathematical model from M.W. Pfaffl (Pfaffl, 2001). This model calculates relative expression by using the equation ratio = [(Etarget)Ct target(control - sample)]/[(Eref)Ct ref(control - sample)]. The ratio of a target gene is expressed in a sample versus a control in comparison to a reference gene. Etarget is the real-time PCR efficiency of a target gene transcript, Eref is the real-time PCR efficiency of a reference gene transcript, Cttarget is the Ct deviation of control - sample of the target gene transcript, and Ctref = Ct deviation of control - sample of reference gene transcript. The reference gene we used is TUB1 because it is a stable unregulated transcript in each of our microarray data sets. Nine target genes belonging to the recombinational pathway were studied in the wild-type and the dna2-1 strains. For the calculation of the ratio old cells/young cells, the individual real-time PCR efficiencies (E) and the deviation Ct must be known. We calculated the efficiencies according to E = 10[-1/slope]. Because each sample was run in triplicate, the mean Ct value was used in the equation and the Ct values were the differences in averaged Ct values between old and young cells for the same gene. The control sample containing the same concentration of cDNA was chosen to be compared with the target gene.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Lin et al., 2001). It is difficult to isolate sufficient quantities of wild-type cells older than 8 generations for studies such as microarray analysis or of chromosomal defects, due to the low yield of cells older than 8 generations by the biotin technique. However, 8-generation wild-type cells do not yet exhibit many of the significant pathologies of old cells (McMurray and Gottschling, 2003). In one previous study of old yeast cells by using microarray analysis, 8-generation wild-type cells showed limited changes in gene expression, and conclusions were mainly extrapolated from results by using a mutant that had a reduced median life span, sip2 (Lin et al., 2001). Because we wanted to work entirely with wild-type cells, we wanted to isolate cells nearer to the median life span of wild type, 21 generations. Based on the fact that a defining distinction between young and old cells is the increase in size of old cells, we reasoned that centrifugal elutriation might allow for enrichment of larger quantities of older cells. The use of size to enrich for old cells was first validated long ago by using a discontinuous centrifugation technique (Jazwinski, 1990). During the course of our studies, another laboratory published the characterization of old cells obtained by an elutriation method similar to the one we describe here, independently establishing the validity of elutriation for isolating aged yeast cells (Laun et al., 2001). Their technique differed in the size of the elutriation rotor with the result that fewer cells could be isolated by their method than by that described here. The most convincing evidence that old cells had been isolated was the demonstration that the residual life span of elutriated cells coincided precisely with the remaining life span of 18-generation-old cells (Laun et al., 2001). Aged cells in exponentially growing cultures were isolated by elutriation as described in MATERIALS AND METHODS. To isolate cells that had reached their median life span, wild-type cells were grown overnight for 10 generations (A600 = 10), and old cells were isolated by elutriation. The large elutriated cells were inoculated into a fresh culture, grown overnight for a further eight generations (A600 = 1.5), and large 18-generation-old cells were isolated by elutriation. Mutant dna2-1 cells were grown overnight for eight generations (A600 = 1.5-2), the number of generations corresponding to their median life span. Using this method, from 12 or 16 l of wild-type or dna2-1 cells, respectively, we were able to reproducibly isolate 3 x 109 aged cells. Thus, the elutriation rotor allows ease of isolation of large quantities of old cells from cultures of both wild-type and mutant cells. Because yeast cells lyse at the end of their life span, there is likely a minimal contamination of our cells by dead cells.
To confirm that the elutriation strategy enriched for aged cells in our hands, we analyzed the composition of the elutriated samples by counting the number of bud scars after Calcofluor staining, and the results for young and old wild-type cells isolated by elutriation are shown in Figure 1, A and B, respectively. The age distribution of old cells is the same as that found using the biotin-streptavidin technique (Figure 1C). At least 75% of the old elutriated wild-type cells have >15 bud scars, and >90% have more than eight bud scars. A small number of cells with less than three bud scars is always present, whether biotin or elutriation is used. This suggests that contamination with a few cells with a small number of bud scars is unavoidable. Such cells may represent daughters of old mothers, because old mothers give rise to large daughters that only return to normal size after two or three divisions (Jazwinski, 1990). Alternatively, these cells may be damaged by some other process unrelated to aging. It is nevertheless unlikely that such a small percentage of cells would significantly contribute to the gene expression profile for the entire population of old cells, because the percentage of these "young" cells is so small. For the dna2-1 strain, bud scar analysis is shown in Figures 1D (young) and 1E (old). Seventy-three to-78% (eight separate elutriations) of the dna2-1 old cells collected showed at least six bud scars. Because life span determinations show that there is stochastic cell cycle cessation throughout the life span, cells that had fewer bud scars were considered to have reached the same physiological state in fewer generations. Thus, although the number of generations is not normalized by this technique, the physiological state of the cells may be normalized by the size isolation.
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We also verified that elutriated cells were old by demonstrating additional phenotypes associated with old cells. Old cells show abnormal nucleolar morphology (Guarente, 1997). Therefore, we monitored the state of the nucleolus in young cells and aged cells after elutriation. Nucleoli were visualized using indirect immunofluorescence and monoclonal antibody to the abundant nucleolar protein Nop1, a protein involved in ribosome biogenesis (Aris and Blobel, 1988). Young elutriated cells showed 100% of cells with a normal nucleolus sitting as a cap (green) on the nucleus (blue stain) (Figure 2, A and B). As expected from the bud scar analysis, most, 90%, of the old dna2-1 cells showed a nucleolus that was either enlarged or extremely fragmented (Figure 2C). Greater than 75% of the sample (50 cells) of old wild-type cells show a fragmented or enlarged nucleolus characteristic of old cells (Figure 2D). The images in Figure 2 reflect the actual relative sizes of the young and old cells. Because both young and old cells were subjected to elutriation, none of these phenotypes is attributable to the elutriation procedure. Elutriation is considered one of the least perturbative methods of isolating cells for cell cycle studies, and this seems to be true for young versus old cell isolation as well.
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Analysis of Cell Cycle State of the Isolated Cells
We next compared the cellular morphology and DNA content of the population of old wild-type and dna2-1 cells isolated by elutriation. We observed by microscopy that the aged wild-type cells represented all stages of the cell cycle, as expected for an exponentially growing culture. By contrast, 75% of the sample of old dna2-1 cells isolated by elutriation showed a dumbbell shape (see Figure 3 for examples). DAPI staining of the nuclei revealed that 70% had a nucleus entirely within one of the cells, although 30% showed some DNA beginning to partition into the daughter (Figure 3, D and F).
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Flow cytometry analysis of DNA content was performed on the same samples of young and old cells. Comparison to the profiles of exponentially growing and stationary phase reference cells (Figure 4, A and B) showed a distribution in the young and old elutriated dna2-1 cells (Figure 4, C and E) and wild-type (Figure 4, D and F) similar to that found in an exponentially growing cell culture, although the peaks were reproducibly broadened in old wild-type cells (Figure 4F). More than 70% of the old dna2-1 cells contain 2C DNA, and 29% of the cells contain 1C DNA (Figure 4E). Our previous life span analysis of numerous dna2 alleles showed that 50% of the cells after six divisions were alive and capable of further division, although the cell cycle was drastically lengthened, with cells dividing only after 6-7 h (Mays Hoopes et al., 2002). We propose that 50% of the elutriated cells analyzed here have ceased division and that the remainder has a significantly lengthened cell cycle. To verify that the aged cells in the dna2-1 culture were capable of further division, we resuspended them in growth medium and monitored further division. After 3 h, 35.5% of the cells divided at least once and after 6 h and 30 min, 52.4% of the population divided at least once (our unpublished data). This shows that a fraction of the elutriated cells has a lengthened cell cycle and confirms that 50% of the elutriated cells have ceased division.
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In summary, the cells isolated are not lysed or damaged by the elutriation method, as shown by light microscopy and fluorescence microscopy. Calcofluor staining of bud scars and Nop1 antibody staining of the nucleolus confirmed the efficient enrichment of old cells in the sample and confirm that the large size of the isolated cells is due to their age. Thus, we have isolated a population of cells that is sufficiently enriched in old cells to enable biochemical analysis, such as microarray analysis, and this method will be valuable in further studies of the pathologies associated with the aging process in yeast.
Because of the short life span of the dna2 mutant, it proved difficult to catch the two different strains at exactly the same point in the life spans. We note that the dna2 cells may be further advanced in their life span than wild type. The old dna2 cells are larger than the old wild-type cells, and the cell cycle distribution is different, as described. This prevents a quantitative, one-to-one comparison between wild type and mutant in some cases. Thus, the microarray studies reported below are interpreted mainly, although not exclusively, as comparison between young and old cells of the same genotype.
Comparison of Global Gene Expression Patterns in Young and Old Cells
We have used microarray analysis to document differences in gene expression in young and old yeast cells isolated by elutriation, both for wild-type yeast and also for the isogenic, prematurely aging mutant dna2-1 (see MATERIALS AND METHODS). Two control experiments, one for each strain, were used to ensure reproducibility, to eliminate bias introduced by use of the two dyes used to label cDNAs, and to set the boundaries for the genes significantly differentially expressed in old versus young cells of each genotype. We labeled reverse transcripts of RNA from young cells of the same strain first with Cy3 and then with Cy5. We then hybridized the Cy3-labeled cDNA and the Cy5-labeled cDNA in the same reaction to a single array. The medians of the distributions of the ratio of the dye intensities were 0.764 and 0.822 for wild type and dna2-1, respectively. After intensity dependent normalization (Figure 5), most of the spot intensities fall along an M = 0 line, demonstrating the reproducibility of the experiment performed with the two dyes. The shift of the low- and high-intensity spots in the raw data closer to the M = 0 line shows that the normalization process was able to remove the bias generated by the dyes. After centering both distributions on 1, the variance of each did not change (1.159 and 0.378 for wild type and dna2-1, respectively). We observed that 94.39 and 94.36% of the ratios of Cy3 to Cy5 in the wild-type and the dna2-1 control experiments, respectively, were between 0.5 and 2. We concluded that a difference in expression level of at least twofold (median centered on 1) in the experiments described below comparing young and old cells could be considered significant with a 94-95% confidence level.
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mRNA levels in young and old cells were then compared by preparing cDNA from young and old cells and determining the mean of the ratios of hybridization intensities for young/old cells in two separate experiments in which the dyes were reversed during preparation of the cDNAs. As just described, for a distribution of ratios centered on 1, genes whose expression changes significantly during aging exhibit ratios >2 or <0.5. Instead of being 1, however, the median of the distribution of the mean of the ratios for the wild-type strain comparing young and old cells is 1.068 and the variance of this distribution is 1.003. The genes differentially expressed in wild type therefore have a ratio of young cells/old cells either <0.568, for RNAs that increase in old cells, or greater than 2.068, for RNAs that decrease. The median of the distribution of the mean of the ratios of the dna2-1 experiment comparing young and old cells is 1.151 and the variance of this distribution is 0.993. Therefore, the genes differentially expressed in strain dna2-1 have a ratio of young cells/old cells either <0.651 (for RNAs that increase) or >2 (for RNAs that decrease). The correlation coefficients are 0.44 for the wild-type experiments and 0.56 for the dna2-1 experiments, which shows their reproducibility (Figure 6). The correlation coefficient was calculated with the linear regression equation CORREL from Excel: 
x,y = cov (X,Y)/(stdev(X) stdev(Y)).
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We found transcription increased for 627 genes in old wild-type cells (10.22% of the genome), and decreased for 387 genes (6.30% of the genome). 898 genes were more highly transcribed (14.63% of the genome) and 656 genes down-regulated (10.69%) in old dna2-1 cells. We also found that 250 genes are commonly up-regulated in the old wild-type cells and the old dna2-1 cells (4.07% of the genome). The lists of all these genes and the raw and normalized data are available as supplementary data on the Web site at http://woldlab.caltech.edu/~lesur/old_and_young_cells/.
Expression Trends in Metabolic Genes
The genes whose levels changed during aging for wild-type and dna2-1, respectively, were put into categories defined in the MIPS database. Several patterns became apparent that overlapped with previous genome-wide responses to environmental or genetic manipulations. One pattern that emerged in both dna2 mutant and wild type as they increased in generational age was an increase in expression of genes involved in energy storage, such as gluconeogenesis and lipid metabolism (Figure 7 and gordon.txt in the supplementary data). Many of these genes overlapped with a set of age-associated changes in gene expression reported previously for wild type and in a mutant affecting the Snf1 pathway, a sip2 strain (a mutant that shows premature aging) (Lin et al., 2001). The genes shown in Figure 7A are limited to those reported by Lin et al. (2001) on which they based their proposal that aging in yeast is associated with a shift away from glycolysis and toward gluconeogenesis and energy storage, a shift otherwise seen in young cells when they detect low glucose levels (DeRisi et al., 1997; Ashrafi et al., 1999; Lin et al., 2001). Although the number of genes is not large, the expression patterns and their interpretation were verified by microbiochemical determination of levels of enzyme activities and of metabolites in the same cells used for the microarray studies (Lin et al., 2001). The mRNA levels of PCK1 (phosphoenolpyruvate carboxykinase), FPB1 (fructose-1,6-bisphosphatase), and BIO2 (biotin synthase), all involved in gluconeogenesis, rise at least twofold in both wild-type cells and in mutant dna2-1 as they age. The expression of genes involved in the glyoxylate cycle, a modified tricarboxylic-acid pathway (TCA) cycle used to make sugars from fat, such as ICL2 (isocitrate lyase) and MLS1 (malate synthase), POT1 (3-oxoacyl-CoA thiolase), ACS1 (acetyl CoA synthetase), SPS19 (2', 4 dienoyl CoA reductase), and CAT2 (carnithine-o-acethyltransferase), and genes involved in lipid metabolism, are also increased. The expression of some genes, such as MDH2 (cytosolic malate dehydrogenase) involved in gluconeogenesis and pH adjustment, GAC1, and SGA1, involved in glycogen production, and ADR1, involved in ethanol/acetate metabolism, increased in the old dna2-1 cells but not in the wild-type strain. The congruence of observed increases in gene expression in wild-type yeast in our study and previous results in wild-type and in the sip2 strain (Lin et al., 2001) provides additional verification that aging cells isolated by elutriation and by the biotin/streptavidin technique reveal similar aging pathways.
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More important than the overlap of our data with the limited sample of genes identified in the 8-generation-old cells observed in the Lin et al. (2001) study, is that a much larger number of genes affecting additional pathways was observed to change in our 18-generation-old wild type. Comparison of our data with data sets describing yeast expression patterns measured under a number of different stress conditions (Gasch et al., 2000) revealed that both wild type and mutant showed a broad metabolic stress response known as the ESR. Figure 7B shows a Venn diagram of the overlap and Figures 8, 10, and 11 show specific genes that change (see ESR_VENN_diagram.txt in the supplementary data). In addition to the genes involved in carbohydrate metabolism already mentioned, DNA repair genes and chaperone genes are induced and ribosomal protein genes are repressed, for instance. These data suggest that dna2-1 and the wild-type strain are responding during aging as if there were external stress even though they are growing in ample glucose at normal pH and ionic strength and in the absence of DNA-damaging agents. Another condition besides aging that induces all these genes is high external osmolarity (HEO), which also extends life span (Kaeberlein et al., 2002). The additional genes identified that were not found to change in the Lin et al. (2001) study of 8-generation cells further demonstrates the utility of the elutriation technique in comparison with the biotin technique for investigating the pathologies of aging.
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In addition to genes in the ESR, genes involved in energy production, in the TCA cycle (CIT1, CIT3, SDH1, SDH2, SDH4, LSC, and LSC2) and in oxidative phosphorylation, increased in dna2-1 but not in wild-type (Figure 7C and TCA.txt in the supplementary data). This suggests that in addition to the shift away from glycolysis, the dna2 cells are also shifting toward energy production. Similar genes are induced during life span-extending caloric restriction (Lin et al., 2002), but not during HEO, during which glycerol synthesis is induced (Kaeberlein et al., 2002). Both respiration and glycerol synthesis increase NAD+/NADPH ratios, however, which have been proposed to affect life span.
We also compared the expression of genes involved in the nuclear NAD+ salvage pathway: NPT1, PCN1, NMA1, NMA2, and QNS1. Additional copies of these genes have been shown to increase replicative life span, and they are thought to be important for fueling SIR2-dependent longevity functions, so they might be expected to change in old cells (Lin et al., 2000; Anderson et al., 2002). Unfortunately, PCN1, NMA1, NMA2, and QNS1 were removed from the dataset during the filtering process. NPT1, however, shows a significant (2.24-fold) increase in the old dna2-1 cells, though only a 1.7-fold increase in wild type, which is slightly less than the twofold significance threshold. This is consistent with the proposal that there may be an attempt to increase flux through the NAD+ pathway to prolong life span in the old dna2-1 cells (Anderson et al., 2002). For SIR2 itself, we found no significant change (WT yc/oc = 1.37 and dna2-1 yc/oc = 1.67). (Additional stress response genes induced during aging are described below).
Although the ESR comprises a set of common up- and down-regulated genes, the transcriptional and signaling pathways that induce the responses are not shared but are specific to each type of stress (Gasch and Werner-Washburne, 2002). Because the cells we study were not subjected to environmental stress in our experimental protocol, it will be interesting to determine what type of internal stress and which regulatory pathways cause the observed response (Gasch and Werner-Washburne, 2002). The data discussed below suggest that DNA damage is among the endogenous inducers of the ESR that we see.
DNA Damage Genes Are Induced during Aging
Genes encoding components of DNA damage repair and checkpoint pathways are activated in both old wild-type and dna2-1 cells (Figures 8 and 9 and repair_table.txt in the supplementary data). In some cases, there are interesting differences in the specific repair genes activated between wild-type and mutant and in other cases interesting similarities.
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Six genes belonging to the homologous recombination pathway that repairs DSBs are activated in wild type (RAD51, RAD52, RAD54, RAD59, RHC18, and RDH54). In addition, several additional genes, required for sister chromatid cohesion but recently implicated also in recombination, SMC1 and SMC3, are induced. Of these recombination genes, only RAD51 is induced in old dna2-1 cells. The induction in wild type and lack of induction in the dna2-1 mutant were confirmed using real-time RT-PCR (Figure 9). The difference could imply that DNA2 is required for induction of these genes; or these genes might be controlled by a clock that measures numbers of divisions and the dna2-1 cells exit the cell cycle before they divide this many times.
A small number of genes are coordinately induced after certain doses of ionizing radiation, MMS, treatment with HO endonuclease to create a double-strand break, and several other types of DNA-damaging agents (Gasch et al., 2001). The induction of these genes is dependent on the ATR-like kinase Mec1, and these genes have been proposed to constitute a "signature" for DNA damage (Gasch et al., 2001). As with other genes in the environmental stress response, the number of these genes induced and the extent of change varies with the type of damaging agent and the dose (Gasch et al., 2001; Gasch and Werner-Washburne, 2002). Among the DNA "signature" genes are RAD51 and RAD54, genes involved in homologous recombination; DUN1, involved in the Mec1-mediated DNA damage checkpoint response; RNR2 and RNR4, affecting ribonucleotide reductase subunits; PLM2, which has homology to the forkhead-associated domain found in a number of transcription factors and kinases; DIN7, encoding a damage inducible mitochondrial protein with homologies to nucleases; YER004w, which has homology to the human protein Tip30, a tumor repressor that mediates apoptosis (Shtivelman, 1997; Xiao et al., 1998); and YBR070c, of unknown function. We find that in old wild-type cells RAD51, RAD54, DUN1, and YBR070c are induced (Figure 8). PLM2 was removed during the filtering process. The fact that not all genes are induced may reflect that old wild-type cells (18-20 generations) are only at 35% of the maximum life span of this strain, and the level of damage may be insufficient for full induction. In dna2-1, six of the nine signature genes for DNA damage (RNR2, RNR4, RAD51, PML2, YER004w, and YBR070c) increased. RAD54, discussed above, DUN1, and DIN7 (a mitochondrial protein) are not overexpressed. dna2 may either be required for their induction, downstream from Mec1, or the level of damage at the permissive temperature in the dna2 mutant may not induce sufficient damage to induce the full set of genes.
Additional differences were found in individual repair genes. It is not clear what significance they have, because it is now well established that the genes induced after ionizing radiation, MMS, and other types of DNA damage show surprisingly little overlap with genes required for repair of the corresponding damage (Birrell et al., 2002). For instance, MSH2 and MSH6, which belong to the mismatch repair pathway; POL30, a replication gene that also belongs to the RAD6 postreplication repair pathway; and CDC9, which belongs to the nucleotide excision repair pathway are induced in the old wild-type cells but not in dna2 cells. In the dna2-1 strain (Figure 8), we found genes involved in the nucleotide excision repair pathway (RAD16 and SSL2) and genes involved in checkpoints overexpressed in old cells. HUG1, involved in the Mec1p-mediated checkpoint response to DNA damage and replication arrest, and perhaps required for recovery from checkpoint arrest (Basrai et al., 1999), and GRH1, involved in the spindle checkpoint, are elevated in old dna2-1 cells. The expression of SGS1, a RecQ helicase that affects life span (Sinclair et al., 1997), is elevated in the old dna2-1 cells (oc/yc = 2.20). This may be of interest in that we have recently shown that the human homologue of Sgs1, human BLM, suppresses the replication and repair defects of yeast dna2 mutants (Imamura and Campbell, 2003). DNA2 itself is induced in old wild-type cells but not in the dna2-1 mutant. No genes involved in the mismatch DNA repair pathway or in the postreplication repair pathway show any variation in expression in the mutant.
In summary, although the DNA repair genes activated in the dna2-1 old cells and in the wild-type old cells are different, activation in both of large numbers of repair genes, including multiple DNA signature genes, suggests that DNA damage is a characteristic of aging cells of both types. The only environmental condition that has been shown to cause such a coordinated induction is treatment of cells with DNA-damaging agents (Jelinsky et al., 2000; Gasch et al., 2001; Gasch and Werner-Washburne, 2002). We therefore suggest that endogenous DNA damage occurs during the aging process in wild type as well as in the dna2-1 mutant.
Response of Genes to Aging in dna2-1 Is Similar to the Telomerase Delete Response (TDR)
The TDR is a genome-wide expression response recently documented in two separate studies of cells lacking telomerase due to a deletion of the telomerase RNA, tlc1 mutants (Nautiyal et al., 2002; Teng et al., 2002). Such mutants undergo a cellular senescence attributed to the shortening of yeast telomeres rather than to deregulation of the normal replicative mother cell aging program. Telomerase senescence differs from mother cell aging in that cells go through 40-60 generations before losing viability and in that it is a symmetric rather than an asymmetric process, leading to cessation of division of both mother and daughter cells inheriting the truncated chromosomes. The senescence-associated TDR is characterized by up-regulation of oxidative phosphorylation and TCA cycle genes, of DNA repair genes, and of a number of genes involved in the ESR. The ESR includes genes involved in carbohydrate metabolism, cell cycle regulation, DNA synthesis, cell wall formation, transcriptional regulation, amino acid biosynthesis, protein degradation and cross-membrane transporters, and a number of unclassified proteins or proteins with no known function (Figure 10 and telomerase_table.txt in the supplementary data). There is significant overlap between the TDR and the genes that change during aging in dna2-1 (Figures 10, 11, and 12; TLC1_VENN_diagram.txt and chance_similarity_tlc1d_ dna2_1.txt in the supplementary data). Almost all of the ESR stress response genes induced in the TDR were identified in our experiments as being up-regulated in old dna2-1 cells, suggesting that there is an integrated cellular reorganization occurring that is similar in the dna2-1 old cells and the senescent tlc1 cells. Yet another aspect of the TDR pattern is the down-regulation of ribosomal genes, which Warner (1999) has suggested may reflect an attempt by cells to conserve energy by slowing growth, reducing protein synthesis and ribosome biogenesis. The down-regulation of ribosomal genes is shared by old wild type (Figure 11 and ribo_table.txt in supplementary data). Among cell cycle genes, the down-regulation of the histone genes is also common to the TDR and to dna2-1 mother cell aging. Wild type actually shows induction of the histone genes, by contrast (Figure 11 and telomerase_table.txt in the supplementary data). Few, if any, of these genes change in our control experiments (e.g., dna2_1 young Cy3/dna2_1 young Cy5). We conclude that similar but not identical stress genes are activated in dna2_1 mutant and in tlc1 mutant with critically short telomeres. The respiration and stress genes were not changed to as significant a degree in the 18-generation-old wild-type cells (Figure 10), perhaps because they were not as far along in their life span as the dna2-1 cells.
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Perhaps the most noteworthy commonality between dna2-1 and the TDR is in the DNA repair genes (Nautiyal et al., 2002; Teng et al., 2002). Five of the DNA damage signature genes are induced during TDR and four of these are among the six induced in old dna2-1. RAD54 and DUN1, recombination and checkpoint genes, are not induced either in the TDR or in aging dna2-1 cells. The recombination genes as a group (RAD54, RAD59) fail to be induced during TDR, just as noted above for old dna2-1, with the exception of RAD51 in both cases (Figures 9 and 10). The similarities in the response of recombination genes in the TDR and the aging dna2-1 cells, and differences from wild type suggest that the conditions leading to the response may be the same in TDR and in aging dna2-1 cells.
In summary, the similarity of genes expressed in old dna2-1 and in TDR supports the idea that the response to replicative mother cell aging in the dna2-1 strain is very similar to that occurring during cellular senescence due to lack of telomerase. This pattern is less obvious in the wild-type strain at the point in the life span we study.
The telomerase-deficient signature (TDS) is a subset of the TDR genes and comprises 12 genes that are induced in telomerase-defective mutants but that are not induced in >20 other conditions of stress and DNA damage (Nautiyal et al., 2002). Despite the large number of genes that respond in the same way to both dna2 aging and telomerase-defective senescence, only two of the TDS genes overlap between the old dna2 gene expression response and the tlc1 mutant: a single replication gene, CRR1, that interacts with cruciform DNA, and a gene of unknown function: YFR018c (Figure 10). However, we do note derepression of the subtelomeric Y' ORF and of HXK1, another subtelomeric gene in the aging dna2-1 cells (Figure 10), suggesting a defect in telomeric silencing and in turn an alteration in telomere structure. We conclude that though the global response is the same in the two conditions, there is still some specialization that is different between mother cell aging and telomerase deficiency.
Cell Cycle-regulated Genes
The levels of numerous genes necessary for normal cell cycle progression are higher in the old wild-type cells. Out of the 627 genes up-regulated in the old wild-type cells, 45 are normally expressed in G2/M phase of the cell cycle (7.18%) (Spellnoon et al., 1998). Only a few are up-regulated in the old dna2-1 cells. (The list of these genes is available in the supplementary data file cycle_repli_table.txt). Only a few of these are common to wild type and the dna2 mutant. Many genes involved in chromosome condensation and segregation were up-regulated at this point in the wild-type life span. BUB2, a checkpoint protein required for cell cycle arrest in response to loss of microtubule function, DBF20, a cell cycle protein kinase involved in termination of M-phase, and FKH1 are more highly expressed in old wild-type cells. In the same way, the expression of APC1, a component of the anaphase promoting complex required for Clb2p degradation and for the metaphase-anaphase transition, increases with aging in the wild-type strain. Genes involved in chromosome condensation and segregation were elevated (CHL4, CIN2, CSE4, IPL1, LOC7, MCD1/SCC1, MIF2, SLI15, SMC1, and SMC3). Many DNA replication genes, such as replication factor A (RFA1, RFA2, and RFA3) and replication factor C (RFC3); CDC54, required for initiation of DNA replication; CDC9; DNA ligase; and RTT104, which promotes replication fork progression through the ribosomal DNA, are up-regulated in the old wild-type cells.
With respect to the old dna2-1 cells, of the 958 genes more highly expressed in the old dna2-1 cells, 147 are cell cycle regulated (15.3%). Forty-nine genes are specifically expressed in G1, 16 in S, 14 in S/G2, 33 in G2/M, and 35 in M/G1 (Spellman et al., 1998). Thus, the cell cycle-regulated genes reflect the distribution of cells with respect to cell cycle position shown in the flow cytometry profile (Figure 4). SRC1, whose expression is cell cycle regulated, ESS1, involved in cell cycle control and HUG1, involved in the Mec1p-mediated checkpoint response to DNA damage and replication arrest, show a higher expression level in the old dna2-1 cells. Interestingly, none of the genes involved in chromosome condensation and segregation and up-regulated in old wild-type cells seems to be up-regulated in the dna2-1 strain.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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