Retrotransposition Is Associated with Genome Instability During Chronological Aging

Retrotransposition is associated with genome instability during chronological aging

Patrick H. Maxwella,1,2, William C. Burhansb, and M. Joan Curcioa,2

aLaboratory of Molecular Genetics, Wadsworth Center, and Department of Biomedical Sciences, School of Public Health, University at Albany, State University of New York, Albany, NY 12201-2002; and bDepartment of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263

Edited by Marlene Belfort, University at Albany, State University of New York, Albany, NY, and approved August 23, 2011 (received for review February 28, 2011) Genetic damage through mutations and genome rearrangements corporated into genomes as processed pseudogenes (12, 13). has been hypothesized to contribute to aging. The specificmecha- Retrotransposon insertions have caused a variety of human dis- nisms responsible for age-induced increases in mutation and eases (14), and a potential role for human retrotransposons in chromosome rearrangement frequencies and a potential causative aging has been recently reviewed (15). It is challenging to study the role for DNA damage in aging are under active investigation. activity of individual endogenous retrotransposons, manipulate Retrotransposons are mobile genetic elements that cause insertion their mobility, and quantitatively assess the consequences of en- mutations and contribute to genome rearrangements through dogenous retrotransposon mobility on genome stability in humans. nonallelic recombination events in humans and other organisms. However, Saccharomyces cerevisiae has proven to be an excellent We have investigated the role of endogenous Ty1 retrotransposons model for studying endogenous retrotransposons (16), because the Saccha- in aging-associated increases in genome instability using the mobility of individual endogenous yeast Ty1 retrotransposons can romyces cerevisiae chronological aging model. We show that age- be routinely quantified, and a variety of means exist to manipulate induced increases in loss of heterozygosity and chromosome loss retrotransposition levels. events are consistently diminished by mutations or treatments that S. cerevisiae has also proven to be a useful model organism for reduce Ty1 retrotransposition. Ty1 mobility is elevated in very old studying aging through two different experimental systems (17, yeast populations, and new retromobility events are often associ- 18). Replicative aging is a measure of the number of times a given ated with chromosome rearrangements. These results reveal a correlation between retrotransposition and genome instability during yeast mother cell can divide to produce daughter cells, whereas yeast aging. Retrotransposition may contribute to genetic damage chronological aging measures the survival of yeast cells main- during aging in diverse organisms and provides a useful tool for tained in saturated cultures that are nutrient-depleted. Numerous studying whether genetic damage is a causative factor for aging. studies have demonstrated that replicative aging and chronological aging in yeast are regulated by conserved growth-signaling and fl long-terminal repeat retrotransposon | mobile element | nutrient-sensing pathways that in uence aging in other organisms, chromosomal rearrangement | lifespan including humans (18, 19). Despite some observations of potential yeast-specific aging factors (18, 20), yeast aging is associated with substantial amount of research has identified associations changes in metabolic and stress response pathways, increases in Abetween aging and genome instability in humans and model oxidative stress, and increases in the accumulation of mutations organisms, but a causative role for DNA damage and genome and chromosome rearrangements, which are commonly associ- – instability in normal aging has not been conclusively demon- ated with aging in diverse organisms (18, 19, 21 24). strated. Increases in frequencies of mutations and chromosome We took advantage of the yeast chronological aging model to rearrangements and in accumulation of DNA repair foci with address whether retrotransposons promote age-associated genome increasing age have been detected in a variety of cells and instability. We show that mutations and treatments that decrease organisms (1). Multiple human syndromes characterized by pre- Ty1 retrotransposition produce corresponding reductions in age- mature symptoms of aging or onset of aging-related diseases, such induced loss of heterozygosity (LOH) and/or chromosome loss in as Werner syndrome and Cockayne syndrome, result from defi- aging diploid yeast populations. Ty1 retromobility was elevated in ciencies in enzymes involved in DNA replication and repair (2, 3). old diploid yeast populations, and cells with new retromobility Elegant work in a Drosophila model has demonstrated that dif- events harbored chromosome rearrangements. These data are ferent DNA repair pathways are used preferentially in old and consistent with the hypothesis that retrotransposons promote ge- young cells to repair DNA damage (1). Continued studies of nome instability as cells age. mechanisms of aging-associated genome instability and the consequences of increasing or decreasing levels of DNA damage on lifespan should help to clarify whether genome instability is an This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, “Telomerase and Retrotransposons: Reverse Transcriptases That Shaped Ge- important causative factor for aging. nomes,” held September 29–30, 2010, at the Arnold and Mabel Beckman Center of the Retrotransposons are ubiquitous eukaryotic mobile DNA ele- National Academies of Sciences and Engineering in Irvine, CA. The complete program and ments that can promote genome instability in a variety of ways. audio files of most presentations are available on the NAS Web site at www.nasonline. Retrotransposition involves transcription of an element, reverse org/telomerase_and_retrotransposons. transcription of the RNA copy into a cDNA by a reverse tran- Author contributions: P.H.M. and M.J.C. designed research; P.H.M. performed research; W.C.B. contributed new reagents/analytic tools; P.H.M., W.C.B., and M.J.C. analyzed data; scriptase encoded by the element, and integration of the cDNA and P.H.M., W.C.B., and M.J.C. wrote the paper. into a new genomic site. Retromobility refers to integration of The authors declare no conflict of interest. cDNA at new sites as well as recombination events between cDNA This article is a PNAS Direct Submission. and preexisting genomic retrotransposon sequences. In addition 1Present address: Center for Biotechnology and Interdisciplinary Studies, Rensselaer Poly- to acting as insertional mutagens, retrotransposons are frequently technic Institute, Troy, NY 12180. present at the sites of chromosome rearrangements, because 2 To whom correspondence may be addressed. E-mail: [email protected] or curcio@wadsworth. DNA repair processes can capture and insert cDNA into the ge- org. – nome at sites of damage (4 11). Additionally, retrotransposons This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. can produce reverse transcripts of cellular mRNA that are in- 1073/pnas.1100271108/-/DCSupplemental.

20376–20381 | PNAS | December 20, 2011 | vol. 108 | no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1100271108 Downloaded by guest on September 27, 2021 FEATURE Results minished in all the mutant strains, except lsm1, and these dif- SACKLER SPECIAL Mutations and Treatments That Reduce Ty1 Retrotransposition ferences were statistically significant for hsx1 and spt3 in the Reduce Levels of LOH and Chromosome Loss During Aging. We de- BY4741/BY4742 background and for dfg10, dhh1, and loc1 veloped a genetic system in two different strains of S. cerevisiae (chromosome loss only for loc1) in the GRF167 background. to quantify genome instability in chronologically aging yeast The similarity between lsm1 and WT strains with regard to aging- populations. Our system consisted of a hemizygous URA3 gene associated increases in LOH could be explained by the relatively insertion at position 522206 on the right arm of chromosome modest decrease in retrotransposition in lsm1 mutants (29); in- VIII and a hemizygous TRP1 gene insertion at position 56054 on deed, we observed minor effects on Ty1 integration in our PCR the left arm of the same copy of chromosome VIII in diploid assay (Fig. S2). strains (Fig. S1). Exposure of cells to medium containing 5-flu- As a further test of the hypothesis that retromobility gives rise to oroorotic acid (5-FOA) selects against the function of URA3 aging-associated genome instability, we exposed cells to the re- (25). Cultures were initiated at low cell densities, allowed to verse transcriptase inhibitor phosphonoformic acid (PFA), which grow to stationary phase, and then maintained in stationary inhibits Ty1 retromobility by reducing levels of Ty1 reverse tran- fi phase for the duration of the experiments. The frequency with scripts (30). Exposure to PFA signi cantly diminished the age- which cells became resistant to 5-FOA was determined during associated increase in LOH from 20-fold to 5.8-fold and the age- the initial growth period and at regular intervals during main- associated increase in chromosome loss from 32-fold to 3.9-fold tenance in stationary phase to measure the frequency of LOH at (Table 1). To identify media conditions that reduce Ty1 retro- different time points during aging. Resistance to 5-FOA could mobility, we used a genetic assay to measure retromobility of arise from small-scale mutations in URA3, gene-conversion re- a single chromosomal Ty1his3AI element. The his3AI retro- combination events between the two copies of chromosome VIII, mobility indicator gene allows Ty1 mobility to be detected through chromosome rearrangements, or loss of the entire marked restoration of a functional HIS3 gene during Ty1 replication (31). The BY4741/BY4742 strain was grown in different media pre- chromosome VIII. We grew 5-FOA–resistant derivatives on viously shown to influence lifespan (20, 32–34). Growth in 2% medium lacking tryptophan to select for the function of TRP1 to glycerol (vol/vol) plus 2% (vol/vol) ethanol decreased Ty1his3AI distinguish chromosome loss events, because these events would mobility fivefold, whereas other media resulted in little or no result in loss of both URA3 and TRP1 function (Fig. S1). change in mobility (<2.5-fold; Fig. S3). Reduced retrotranspo- We decreased Ty1 retromobility in our strains by deleting or

sition in 2% (vol/vol) glycerol plus 2% (vol/vol) ethanol medium GENETICS disrupting the DHH1, DFG10, HSX1, LOC1, LSM1,orSPT3 was accompanied by significantly smaller age-associated increases gene and then measured genome instability in the chronologi- in LOH (36-fold vs. 5.1-fold) and chromosome loss (98-fold vs. cally aging populations (Fig. 1 and Table 1). Mutation of SPT3 9.0-fold; Table 1). Overall, these results are consistent with the substantially reduces Ty1 transcription (26); mutation of HSX1 idea that retrotransposition promotes genome instability during tR CCU J [ ( ) ] disrupts the normal translational frame-shifting that yeast aging. produces Ty1 proteins (27); mutations in DHH1 and LSM1 de- It could be argued that the aging-associated increase in LOH crease Ty1 retromobility at posttranscriptional steps (28, 29); fl and chromosome loss frequencies we observed in the WT pop- and the steps of retromobility in uenced by mutations in DFG10 ulations result from improved survival of mutant cells present and LOC1 are under active investigation. We used a semiquan- fi early in the growth of a culture, rather than true increases in the titative Ty1 integration PCR assay to con rm that the mutants frequencies of these events in a cell population during aging. To tested had lower levels of Ty1 mobility (Fig. S2 and SI Results). address this issue, we compared the chronological aging of the Initial LOH and chromosome loss frequencies were similar to parent strains with that of derivatives that had undergone LOH or − WT frequencies or elevated in the mutants (Fig. 1 and Table 1). chromosome loss events. Independent 5-FOA–resistant (Ura ) As expected, LOH frequencies and chromosome loss frequencies derivatives with LOH or chromosome loss events were obtained were substantially elevated in the WT strains, with age-induced from populations aged until cells exhibited <25% viability. The − increases of 20- and 32-fold (BY4741/BY4742) or 14- and 72- Ura isolates were mixed at equal cell densities with the corre- fold (GRF167), respectively (Table 1). The age-associated sponding Ura+ parent strains to initiate competitive chronological increases in LOH and chromosome loss frequencies were di- aging experiments. We determined the relative number of Ura+ − and Ura cells at different time points during aging. Note that the frequency of losing URA3 function in the parent strain was too low 100 to influence the results of this assay. The results of four independent experiments indicate that the Ty1 retrotransposition − Ura cells were losing viability at a rate similar to the parental 10 initial LOH − initial chr loss cells. The median values for the initial percentage of Ura cells

aging LOH present in the cell populations were variable for derivatives with

1 aging chr loss LOH or chromosome loss events for each of the four experiments, − reﬂecting different initial growth rates between the Ura deriva-

Relative Frequency − tives and the parent strains (Fig. 2). The percentage of Ura cells 0.1 in each experiment remained relatively constant during aging. The WT hsx1 loc1 spt3WT dhh1 loc1 lsm1 WT dfg10 − ADE2 ratio of the percentage of Ura cells at the ﬁnal time point to the − – BY4741/BY4742 GRF167 percentage of Ura cells at the initial time point was 0.76 1.27 for derivatives with LOH events and 0.42–4.1 for derivatives with Fig. 1. Ty1 retrotransposition levels and aging-associated increases in ge- chromosome loss events. This set of results is consistent with age- nome instability are frequently correlated. Relative mean (±SD) of median dependent increases in LOH and chromosome loss frequencies values for initial and aging-associated LOH and chromosome (chr) loss fre- resulting from new events that occur in old cells, rather than se- quencies was calculated for the indicated WT and mutant strains using the data presented in Table 1. Relative retrotransposition for each mutant was lection for cells with preexisting genetic changes. determined using Ty1 integration PCR data, as described in SI Materials and Methods. All WT values were set to 1, and values for mutants were nor- Most LOH Events Result from Large-Scale Recombination or Deletion malized to the corresponding WT value. A star over a bar for aging LOH or Events. We further analyzed LOH events in two WT and three aging chr loss indicates P < 0.05 using a two-tailed t test assuming unequal mutant strains (dfg10, hsx1, and loc1) making use of a second variance. hemizygous gene, HIS3, located ∼15 kb telomere-distal to URA3

Maxwell et al. PNAS | December 20, 2011 | vol. 108 | no. 51 | 20377 Downloaded by guest on September 27, 2021 Table 1. Mutations and treatments that decrease Ty1 mobility tend to diminish age-associated increases in LOH and chromosome loss frequencies Days until Days until LOH frequency, LOH frequency, Chromosome loss Chromosome loss Genotype 50% viability 10% viability initial (×10−5) fold increase frequency, initial (×10−6) frequency, fold increase

WT (BY) 12 ± 0.88 24 ± 1.3 3.0 ± 1.5 20 ± 4.6 2.1 ± 1.4 32 ± 21 hsx1::LEU2 13 ± 3.8 35 ± 3.6* 4.5 ± 0.40 3.2 ± 0.10* 5.1 ± 0.61 3.6 ± 0.66* loc1::KanMX 16 ± 0.15* 26 ± 1.3 4.88 ± 1.7 13 ± 6.1 6.1 ± 2.3 11 ± 7.9 spt3::KanMX 12 ± 0.04 15 ± 0.60* 12 ± 2.8 1.6 ± 0.43* 28 ± 1.3 4.8 ± 4.1* WT + PFA 19 ± 0.1* 38 ± 9.3 5.3 ± 4.9 5.8 ± 0.92* 25 ± 23 3.9 ± 1.4* WT (BY) glu 12 ± 0.9 26 ± 4.5 1.9 ± 0.43 36 ± 13 1.1 ± 0.58 98 ± 28 WT eth/gly 19 ± 0.73* 56 ± 4.5* 2.3 ± 0.66 5.1 ± 1.6* 2.2 ± 0.91 9.0 ± 0.25* WT (GRF) 17 ± 4.3 32 ± 5.7 2.2 ± 0.69 14 ± 4.8 0.53 ± 0.27 72 ± 13 dhh1::KanMX 15 ± 2.0 27 ± 8.3 3.0 ± 1.1 1.8 ± 0.18* 1.1 ± 0.87 2.8 ± 0.89* loc1::KanMX 26 ± 3.7* 41 ± 8.2 2.1 ± 1.4 8.2 ± 3.1 1.1 ± 0.73 18 ± 15* lsm1::KanMX 13 ± 5.4 22 ± 6.4 2.6 ± 1.7 28 ± 7.2 1.6 ± 1.2 58 ± 37 WT (ADE2)9.1± 0.2 24 ± 1.1 9.3 ± 7.1 19 ± 1.8 0.49 ± 0.38 91 ± 11 dfg10::ADE2 19 ± 0.28* 27 ± 0.88 5.1 ± 1.1 1.3 ± 0.04* 1.8 ± 0.88 7.0 ± 2.7*

Mean (±SD) of median values for each category from two or more independent experiments each using three to four replicates for the BY4741/BY4742 background (BY, first 7 rows) or GRF167 background (GRF, last 6 rows). WT glu or WT eth/gly refers to strains grown in medium containing 2% (wt/vol) glucose or 2% (vol/vol) ethanol plus 2% (vol/vol) glycerol, respectively. WT (ADE2) refers to ADE2 derivatives in the GRF167 background used for comparison with dfg10::ADE2 mutants. Days to 50% or 10% viability were determined from trend lines for each dataset, and LOH and chromosome loss were measured as frequencies of obtaining 5-FOAr and 5-FOAr Trp− derivatives, respectively. Initial values were obtained from the first time point or the first time point at which cfu/mL was at least 33% of the initial control cfu/mL. Fold increase was determined by dividing the peak value obtained over the course of a chronological aging experiment by the initial value. *P < 0.05 using a two-tailed t test assuming unequal variance.

on the same copy of chromosome VIII so as to group LOH tions/deletions. The percentage of events in each class was events into three classes (Fig. S4 and SI Materials and Methods). generally similar in WT and mutant strains, and also over the In all strains analyzed, most (82–99%) LOH events were asso- course of aging (Fig. 3). ciated with loss of HIS3 function but retention of TRP1 function (Fig. 3). Such events are likely the result of recombination, Ty1 Mobility Is Not Always Correlated with Lifespan. An inﬂuence of break-induced replication, or chromosome deletions that either retrotransposition on yeast lifespan during chronological aging replaced the marked chromosome arm sequences with the cor- was supported by some but not all of the results for different responding sequences of the unmarked copy of chromosome mutant strains and treatment conditions. It is worth noting that VIII or caused the loss of the genomic region containing URA3 the lifespan of yeast strains grown at 20 °C, the optimal tem- and HIS3. Loss of both TRP1 and HIS3 functions attributable to perature for Ty1 mobility (35, 36), was substantially longer than chromosome loss was the next most frequent class of events. the lifespan typically observed for strains grown at 30 °C (17). Local changes in the URA3 locus (retention of TRP1 and HIS3 Populations of hsx1, loc1, and dfg10 mutants and cells exposed to function) were only observed in the BY4741/BY4742 back- PFA or grown in 2% (vol/vol) glycerol plus 2% (vol/vol) ethanol ground (Fig. 3). PCR analysis of these local mutations indicated medium showed improved survival and took longer to reach 50% that ∼10% (∼0.2–0.5% of total events) were attributable to point mutations in URA3, indicating that most local changes were gene-conversion recombination events or relatively large inser- chromosome loss large-scale recombination or deletion local mutation 100%

LOH derivatives Chromosome loss derivatives 80% 35 30 60% % 25 Ura 20 40%

cells 15 LOH Events 10 20% 5 0 0% Initial >50 50-21 20-10 9-1 <1 viability % of initial cfu/ml BY loc1 hsx1 GRF dfg10

Fig. 2. Survival of cells with chromosome VIII LOH or chromosome loss Fig. 3. LOH events primarily arise through large-scale recombination or events during chronological aging is similar to survival of parental strains. deletion events. Percent of LOH events that arose attributable to the in- − Ura cells with chromosome VIII LOH or chromosome loss events were mixed dicated classes of events is shown for two WT strains (BY, BY4741/BY4742 at equal starting densities with Ura+ parental strains, and the relative frac- background; GRF, GRF167 ADE2 background) and for three mutants with − tions of Ura cells were measured at various time points during chronolog- reduced Ty1 retrotransposition (hsx1, loc1, dfg10). The three bars for each − ical aging. Median percent Ura cells for all time points that fell within the genotype show the results for LOH events that occurred in populations as indicated range of the initial cfu/mL for each of four experiments performed viability decreased from 80% to 100%, to 40–80%, and then to less than with three independent derivatives with LOH (black lines) or chromosome 15%. The number of events tested for each genotype for each viability range loss (gray lines) events is plotted. Dotted lines indicate categories of cfu/mL was as follows: BY: 513, 2,208, 1,371; loc1: 596, 960, 723; hsx1: 522, 703, 487; for which no data points were obtained for a particular experiment. GRF: 1,153, 94, 382; dfg10: 380, 280, 59.

20378 | www.pnas.org/cgi/doi/10.1073/pnas.1100271108 Maxwell et al. Downloaded by guest on September 27, 2021 FEATURE or 10% viability (Table 1). The dhh1 mutants had no change in JC5357 JC5390

A SACKLER SPECIAL viability, and the spt3 mutants reached 10% viability more quickly than the WT strain, however, despite low levels of Ty1 mobility 1.E+09 and aging-associated LOH and chromosome loss in these mutant 1.E+08 strains. Analysis of the data for an influence of Ty1 on lifespan is 1.E+07 complicated by potential gene- or treatment-specific effects on 1.E+06 lifespan that are independent of Ty1 retromobility. 1.E+05 An experiment comparing the lifespan of a Saccharomyces 1.E+04 0 4 8 12 16 20 24 28 32 36 40 44 48 paradoxus strain completely lacking Ty1 elements with that of Colony forming units/ml derivatives harboring a single Ty1 element did not support a direct Days role for Ty1 retrotransposition in cell survival during chronological aging (Fig. S5 and SI Results). However, we previously found B 1.E-05 that genomic copies of Ty1 can participate in recombination events with Ty1-generated reverse transcripts to cause chromo- 1.E-06 Mobility some rearrangements in S. cerevisiae (37), and the absence of 1.E-07 dispersed genomic copies of Ty1 in the S. paradoxus strains may limit the potential for Ty1 to cause genome rearrangements. his3AI 1.E-08 Ty1 1.E-09 Ty1 Mobility Frequency Is Elevated in Old Yeast Populations and Is 0 4 8 12 16 20 24 28 32 36 40 44 48 Associated with the Appearance of Chromosome Rearrangements. Days We used two diploid strains homozygous for independently in- tegrated chromosomal Ty1his3AI elements to measure retro- C old His- young His+ old His+ mobility during chronological aging. The frequency of Ty1 P mobility differed slightly between the two strains and remained stable or slightly decreased during the first half of these experiments (Fig. 4). Retromobility was greatly elevated at late stages

of three of four experiments, reaching levels 24-, 46-, or 77-fold GENETICS higher than the initial value for each experiment (Fig. 4). Sub- stantial increases in retromobility were not observed until populations reached ∼1 × 106 cfu/mL or less. Interestingly, the populations in the one experiment in which an increase in retromobility was not observed exhibited relatively steady densities of colony-forming units that were greater than 1 × 106 over the course of the experiment. Pulsed-field gel electrophoresis identified novel chromosome bands and the absence of one or more parental chromosome bands in the electrophoretic karyotypes of His+ cells obtained from aged populations (Fig. 4C). Novel and/ or missing chromosome bands were identified by pulsed-field gel + electrophoresis in 6 (50%) of 12 His derivatives from old popu- Fig. 4. Ty1 mobility is elevated and associated with chromosome rear- − lations (≥24 d), in 1 (7.1%) of 14 His derivatives from old rangements in old cells. (A) Each line shows the mean cfu/mL for an in- populations, and in 4 (29%) of 14 His+ derivatives of young dependent chronological aging experiment with three replicates performed populations (3–7 d) from the BY4741/BY4742 background (P < using one of the two indicated diploid strains. (B) Mean of His+ prototroph − 0.05 for old His+ vs. old His , Fisher’s exact test). The chro- frequencies as a measure of Ty1his3AI mobility corresponding to the strains and aging experiments shown in A.(C) Intact yeast chromosomes from pa- mosome rearrangements are therefore specifically associated − rental strain JC5357 (P) and three His isolates lacking retromobility events with retromobility in these populations. from old populations, three His+ isolates harboring new Ty1 retromobility + Discussion events from young populations, and three His isolates harboring new Ty1 retromobility events from old populations separated by clamped homoge- We have demonstrated an association between retromobility and neous electrical field gel electrophoresis and visualized with ethidium bro- genome instability during chronological aging of S. cerevisiae. mide. White arrows indicate bands not present in the parental lane. Mutations and treatments that reduce Ty1 retrotransposition resulted in corresponding decreases in age-induced LOH and chromosome loss frequencies in diploid yeast strains from two integrase (Fig. S6). Interaction of DNA repair and recombi- genetic backgrounds. Ty1 could potentially play a role in for- nation proteins with reverse transcripts during integration could mation of the LOH events we observed, because the majority of lead to recombination or break-induced replication events that LOH events resulted from large-scale recombination/deletion involve allelic or nonallelic genomic sites that have Ty1 se- events rather than point mutations. Ty1 retromobility was greatly quences. The sequenced yeast genome contains three Ty1 solo elevated in old yeast populations and correlated with high fre- LTRs and one full-length element within 60 kb of the position of quencies of chromosome rearrangements. This work suggests the hemizygous marker we introduced into our strains (www. that retrotransposition could be a useful target to modulate yeastgenome.org). Failure to repair the single-stranded DNA levels of age-induced genome instability and to examine whether gaps that flank Ty1 integration events could stimulate re- genome instability plays a causative role in aging. combination events or give rise to DNA breaks during replica- The correlation between Ty1 mobility and age-related genome tion, thereby activating DNA damage signaling activities that instability established by our results is consistent with the pos- lead to break-induced replication or chromosome rearrange- sibility that Ty1 mobility is one of the causes of age-related ge- ments. Stimulation of such events could also occur in the absence nome instability. Ty1 elements could contribute to LOH and of integration if Ty1 integrase possesses a nonspecific endonu- chromosome loss events by integrating into one of two hetero- clease activity similar to what has been observed for some ret- zygous alleles, by triggering allelic or nonallelic recombination, roviral integrases (38, 39). Retrotransposition into replicating and by causing chromosome breaks through the action of Ty1 DNA could stimulate recombination events or impair replication

Maxwell et al. PNAS | December 20, 2011 | vol. 108 | no. 51 | 20379 Downloaded by guest on September 27, 2021 fork progression. Failure to repair chromosome breaks or to of the insertions could represent recent events, based on very low complete replication could result in chromosome loss. In addi- allele frequencies (51–53). Our results linking retrotransposition tion, replication stress has been linked to yeast chronological to aging-related genome instability in yeast and the growing aging (34, 40), and Ty1 retromobility is activated by hydroxyurea appreciation of how dynamic these elements are in human (41), an agent that causes replication stress. Therefore, replica- genomes emphasize the importance of investigating the contri- tion stress could promote aging-associated genome instability by bution of retrotransposition to aging in humans. activating Ty1. Retrotransposons could promote age-associated genome in- Materials and Methods stability in many organisms, based on similarities in the mechanism Yeast Strains and Media. Yeast strains were grown using standard media. of retrotransposition and regulation through common cellular Most S. cerevisiae strains were diploids made by mating derivatives of stress conditions (42). Yeast, mammalian, fungal, and plant retro- BY4741 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0) and BY4742 (MATα ura3Δ0 transposons can be activated by oxidative stress and/or DNA dam- leu2Δ0 his3Δ1 lys2Δ0) from the S288c background or JC2980 (MATα his3Δ200 Δ age (10, 43–47). Oxidative stress and DNA damage are associated ade2::hisG ura3-167 leu2::hisG trp1::hisG) and JC4545 (MATa his3 200 ade2:: with aging in a variety of organisms (19). In addition, human L1 hisG ura3-167 leu2::hisG trp1::hisG) from the GRF167 background. Ty1 mobility assays were performed using diploids JC5357 (MATa/α his3Δ1/his3Δ1 retrotransposons generate DNA breaks at frequencies greater than leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0 Ty1his3AI/Ty1hi- the frequency of retrotransposition (48). Increases in mutations and s3AI) and JC5390 (MATa::URA3/MATα ade2::hisG/ADE2 his3Δ200/his3Δ200 genome rearrangements caused by retrotransposon insertions, en- TRP1/trp1::hisG ura3-167/ura3-167 Ty1588:NEO/+ Ty1his3AI/Ty1his3AI) from donuclease activities of retrotransposon proteins, and interactions the BY4741/BY4742 and GRF167 backgrounds, respectively. S. paradoxus between DNA repair proteins and reverse transcripts could there- strains were derivatives of DG1768 (54). Details of strain constructions are fore be common occurrences during aging of many species. provided in SI Materials and Methods. Because we did not always observe an inverse correlation between Ty1 retromobility and survival during stationary phase for Chronological Aging, Viability, and LOH Assays. Yeast strains were inoculated the different mutants and conditions, we cannot conclude that Ty1 at densities of 5 × 103 cells/mL into 25 or 50 mL of broth in 125- or 250-mL activity directly reduces lifespan. However, changes in cellular flasks, respectively, and grown with shaking at 20 °C in synthetic complete processes that influence lifespan independent of retrotransposition (SC) medium-uracil plus 2% (wt/vol) glucose plus fourfold excess of histidine, might obscure an effect of Ty1 on lifespan. Mutations in DHH1, leucine, lysine, methionine, and tryptophan, unless otherwise noted. We used medium lacking uracil to reduce the proliferation of cells lacking URA3 LOC1, LSM1,andSPT3 result in a wide variety of phenotypes, function during the initial growth to saturation. After 3–4 d of incubation, whereas mutations in DFG10 and HSX1 result in limited pheno- and thereafter at intervals of ∼3–7 d, aliquots of cells were removed and types beyond their roles in regulating Ty1 (www.yeastgenome.org). analyzed. Cells were diluted in water, mixed with one or two volumes of For instance, although Ty1 mobility is somewhat reduced in lsm1 0.4% trypan blue in PBS, and incubated for 1 h before scoring the number of mutants, these strains exhibit a reduced chronological lifespan (49) live and dead cells in a population of ≥200 cells using a hemacytometer to and elevated genome instability attributable to changes in the determine viability of populations. Cells were diluted in water and spread regulation of histone gene expression (50). onto rich medium to determine the number of cfu/mL. To measure LOH, cells We also expected that survival during stationary phase and were diluted in water or centrifuged and washed in water and spread onto levels of genome instability would show an inverse correlation if SC medium containing 5-FOA. LOH frequencies were calculated by dividing the accumulation of mutations and genome rearrangements di- the number of resistant colonies by the total number of colony forming units rectly contributes to aging. Previous studies have found LOH plated. All plates were incubated at 30 °C for up to 5 d. Chromosome loss frequencies were determined by replicating 5-FOA–resistant colonies to SC increases during replicative and chronological aging in yeast (21, fi medium lacking tryptophan and dividing the number of colonies that failed 22, 24). One of these studies identi ed a correlation between to grow by the total number of colony forming units originally plated on 5- natural variation in replicative lifespan and the increase in LOH FOA medium. Initial values were those obtained after the initial 3–4dof during chronological aging, although the increase in LOH during growth or from the first time point when cfu/mL was at least 33% of the chronological aging was observed to lag slightly behind the drop in initial WT cfu/mL. Fold increase during aging was determined as the peak viability (22). We did not always observe a longer lifespan for value during the course of the aging experiment divided by the initial value. strains with lower frequencies of LOH or chromosome loss. For competitive aging experiments, we mixed 5 × 104 cells/mL each of a Ura+ − Therefore, our results are not fully consistent with yeast chrono- parent strain and a Ura (5-FOAr) derivative in medium containing uracil. We + r logical aging resulting solely from an increase in the accumulation determined the relative number of cells from Ura parent strains and 5-FOA of deleterious genetic changes. However, cells have to survive derivatives at different time points during aging by spreading cells onto rich medium and then replicating colonies to medium lacking uracil, on which DNA damage to exhibit an increased frequency of mutations, and only the parental Ura+ colonies could grow. Three independent 5-FOAr preferential death of cells experiencing DNA damage and repli- derivatives obtained from aging cultures at <25% viability were used for cation stress during yeast aging has been observed (34). Such each of four experiments. preferential cell death before mutagenic repair events occur could mask a correlation between lifespan and indicators of genome Pulsed-Field Gel Electrophoresis to Detect Chromosome Rearrangements. Yeast instability. Future work will need to address carefully whether cell chromosomal DNA was prepared, and clamped homogeneous electrical field death from DNA damage before the accumulation of mutations gels were prepared and run essentially as described previously (37), except has a substantial impact on chronological lifespan. that gels were run at 4.5 V/cm for 51 h with switch times of 50–135 s. The frequency of retrotransposition in human populations may be greater than originally suspected, based on a few recent ACKNOWLEDGMENTS. We thank D. Garfinkel for providing the Ty1-less reports of genome-wide studies of individual human genomes. haploid S. paradoxus strain. This work was supported, in part, by Grant fi K99AG031911 from the National Institute on Aging (to P.H.M., Grant These reports identi ed many polymorphic retrotransposon CA016056 from the National Cancer Institute to Roswell Park Cancer Insti- insertions using independent methods, and a substantial fraction tute) and Grant GM52072 from the National Institutes of Health (to M.J.C.).

1. Engels WR, Johnson-Schlitz D, Flores C, White L, Preston CR (2007) A third link con- 5. Teng SC, Kim B, Gabriel A (1996) Retrotransposon reverse-transcriptase-mediated necting aging with double strand break repair. Cell Cycle 6:131–135. repair of chromosomal breaks. Nature 383:641–644. 2. Andressoo JO, Hoeijmakers JH, Mitchell JR (2006) Nucleotide excision repair disorders 6. Dunham MJ, et al. (2002) Characteristic genome rearrangements in experimental and the balance between cancer and aging. Cell Cycle 5:2886–2888. evolution of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 99:16144–16149. 3. Brosh RM, Jr., Bohr VA (2007) Human premature aging, DNA repair and RecQ heli- 7. Morrish TA, et al. (2002) DNA repair mediated by endonuclease-independent LINE-1 cases. Nucleic Acids Res 35:7527–7544. retrotransposition. Nat Genet 31:159–165. 4. Moore JK, Haber JE (1996) Capture of retrotransposon DNA at the sites of chromo- 8. Umezu K, Hiraoka M, Mori M, Maki H (2002) Structural analysis of aberrant chro- somal double-strand breaks. Nature 383:644–646. mosomes that occur spontaneously in diploid Saccharomyces cerevisiae: Retro-

20380 | www.pnas.org/cgi/doi/10.1073/pnas.1100271108 Maxwell et al. Downloaded by guest on September 27, 2021 FEATURE transposon Ty1 plays a crucial role in chromosomal rearrangements. Genetics 160: 32. Smith DL, Jr., McClure JM, Matecic M, Smith JS (2007) Calorie restriction extends the

97–110. chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins. SACKLER SPECIAL 9. Abeysinghe SS, Chuzhanova N, Krawczak M, Ball EV, Cooper DN (2003) Translocation Aging Cell 6:649–662. and gross deletion breakpoints in human inherited disease and cancer I: Nucleotide 33. Murakami CJ, Burtner CR, Kennedy BK, Kaeberlein M (2008) A method for high- composition and recombination-associated motifs. Hum Mutat 22:229–244. throughput quantitative analysis of yeast chronological life span. J Gerontol A Biol Sci 10. Rockwood LD, Felix K, Janz S (2004) Elevated presence of retrotransposons at sites of Med Sci 63(2):113–121. DNA double strand break repair in mouse models of metabolic oxidative stress and 34. Weinberger M, et al. (2010) Growth signaling promotes chronological aging in – MYC-induced lymphoma. Mutat Res 548(1-2):117 125. budding yeast by inducing superoxide anions that inhibit quiescence. Aging (Albany 11. Morrish TA, et al. (2007) Endonuclease-independent LINE-1 retrotransposition at NY) 2:709–726. mammalian telomeres. Nature 446:208–212. 35. Paquin CE, Williamson VM (1984) Temperature effects on the rate of ty transposition. 12. Derr LK, Strathern JN (1993) A role for reverse transcripts in gene conversion. Nature Science 226:53–55. 361:170–173. 36. Lawler JF, Jr., Haeusser DP, Dull A, Boeke JD, Keeney JB (2002) Ty1 defect in pro- 13. Esnault C, Maestre J, Heidmann T (2000) Human LINE retrotransposons generate teolysis at high temperature. J Virol 76:4233–4240. processed pseudogenes. Nat Genet 24:363–367. 37. Maxwell PH, Curcio MJ (2007) Retrosequence formation restructures the yeast ge- 14. Belancio VP, Deininger PL, Roy-Engel AM (2009) LINE dancing in the human genome: nome. Genes Dev 21:3308–3318. Transposable elements and disease. Genome Med 1:97. 38. Caumont AB, et al. (1996) Expression of functional HIV-1 integrase in the yeast 15. St Laurent, G, 3rd, Hammell N, McCaffrey TA (2010) A LINE-1 component to human aging: Do LINE elements exact a longevity cost for evolutionary advantage? Mech Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: Potential use – Ageing Dev 131:299–305. for inhibitor screening. Curr Genet 29:503 510. ﬁ 16. Lesage P, Todeschini AL (2005) Happy together: The life and times of Ty retro- 39. Katzman M, Sudol M (1996) Nonspeci c alcoholysis, a novel endonuclease activity of ﬁ transposons and their hosts. Cytogenet Genome Res 110(1-4):70–90. human immunode ciency virus type 1 and other retroviral integrases. J Virol 70: 17. Madia F, Gattazzo C, Fabrizio P, Longo VD (2007) A simple model system for age- 2598–2604. dependent DNA damage and cancer. Mech Ageing Dev 128:45–49. 40. Weinberger M, et al. (2007) DNA replication stress is a determinant of chronological 18. Kaeberlein M (2010) Lessons on longevity from budding yeast. Nature 464:513–519. lifespan in budding yeast. PLoS ONE 2:e748. 19. Burhans WC, Weinberger M (2007) DNA replication stress, genome instability and 41. Curcio MJ, et al. (2007) S-phase checkpoint pathways stimulate the mobility of the aging. Nucleic Acids Res 35:7545–7556. retrovirus-like transposon Ty1. Mol Cell Biol 27:8874–8885. 20. Burtner CR, Murakami CJ, Kennedy BK, Kaeberlein M (2009) A molecular mechanism 42. Beauregard A, Curcio MJ, Belfort M (2008) The take and give between retro- of chronological aging in yeast. Cell Cycle 8:1256–1270. transposable elements and their hosts. Annu Rev Genet 42:587–617. 21. McMurray MA, Gottschling DE (2003) An age-induced switch to a hyper-re- 43. Mhiri C, et al. (1997) The promoter of the tobacco Tnt1 retrotransposon is induced by combinational state. Science 301:1908–1911. wounding and by abiotic stress. Plant Mol Biol 33:257–266. 22. Qin H, Lu M, Goldfarb DS (2008) Genomic instability is associated with natural life 44. Ikeda K, Nakayashiki H, Takagi M, Tosa Y, Mayama S (2001) Heat shock, copper sul- span variation in Saccharomyces cerevisiae. PLoS ONE 3:e2670. fate and oxidative stress activate the retrotransposon MAGGY resident in the plant 23. Burhans WC, Weinberger M (2009) Acetic acid effects on aging in budding yeast: Are pathogenic fungus Magnaporthe grisea. Mol Genet Genomics 266:318–325.

– GENETICS they relevant to aging in higher eukaryotes? Cell Cycle 8:2300 2302. 45. Scholes DT, Kenny AE, Gamache ER, Mou Z, Curcio MJ (2003) Activation of a LTR- 24. Lindstrom DL, Leverich CK, Henderson KA, Gottschling DE (2011) Replicative age in- retrotransposon by telomere erosion. Proc Natl Acad Sci USA 100:15736–15741. duces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces 46. Stribinskis V, Ramos KS (2006) Activation of human long interspersed nuclear element cerevisiae. PLoS Genet 7:e1002015. 1 retrotransposition by benzo(a)pyrene, an ubiquitous environmental carcinogen. 25. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking Cancer Res 66:2616–2620. orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. 47. Stoycheva T, Pesheva M, Venkov P (2010) The role of reactive oxygen species in the Mol Gen Genet 197:345–346. induction of Ty1 retrotransposition in Saccharomyces cerevisiae. Yeast 27:259–267. 26. Winston F, Durbin KJ, Fink GR (1984) The SPT3 gene is required for normal tran- 48. Gasior SL, Wakeman TP, Xu B, Deininger PL (2006) The human LINE-1 retrotransposon scription of Ty elements in S. cerevisiae. Cell 39:675–682. creates DNA double-strand breaks. J Mol Biol 357:1383–1393. 27. Kawakami K, et al. (1993) A rare tRNA-Arg(CCU) that regulates Ty1 element ribo- 49. Palermo V, Cundari E, Mangiapelo E, Falcone C, Mazzoni C (2010) Yeast lsm pro- somal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cer- apoptotic mutants show defects in S-phase entry and progression. Cell Cycle 9: evisiae. Genetics 135:309–320. – 28. Checkley MA, Nagashima K, Lockett SJ, Nyswaner KM, Garfinkel DJ (2010) P-body 3991 3996. components are required for Ty1 retrotransposition during assembly of retro- 50. Herrero AB, Moreno S (2011) Lsm1 promotes genomic stability by controlling histone – transposition-competent virus-like particles. Mol Cell Biol 30:382–398. mRNA decay. EMBO J 30:2008 2018. 29. Dutko JA, Kenny AE, Gamache ER, Curcio MJ (2010) 5′ to 3′ mRNA decay factors 51. Beck CR, et al. (2010) LINE-1 retrotransposition activity in human genomes. Cell 141: colocalize with Ty1 gag and human APOBEC3G and promote Ty1 retrotransposition. 1159–1170. J Virol 84:5052–5066. 52. Huang CR, et al. (2010) Mobile interspersed repeats are major structural variants in 30. Lee BS, Bi L, Garfinkel DJ, Bailis AM (2000) Nucleotide excision repair/TFIIH helicases the human genome. Cell 141:1171–1182. RAD3 and SSL2 inhibit short-sequence recombination and Ty1 retrotransposition by 53. Iskow RC, et al. (2010) Natural mutagenesis of human genomes by endogenous similar mechanisms. Mol Cell Biol 20:2436–2445. retrotransposons. Cell 141:1253–1261. 31. Curcio MJ, Garfinkel DJ (1991) Single-step selection for Ty1 element retro- 54. Garfinkel DJ, Nyswaner K, Wang J, Cho JY (2003) Post-transcriptional cosuppression of transposition. Proc Natl Acad Sci USA 88:936–940. Ty1 retrotransposition. Genetics 165:83–99.

Maxwell et al. PNAS | December 20, 2011 | vol. 108 | no. 51 | 20381 Downloaded by guest on September 27, 2021