Telomeres protect the chromosome ends from degradation and play crucial roles in cellular aging and disease. Recent studies have additionally found a correlation between psychological stress, telomere length, and health outcome in humans. However, studies have not yet explored the causal relationship between stress and telomere length, or the molecular mechanisms underlying that relationship. Using yeast as a model organism, we show that stresses may have very different outcomes: alcohol and acetic acid elongate telomeres, whereas caffeine and high temperatures shorten telomeres. Additional treatments, such as oxidative stress, show no effect. By combining genome-wide expression measurements with a systematic genetic screen, we identify the Rap1/Rif1 pathway as the central mediator of the telomeric response to environmental signals. These results demonstrate that telomere length can be manipulated, and that a carefully regulated homeostasis may become markedly deregulated in opposing directions in response to different environmental cues.

Over 70 years ago, Barbara McClintock described telomeres and hypothesized about their role in protecting the integrity of chromosomes. Since then, scientists have shown that telomere length is highly regulated and associated with cell senescence and longevity, as well as with age-related disorders and cancer. Here, we show that despite their importance, the tight, highly complex regulation of telomeres may be disrupted by environmental cues, leading to changes in telomere length. We have introduced yeast cells to 13 different environmental stresses to show that some stresses directly alter telomere length. Our results indicate that alcohol and acetic acid elongate telomeres, while caffeine and high temperatures shorten telomeres. Using expression data, bioinformatics tools, and a large genetic screen, we explored the mechanisms responsible for the alterations of telomere length under several stress conditions. We identify Rap1 and Rif1, central players in telomere length maintenance, as the central proteins directly affected by external cues that respond by altering telomere length. Because many human diseases are related to alterations in telomere length that fuel the disease's pathology, controlling telomere length by manipulating simple stressing agents may point the way to effective treatment, and will supply scientists with an additional tool to study the machinery responsible for telomere length homeostasis.

Funding: MK was supported by grants from the Israel Ministry of Science and Technology, the Israel Cancer Research Fund and the Israel Cancer Foundation. GHR was supported by the Machiah foundation and by the Safra Center for Bioinformatics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2013 Romano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Environmental stresses affect the regulation and the activity of many genes and accordingly may perturb telomere length homeostasis by altering the expression or activity of genes in the TLM network described above. Previous studies have suggested that emotional stress in humans is associated with telomere shortening, presumably through its effect on oxidative stress [14] , [15] . These studies, however, establish a correlation, but not causality. Here, we use controlled experimental approaches to explore a possible effect of the environment on yeast telomere length, and to identify the molecular mechanisms by which external signals exert their effect.

Three systematic genome-wide surveys in the yeast Saccharomyces cerevisiae [9] – [11] have revealed that mutations in at least 6% of the genes lead to alterations of telomere length. These TLM (Telomere Length Maintenance) genes span a broad range of functional categories and different cellular compartments. Integration of data from these large-scale mutant screens with information about protein–protein interactions has further permitted charting of the cellular sub-network underlying telomere length regulation in yeast [12] , [13] , revealing a complex set of interactions responsible for a very tight length homeostasis.

Telomeres are nucleoprotein structures located at the ends of chromosomes. Telomeres are essential for chromosome replication and stability [1] , and protect chromosome ends from degradation and deleterious chromosomal rearrangements [1] , [2] . In human embryonic cells, telomeres are elongated by the enzyme telomerase [3] . In somatic cells, however, telomerase expression is low, and telomeres shorten with each cell division due to the incomplete replication of the linear chromosome ends by conventional DNA polymerases. This progressive telomere shortening constitutes a “molecular clock” that underlies cellular aging [4] . Accordingly, telomere length is associated with cell senescence and longevity [5] , as well as with age-related disorders and cancer [6] . While short telomeres have been reported to predict early mortality [7] , recent work has shown that telomerase reactivation may reverse tissue degeneration in aged telomerase-deficient mice [8] .

Results and Discussion

Telomere length alteration under stress is not recombination-dependent Under unperturbed conditions, telomere length can be modified either by disrupting the regulation of telomerase/telomere-associated nucleases or by recombination. To distinguish between these two mechanisms, we analyzed the response to stresses of cells unable to carry out homologous recombination due to a deletion of the RAD52 gene. rad52 cells responded to the stresses much as would a wild type strain, indicating that telomere length alteration in response to these stresses is not recombination-dependent (Figure S2) and that the external signals affect telomerase or telomere-associated nucleases.

Exploring the mechanisms in which stress affect telomere length To understand how external signals affect telomere length and to identify the mechanism behind this telomeric response to stress, we measured genome-wide transcript levels in yeast cells grown for 20 generations in the presence of stresses that showed an effect on telomere length (ethanol, caffeine or high temperature), as well as in the presence of H 2 O 2 , a stress that does not alter telomere length. The results were compared to genome-wide transcript levels of the same strain grown under standard conditions (YEPD medium, 30°C). Using Significance Analysis of Microarrays (SAM) [17] with a false discovery rate (FDR) below 0.01, we obtained a set of 1,744, 1,404, 1,670 and 1,019 differentially expressed genes for caffeine, 37°C, ethanol and H 2 O 2 , respectively. General environmental stress responding (ESR) genes were not induced under these conditions, as expression level was measured after a long-term exposure to the stresses while ESR genes are induced for a short time period [18]. To identify the mechanisms responsible for telomere elongation and shortening, we sought genes that were differentially expressed only under shortening or only under elongating conditions (Figure S3). We integrated transcript abundance data with the known TLM network [13] that uses protein-protein interactions data, connecting TLM genes to the telomere maintenance machinery. The (unweighted) pairwise distances between stress-specific differentially expressed TLM genes were compared with pairwise distances of other TLM genes. This revealed that stress-specific, differentially expressed TLM genes lie significantly closer to each other for ethanol, caffeine and 37°C (p<2E-33,p<3E-27 and p<3E-50, respectively), but not for hydrogen peroxide stress, which does not affect telomere length (Materials and Methods). This phenomenon was unique to TLM genes under stresses that affect telomere length, suggesting that the differentially expressed TLM genes may be involved in transducing the external signals and disrupting telomere length homeostasis. Based on the analysis above, we generated a list of candidate genes for further analysis. Using strains from the yeast deletion library [19] and the DAmP library of hypomorphic mutants [20] we screened mutants in this list to identify genes important for telomere length maintenance under stress conditions. Strikingly, we found a strong correlation between the rate of change in telomere length and the initial length of the mutant: in ethanol, long tlm mutants elongate more rapidly than the wild type, while short tlm mutants elongate more slowly (Pearson correlation, r = 0.61, p<E-12, Figure 3A). Similarly, in caffeine and at 37°C long tlm mutants shorten more rapidly, while short tlm mutants shorten more slowly than does the wild type (Pearson correlation, r = −0.78, p<2E-22 and r = −0.96, p<9E-34, respectively; Figure 3B–C). This correlation between abnormal telomere length and response magnitude to the stresses suggests that telomere elongation/shortening in the presence of external cues is carried out by the same basic mechanisms that maintain telomere length under unperturbed conditions. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Different stresses affect telomere length via different genes. The effect of ethanol, caffeine and high temperature on telomere length was tested on strains carrying individual gene deletions/hypomorphic mutations. Each mutant was grown under the relevant stress for 100 generations and its telomere length was measured using Southern blot analysis. A–C. The X-axis shows the initial length of each mutant and the Y-axis shows the elongation or shortening after 100 generations. Each strain analyzed is represented by a circle (wt in green). A strong correlation (demarked by a red line; ±5% SD) was seen between the initial length and the effect of the stress. A. Ethanol. B. Caffeine. C. 37°C. D. Each bar represents the ratio between the initial telomere length and the elongation after 100 generations in ethanol. Very short tlm mutants (below 200 nt long) could be clearly separated into two groups: mutants of the Tel1 pathway (tel1Δ, mre11Δ, rad50Δ, xrs2Δ) were highly responsive to ethanol stress, while mutants of the NMD (nam7Δ, upf3Δ, nmd2Δ) and Ku (yku70Δ, yku80Δ) pathways show little telomeric elongation under ethanol stress. https://doi.org/10.1371/journal.pgen.1003721.g003

Telomere length alteration under stress is Rif1, but not Rif2 dependent To identify the genes that mediate the telomeric response to stress and to understand how external signals are transduced to altering telomere length, we focused on mutants that disrupt this transduction and, therefore, show an atypical response to each stress (Figure 3). A remarkable such tlm mutant is rif1Δ, which exhibited a reduced response to ethanol and caffeine but normal response to 37°C (Figures 3A and 4), indicating that elongation by ethanol and shortening by caffeine are Rif1-dependent, while telomere shortening by high temperature relies on a different mechanism. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Telomere elongation of different mutants grown in the presence of ethanol. A. The level of Rap1 protein is reduced upon exposure to ethanol in wt, rif1Δ and rif2Δ mutants, but not in a strain in which the RAP1 gene is under the Tet promoter. B. The initial telomere length and the elongation after 100 generations in ethanol were measured by Southern blot. A deletion of RIF1 (two independent cultures) inhibits the response to ethanol while deletion of RIF2 (two independent cultures) increases it. A rap1-17 strain, unable to bind Rif1 or Rif2, behaves similarly to the double mutant rif1Δ rif2Δ. Expressing Rap1 under the tetracycline promoter (which is not affected by ethanol) prevents the telomeric elongation under ethanol stress. C. Chromatin Immunoprecipitation (ChIP) analysis for the recruitment levels of Rif1 and Rif2 proteins to telomeres, in the absence and in the presence of ethanol. The non-telomeric ARO1 locus was used to normalize the relative levels. https://doi.org/10.1371/journal.pgen.1003721.g004 The Rif1 and Rif2 proteins are negative regulators of telomerase that interact with the C-terminus of Rap1, an essential protein that binds to the telomeric repeats [21]. Under normal growth conditions, short telomeres are preferentially elongated by a mechanism that depends on Rap1. Mutations in the carboxy-terminus of RAP1 or down-regulation of the RAP1 gene lead to extreme telomere elongation and to an increase in telomere length variability, similar to what we observed in the presence of ethanol ([22], [23]; Figure 2). Our transcript measurements detected a reduction in the level of Rap1 expression in cells grown in the presence of ethanol [(Table S2); and [24]]. These results suggest a model in which telomere elongation under ethanol stress is primarily due to reduced levels of Rap1, which reduce Rif1 recruitment to telomeres. To test this hypothesis, we used a strain in which RAP1 was expressed from a Tetracycline-inducible promoter [25]. In this strain the level of Rap1 remained unchanged in the presence of ethanol (Figure 4A) and only a slight telomere elongation was observed (Figure 4B). Also consistent with the model, a rap1-17 strain (deleted for the C terminus of Rap1), a rif1Δ single mutant and a rif1Δ rif2Δ double mutant exhibited attenuated responses to ethanol (Figure 4B). Thus, the telomere elongation response to ethanol was abolished when a steady level of Rap1 protein was maintained or when Rif1 activity was eliminated, indicating that the Rap1- Rif1 pathway is central to telomere elongation in response to ethanol. Consistent with this hypothesis, chromatin immunoprecipitation (ChIP) experiments showed that upon exposure to ethanol there is a two-fold reduction in the level of Rif1 at telomeres, as well as a slighter reduction in the level of Rif2 (Figure 4C). Since it is necessary for both elongation and shortening responses, Rif1 may play a general sensing/structural/regulatory role, rather than a catalytic one, in the telomeric response to environmental signals. This is consistent with recent studies that found a role for Rif1 in the regulation of chromatin structure and of DNA replication origin firing [26], [27]. Remarkably, rif2Δ cells exhibited a strong response to ethanol (Figure 3A), underscoring the different roles of Rif1 and Rif2 in telomere length maintenance [28]–[32]. We suggest that exposure to ethanol reduces the recruitment of the Rif proteins at the telomere ends, resulting in conditions permissive for indiscriminate telomerase recruitment, elongating both short and long telomeres, and yielding a broad distribution of telomere lengths (Figure 2A). The insensitivity of rif1Δ mutants to ethanol could be due to the importance of Rif1p for the telomere elongation response, and/or the increased binding of Rif2 to telomeres in the absence of Rif1. In agreement with this model, deletion of RIF2 caused over-extension of telomeres in ethanol (Figure 3A); a reduction of Rif1 telomere recruitment by ethanol in the strain deleted for RIF2 mimics a rif1Δ rif2Δ double mutant, which exhibits increased levels of telomere elongation. In contrast to these results, the RIF2 deletion had no effect on the reduction in telomere length upon exposure to caffeine or 37°C (Figure 3B,C).

The Tel1 and NMD pathways have separate roles in telomere elongation under ethanol stress Mutations in the TEL1 gene, which encodes the yeast ortholog of the mammalian ATM protein kinase, result in very short telomeres. Tel1 regulates the preferential elongation of short telomeres [33] by a pathway that also includes the MRX complex (Mre11, Rad50, Xrs2; [34]). A separate regulatory branch includes the yeast Ku proteins [35]. Figure 3D shows that the tlm mutants with very short telomeres could be clearly separated into two groups: telomeres of mutants of the Tel1 pathway (tel1Δ, mre11Δ, rad50Δ, xrs2Δ) were hyper-responsive, while mutants of the NMD (nonsense mediated decay, nmd2Δ, nam7Δ and upf3Δ) and Ku pathways had only a mild response to ethanol. The fact that telomeres can be elongated by ethanol in the absence of Tel1 or of components of the MRX complex is surprising; notably, the wide size distribution observed upon exposure to ethanol (Figures 1, 2), is consistent with a mechanism independent of the one that preferentially elongates the shortest telomeres, which depends on the Tel1 pathway [16]. The NMD pathway degrades mRNAs carrying nonsense mutations. In addition, it affects the steady state level of hundreds of mRNAs, including those known to act at telomeres (e.g., Est1, Est2, and two components of the CST telomeric capping complex, Stn1 and Ten1 [36]). Mutations in the NMD machinery lead to higher mRNA levels of these proteins and to short telomeres [37]. The NMD pathway has been recently shown to affect the fitness of cdc13-1 and yku70 mutants by controlling the expression of Stn1, an essential telomere capping protein, which interacts with Cdc13 and participates in the recruitment of telomerase [38]. In nmd mutants, the response of telomeres to ethanol stress is reduced relative to wild-type strains, indicating that the NMD pathway is involved in telomere elongation during ethanol stress. We asked if upregulation of Ten1 and Stn1 is involved in this effect by overexpressing these genes in naïve cells and measuring the effect of ethanol on telomere length in these cells (Figure S4). Overexpression of Stn1 reduced the ethanol response and overexpression of both Stn1 and Ten1 completely abolished the telomere length response to ethanol. These results suggest that the level of CST activity, controlled by the NMD pathway, plays an important role in the telomere elongation response to ethanol. This is consistent with the proposed role of the CST complex in telomerase activation. Interestingly, mutations in the CST proteins are lethal when combined with a deletion of RIF1 [28]–[32], indicating the existence of an essential overlapping function between the two telomere regulatory components. The roles of the CST and Rif1 in transducing the ethanol signal to the telomeres will be the subject of future research.

Additional mutants affecting telomere response to ethanol Among the additional mutants with a reduced response to ethanol were doa4Δ, snf7Δ and did4Δ (Figure 3A). DOA4 encodes an enzyme that removes ubiquitin from membrane proteins destined for vacuolar degradation. The Doa4 protein resides in the late endosome, where it interacts with the ESCRT-III machinery, which includes Did4 and Snf7 [39]. A role was previously observed for vacuolar traffic proteins in telomere length maintenance [40]; however, the precise mechanism remains enigmatic. Another mutant that shows apathy towards ethanol is hpr1Δ, defective for a component of the THO complex. Consistent with these results, mutations in HPR1 were recently shown to affect the expression levels of RIF1 [41]. In contrast to these genes, a deletion of HSP104 was hyper-responsive to ethanol. Hsp104 is a stress chaperone that plays an important role in maintaining prion particles in the cell [42]. It is unclear whether its role in telomere length regulation is related to its role in prion maintenance.