Tethering telomerase to telomeres increases genome instability and promotes chronological aging in yeast

Results

Genetic strategy to obtain the cells with overlong telomeres

In the budding yeast S. cerevisiae, telomeres are usually 350 ± 75 bp long. The telomere length is mainly determined by two opposite activities: telomerase-mediated elongation and nuclease-mediated degradation. In order to obtain the strains that have overlong telomeres, we employed the CDC13-EST2 fusion gene, whose expression can force enhanced association of telomerase at telomeres and produce long telomeres after successive passages [41]. We transformed either the CEN plasmid pRS316 (control) or the plasmid containing the CDC13-EST2 fusion gene, namely pRS316-CDC13-EST2, into the wild-type BY4742 cells. The individual transformants were passaged on selective plates every 72 hrs for thirteen times. The telomere length was examined by Southern blot using a telomeric TG-probe. The result showed that the ectopic expression of Cdc13-Est2 fusion protein led to gradual elongation of all telomeres (Fig. 1A). At the 5^th and 13^th streakout of transformants with CDC13-EST2 fusion gene, the size of the telomeric terminal-restriction-fragments (TRFs) digested by restriction endonuclease XhoI reached about 2.0 and 2.6 kb, respectively, which was 0.7 and 1.3 kb longer than that of the vector control, and 0.5 and 1.1 kb longer than that of the 1^st streakout. The telomere TRFs longer than 2.0 kb were designated “overlong telomeres”. Because the TRFs contain about 0.95 kb subtelomeric sequence, and the telomeric TG_1-3-tracts of wild-type cells are about 0.35 kb, the TG_1-3-tracts of the Cdc13-Est2 expressing cells are approximately two (the 5^th streakout) to four (the 13^th streakout) folds longer.

Figure 1. Expression of CDC13-EST2 promotes yeast chronological aging. (A) Telomere length analysis by Southern blot. The cells that contain pRS316 (Vector) or pRS316-CDC13-EST2 plasmid were passaged, and the genomic DNA from cells of different streakouts (labeled on top) was subjected to Southern blot assay using a telomeric TG_1-3 probe. (B) Flow chart of CLS assay. (C) Spot assay of CLS. Both the normal-telomere and overlong-telomere cells of streakouts 4^th and 5^th were used to perform semi-quantitative CLS assay. The time of cultures in the CLS assay was labeled on the left (e.g. D3 means day 3). (D) Telomere length of cells used in (C) at D3 and D38 was examined by Southern blot with telomeric TG_1-3 probe. (E) Colony formation unit (CFU) assay of CLS. CLS of cells BY4742-pRS316 (streakout 8^th, normal telomeres) and BY4742 -pRS316-CDC13-EST2 (streakout 8^th, overlong telomeres) was quantitatively examined. Survival (viable colonies) values at the indicated days of culture were normalized to CLS D1. Values are the averages of 6-10 cultures ± SEM. * denotes p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001. (F) Telomere length of cells in (E) at CLS D1 and CLS D27 was examined by Southern blot.

Expression of Cdc13-Est2 promotes yeast chronological aging

To evaluate the effect of Cdc13-Est2 expression on CLS, we randomly chose multiple single colonies to perform CLS assay. The procedures for a CLS experiment were schematically shown in Fig. 1B (see Materials and Methods for the details) [58,59]. The results showed that along the extension of culture time (day 3 to day 52), the viability of the cells with Cdc13-Est2 expression dropped much faster than that of the cells without Cdc13-Est2 expression (Fig. 1C). Because the CLS assay involved many steps, as well as many days of culturing (Fig. 1B and C), there was a possibility that telomere length had changed in the long process of culture. Therefore, we examined the telomere length in the cells of day 3 and day 38 samples by Southern blot (D3 and D38 in Fig. 1D). The results showed that the telomeres in the cells of day 38 had not undergone lengthening or shortening (Fig. 1D), and the telomeres in the Cdc13-Est2-expressing cells were substantially longer than that in the cells containing the control-vector. These data suggested that overlong telomeres induced by Cdc13-Est2 expression led to shorter CLS.

To quantify the survival rate of the aging cells, we carried out the colony formation assay (see the Materials and methods for the details) [7,59]. Multiple single colonies of the 8^th streakout were used to perform the CLS assay. The survival rate of the cells expressing CDC13-EST2 fusion gene was significantly lower than that of the cells containing the control vector (Fig. 1E). The telomere length in the tested cells did not change during the long-time culture (e.g. 27 days, D27 in Fig. 1F). These results consistently supported the notion that Cdc13-Est2 expression results in overlong telomeres and promotes yeast chronological aging.

Expression of Cdc13-Est2 enhances ERC accumulation

Genome instability is one of the major regulators of CLS. Constitutively active telomerase at telomeres might have led to genome instability that shortens CLS. To test this hypothesis, we firstly examined the recombination activity of rDNA loci because the production of extra-chromosomal rDNA circles (ERCs) is an indication of rDNA stability [27]. The fob1Δ and sir2Δ cells were used as the negative and positive controls, respectively (see Materials and Methods for detail). The Southern blot results showed that deletion of FOB1 and SIR2 reduced and increased the ERCs, respectively (Fig. 2A). Six to ten independent cultures of both the young (CLS D1) and old (CLS D27) cells were subjected to ERC detection (Fig. 2B and C). The ERCs level in the CDC13-EST2 expressing cells is significantly higher than that in the vector-control cells (Fig. 2B and C). One species of multimer ERCs (indicated by solid arrow) in either the young (CLS D1) or old CDC13-EST2 expressing cells (CLS D27) was approximately 60% higher than that in the vector-control cells (Fig. 2B and C). The enhanced ERCs levels in young (CLS D1) and old (CLS D27) cells expressing CDC13-EST2 were comparable, we speculated that ERCs accumulation might take place in the proliferation stage of the culture (Fig. 2B and C). These results suggested that Cdc13-Est2 expression perturbed genome stability.

Figure 2. CDC13-EST2 expression enhances genome instability. (A) ERC level in fob1∆ and sir2∆ mutant cells was examined by Southern blot with a probe of 25S rDNA sequence (upper panel). One species of multimer ERCs (indicated by solid arrow) was quantified and normalized to genomic rDNA signal. Lower panel is the quantification of the ERC level in fob1∆ and sir2∆ mutant cells. The ERC level in wild-type cells was set as “1”, and the ERC value in each strain was normalized to the genomic rDNA. (B) and (C) Detection of ERC level in young cells of CLS D1 (B) and old cells of CLS D27 (C) used in Fig. 1E by Southern blot (upper panel). One species of multimer ERC (indicated by solid arrow) was quantified (lower panel). POL1 gene was used as an internal loading control. Values in the quantification are normalized to POL1 level ± SEM. (D) Chronological viability of cells carrying CAN1-URA3 cassette at CLS D1, D15 and D29. The cells of streakout 8^th containing pRS315 (control) and pRS315-CDC13-EST2 was examined. Survival (viable colonies) values were normalized to CLS D1. Values are the averages of 6-10 cultures ± SEM. (E) and (F) CAN1 mutation frequency (E) and GCRs frequency (F) in overlong- and normal-telomere cells at CLS D1, D15 and D29 were examined. Values in (E) and (F) are the averages of 6-10 cultures ±SEM. (G) Telomere length analysis of cells at CLS D1 and D29. The cells of streakout 8^th that containing pRS315 (normal telomere) and pRS315-CDC13-EST2 (overlong telomere) were examined.

Expression of Cdc13-Est2 increases age-dependent CAN1 marker-gene mutation frequency and GCR frequency

In order to validate further our hypothesis that expression of Cdc13-Est2 promotes yeast chronological aging by increasing genome instability, we next examined the CAN1 marker-gene mutation frequency and gross chromosomal rearrangement (GCR) frequency in the CDC13-EST2 expressing cells. These two assays have been widely used to evaluate age-dependent genome instability in yeast [8,22,60,61]. The CAN1 gene encodes an arginine permease, which controls the uptake of arginine by yeast cells. Detection of the CAN1 mutation frequency uses an arginine analogue, canavanine, which cells intake under arginine deficient conditions. Canavanine is toxic to yeast cells and leads to cell death. Unless CAN1 mutation (such as point mutation, insertion or deletion) inactivates CAN1, cells cannot survive when canavanine is supplemented in the medium lack of arginine [60]. Different from the CAN1 marker-gene mutation frequency assay, the GCR assay detects the loss of both CAN1 and URA3 marker genes simultaneously [60]. To measure GCR, the CAN1 proximal to HXT13 gene was substituted by URA3 marker-gene. 5-fluoroorotic acid (5-FOA) was used to counter-select yeast cells expressing URA3. In the presence of both 5-FOA and canavanine, cells must lose the functions of both CAN1 and URA3 to survive. We firstly examined the survival rate of the cells that contained the CAN1-URA3 marker cassette. The results showed that CDC13-EST2 expressing cells had significantly shortened CLS compared with vector control cells at CLS D15 (p value <0.05, n=6-10) and CLS D29 (p value <0.0001, n=6-10) (Fig. 2D). Consistently, CDC13-EST2 expression significantly increased the CAN1 marker-gene mutation frequency at CLS D15 (p value <0.01, n=6-10) and CLS D29 (p value <0.01, n=6-10) (Fig. 2E), and the GCR frequency at CLS D15 (p value <0.05, n=6-10) and CLS D29 (p value <0.05, n=6-10) (Fig. 2F). The cells used to detect CAN1 mutation frequency and GCR frequency did have overlong telomeres, and their long telomeres were stably maintained during chronological aging (Fig. 2G). Altogether, our results indicated that Cdc13-Est2 expression caused genome instability, and accelerated chronological aging.

Deletion of SCH9 reduces genome instability and suppresses chronological aging mediated by expression of Cdc13-Est2

Previous study has shown that deletion of SCH9 in yeast significantly decreases genome instability during chronological aging [22]. Now that Cdc13-Est2 expression led to enhanced genome instability and resulted in accelerated chronological aging, could calorie restriction (CR) or deletion of SCH9 slow down the rate of chronological aging mediated by Cdc13-Est2 expression? To address this question, we examined whether reducing glucose concentration from 2% to 0.5%, which mimics moderate CR conditions, could restore the CLS of the CDC13-EST2 expressing cells. The results showed that moderate CR did not significantly affect telomere length (Fig. S1A), nor recover the CLS of CDC13-EST2 expressing cells (Fig. S1B and C). This result is consistent with previous report that buffering culture medium to pH 6.0 by citrate/phosphate acts similarly with moderate CR in lengthening CLS [62], and application of moderate CR to synthetic complete medium buffered to pH 6.0 with citrate/phosphate cannot further extend CLS [62].

We next examined the effect of deletion of SCH9 on chronological aging in CDC13-EST2 expressing cells. Deletion of SCH9 had little effect on telomere length in either vector-control or CDC13-EST2 expressing cells (Fig. 3A), but significantly extended CLS in both cells (Fig. 3B). This is consistent with published data that sch9∆ increases CLS [14]. In order to address whether the CLS recovery is attributed to the suppression of genome instability, we examined CAN1 marker-gene mutation frequency, GCR frequency and ERCs accumulation in the sch9Δ cells with CDC13-EST2 expression. The results showed that deletion of SCH9 significantly lowered both the CAN1 marker-gene mutation frequency and the age-dependent GCR frequency mediated by CDC13-EST2 expression during chronological aging (Fig. 4A and B). The cells in the genome instability assay did have overlong telomeres and the telomere length did not exhibit a significant change during chorological aging (e.g. CLS D29) (Fig. 4C). In addition, deletion of SCH9 also lowered ERC accumulation mediated by CDC13-EST2 expression at CLS D1 (p value < 9.2E-07, n=6) and CLS D15 (p value < 8E-05, n=6) (Fig. S2A and B). CDC13-EST2 expressing cells stably maintained overlong telomeres during chronological aging (Fig. S2C). These data suggested that deletion of SCH9 contributed to the maintenance of genome stability in Cdc13-Est2 expressing cells, and inhibition of genome instability by deletion of SCH9 slowed down chronological aging process promoted by constitutively active telomerase.

Figure 3. CLS of sch9∆ cells. (A) Telomere length of cells in (B) at CLS D1 and CLS D29 was examined by Southern blot. (B) CLS assay was done using cells WT-pRS316/sch9∆-pRS316 (streakout 13^th, normal telomeres), WT-pRS316-CDC13-EST2/sch9∆-pRS316-CDC13-EST2 (streakout 13^th, overlong telomeres). Survival (viable colonies) values are normalized to CLS D1 and are the averages of 6-10 cultures ± SEM.

Figure 4. Detection of CAN1 mutation frequency and GCR frequency in sch9∆ cells during chronological aging. (A) Cells of hxt13∆-pRS315/ hxt13∆sch9∆-pRS315 (streakout 14^th, normal telomeres), hxt13∆-pRS315-CDC13-EST2/ hxt13∆sch9∆-pRS315-CDC13-EST2 (streakout 14^th, overlong telomeres) were used to assay CAN1 marker-gene mutation frequency during chronological aging. (B) The same cells in (A) were used to examine GCRs frequency during chronological aging. The values (viable colonies) are the averages of 6-10 cultures ± SEM. (C) Telomere length of cells of CLS D1 and D29 in (A) and (B) was examined by Southern blot.

Loss of CDC13-EST2 fusion gene restores regular CLS

We used a genetic strategy to force enhanced association of telomerase at telomeres through expression of CDC13-EST2. The expression of Cdc13-Est2 fusion protein resulted in overlong telomeres and shortened CLS (Fig. 1). However, it was unclear whether the shortened CLS was attributed to overlong telomeres or constitutive association of telomerase at telomeres. To discriminate these two possibilities, the pRS316 (control) and pRS316-CDC13-EST2 plasmids in the normal- and overlong-telomere cells were evicted, and CLS assays were performed (See Fig. S3 and S4 for the details). The results showed that the CLS of cells with overlong telomeres was not different from that of cells with normal telomeres (Fig. 5A and S4C). The cells used in the CLS assays maintained either normal or overlong telomeres during their long-term chronological aging (Fig. 5B and Fig. S4D). These results suggested that it was not the overlong telomeres, but rather the constitutive association of telomerase at telomeres that affected CLS. Following the notion that overlong telomeres might not affect CLS, we examined CLS of the rif1∆ mutant cells that have overlong telomeres (Fig.5D) [42,43]. The results showed that the rif1∆ mutant cells exhibited nearly the same chronological aging profile as the wild-type cells (Fig. 5C). The telomere length of wild-type and rif1∆ cells was stably maintained during chronological aging (Fig. 5D). Thus, constitutively tethering telomerase to telomeres causes genome instability and promotes chronological aging.

Figure 5. CLS assay of cells with overlong telomeres but no CDC13-EST2 fusion gene (A) CLS of normal- and overlong-telomere cells after plasmids eviction (21^st streakout) (B) Southern blot analysis of telomere length using telomeric TG_1-3 probe. (C) CLS of wild-type (4^th streakout) and rif1∆ (4^th streakout). (D) Telomere length detection by Southern blot at CLS D1 and D15.

Discussion

Previous studies have indicated that the replicative aging and the chronological aging are quite different [4,63]. However, numerous lines of evidence have pointed to a consensus that genome instability is one of the major culprits of aging (Fig.6) [1]. Telomeres reside at the end of the chromosomes, and their structural integrity is essential for genome stability [37,64]. Telomerase mediated telomere elongation usually takes place in the late S-phase of the cell cycle, and plays important roles in both telomere length and structure regulation [65,66]. Intact telomeres are required for genome stability (Fig. 6). In the absence of telomerase, following every cell division telomere attrition occurs because of the end replication problem [44,48,67]. When telomeres become critically short, cells could either undergo senescence [45], or repair telomeres through homologous recombination that promote replicative aging (Fig. 6) [23]. Consistently, long telomeres potentiate longer RLS in human somatic cells that have inactive telomerase [52,68]. Therefore, longer telomeres appear to be beneficial to lifespan extension when telomerase is not active. In the presence of telomerase, the telomere length and the RLS seems not to have consistent positive correlation. For examples, the mutant cells containing a truncated tlc1 allele have shortened telomeres but increased RLS, whereas rif1∆ mutant cells with long telomeres had shortened RLS [55]. Additionally, overlong telomeres induced by expressing CDC13-EST2 fusion gene do not affect RLS [23]. In the current work, we have evaluated the effect of overlong telomeres on yeast CLS. Our data suggest that overlong telomeres per se do not affect chronological aging (Fig. 5).

Figure 6. A model of telomere/telomerase associated genome instability affects yeast aging. Wild-type yeast cells have evolved to have regulated telomerase activity to maintain telomere homeostasis. In the absence of telomerase, telomeres gradually shrink to critically short ones that lead to either checkpoint activation and replicative senescence, or telomere recombination and accelerated replicative aging. When telomerase is constitutively active, the genome becomes less stable, and promotes chronological aging, which can be suppressed by down-regulation of TORC1/Sch9 pathway.

Interestingly, constitutively active telomerase mediated by expression of Cdc13-Est2 promotes chronological aging (Fig. 1). The underlying reason appears to be the enhanced genome instability (Fig. 6), exemplified by increased accumulation of ERCs (Fig. 2A-C), enhanced age-dependent spontaneous CAN1 marker-gene mutation frequency and GCR frequency in CDC13-EST2 expressing cells (Fig. 2D-G). How the enhanced association of telomerase at telomeres causes the effect on other genomic loci remains unknown. One possibility is that Cdc13-Est2 fusion protein not only targets to telomeric G-rich overlong, but also spontaneously binds other genomic loci where double strand breaks occur. This speculation is supported by the previous observation that Cdc13 can act on double strand breaks (DSBs) at low frequencies [69,70]. Under normal situation, Est2 binding to DSBs requires the interaction between Cdc13 and Est1 [71]. Cells seem to have evolved mechanisms that prevent the pervasive action of telomerase on DSBs. For example, Mec1 inhibits telomere healing by phosphorylating Cdc13 on its S306 residue, which suppresses Cdc13 accumulation at DSBs [72]. Est1 abundance is cell cycle regulated and peaks in S-phase, which may limit the activation of Cdc13-bound telomerase Est2 [65]. While under the scenario of Cdc13-Est2 fusion, wherever Cdc13 binds, Est2 may pose an effect on these loci. Telomerase mediated chromosomal healing on one hand repairs the DSBs, but on the other hand may endanger genome stability by loss of chromosomal arms.

The genome alterations observed in the CDC13-EST2 expressing cells are the accumulation of ERCs (Fig. 2B and C), enhanced spontaneous CAN1 mutation frequency and GCR frequency (Fig. 2E and F). These changes may exacerbate the chronological aging when the cells are challenged by nutrient depletion (Fig. 1), supporting the argument that genome instability is a direct causal factor for the accelerated chronological aging [1,22]. In agreement with this hypothesis, deletion of SCH9 did not shorten the overlong telomeres (Fig. 3A, 4C and S2C), but significantly suppressed the phenotypes of genome instability (Fig. 4A-B and Fig. S2A-B), and accordingly restored the lifespan of CDC13-ETS2 expressing cells (Fig. 3B). The accelerated chronological aging seen in CDC13-EST2 expressing cells is very similar to the case of sgs1Δ mutant cells. Deletion of SGS1 results in premature age-related changes including reduced CLS, elevated recombinant errors and age-dependent increase in DNA mutations [22]. Additionally, deletion of SCH9 in sgs1∆ mutant cells suppresses recombination and DNA damage, and slows down the chronological aging [22]. Thus, it is significant for the cells to regulate telomerase activity in a properly spatio-temporal manner to avoid genome instability and promote cell longevity.

Author Contributions

JL and JQZ developed the ideas and designed the study. JL performed most of experiments. MHH, JP, YMD, YSL, ZW, TG and HTL provided the reagents and carried out some of the experiments. JL and JQZ wrote the manuscript.

Acknowledgements

We thank Dr. V. Lundblad for plasmid expressing fusion gene CDC13-EST2.

Conflicts of Interest

There is no conflict of interest.

Funding

JL was funded by grants from China Postdoctoral Science Foundation (2014M551470), Postdoctoral Research Program of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (2014KIP305) and the National Natural Science Foundation of China (NSFC 31501110). The research in JQZ’s lab was supported by the grants from NSFC (31230040/31461143003/31521061) and from Ministry of Science and Technology of China (MoST 2013CB910400). This work was also supported in part by the Joint NSFC-ISF (Israel Science Foundation) Research Program (NSFC 31461143003).

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