Telo-3C detects telomere folding in S. cerevisiae

To gain insights into the dynamic regulation of telomere folding in S. cerevisiae, we developed a telomere chromosome conformation capture (3C) approach [45] (Telo-3C). This provides a direct readout to quantitatively assess the interaction of one telomere (left telomere of chromosome I) with its adjacent subtelomeric region. As illustrated in Fig 1A, chromosome contacts were crosslinked in vivo using formaldehyde and the extracted chromatin was digested with a suitable restriction enzyme (NcoI for telomere 1L) (see Fig 1B for precise restriction sites). The compatible DNA ends were ligated under very dilute conditions, resulting in the ligation of only those loci that were in close proximity at the time of the crosslink. After reversing the crosslink, a specific 3C ligation product can be amplified from primers that were initially co-directionally oriented on genomic DNA (no PCR product) but get converted through the 3C reaction to being convergent (giving a PCR product, see blue arrows Fig 1A). The abundance of this specific PCR product can be quantified by quantitative PCR (qPCR).

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Fig 1. Telo-3C detects telomere folding in S. cerevisiae.

A. Schematic representation of the Telo-3C method. Formaldehyde was used to crosslink chromosome interactions such as telomere folding. DNA was digested with the restriction enzyme NcoI and DNA ends were ligated under diluted DNA conditions. The tandem primer pairs on linear DNA (blue arrows) got converted to convergent primer pairs by ligation, and telomere folding was detected by qPCR. Red arrowheads = telomere repeats; Scissors = NcoI restriction sites B. Illustration of NcoI cut sites along the subtelomere 1L and the primers (blue arrows) used to detect interaction frequencies between the respective sites. P5 was used as anchor primer in close proximity to the telomeric repeats. Genomic location of primers and cut sites (scissors) were calculated from the end of telomere 1L (depicted in bp) as annotated in the SGD (Saccharomyces genome database). C. The Telo-3C protocol was performed without digesting or ligating the chromatin as indicated. All interaction frequencies were normalized to a control qPCR product. Relative interaction frequencies were calculated by setting the normalized interaction frequency of the sample which was performed with the complete Telo-3C protocol to 1. Mean +/- SD of 4 independent experiments. Adjusted p-values were obtained from one-way ANOVA with Dunnett’s multiple comparisons test (****p ≤ 0.0001). D. The Telo-3C protocol was performed on increasing amounts of chromatin without formaldehyde crosslinking (no x-link). All interaction frequencies were normalized to a control qPCR product. Relative interaction frequencies were calculated by setting the normalized interaction frequency of the sample which was performed with the complete Telo-3C protocol to 1. Mean +/- SD of 3 independent experiments. Adjusted p-values were obtained from one-way ANOVA with Dunnett’s multiple comparisons test (**p ≤ 0.01, ***p ≤ 0.001). E. Relative interaction frequencies of telomere 1L with loci of increasing distance from the telomeric repeats (primers P6, P7 and P9). All interaction frequencies were normalized to a control qPCR product. Relative interaction frequencies were calculated by setting the normalized interaction frequency of P5 + P6 to 1. Mean +/- SD of 6 independent experiments. Adjusted p-values were obtained from one-way ANOVA with Tukey’s multiple comparisons test (****p ≤ 0.0001, ***p ≤ 0.001). F. Illustration of NcoI cut sites at a non-telomeric, internal locus on chromosome I and the primers (green arrows) used to detect interaction frequencies between the respective sites. Genomic location of primers and cut sites were calculated from the end of telomere 1L as annotated in the SGD. G. 3C results comparing the relative interaction frequencies of telomeric with non-telomeric loci on chromosome I using the indicated primer pairs. The interaction frequencies of telomeric and non-telomeric 3C were normalized to a control qPCR product. The relative interaction frequencies were calculated by setting the normalized interaction frequency of P5 + P6 to 1. Mean +/- SD of 4 independent experiments. Adjusted p-values were obtained from one-way ANOVA with Tukey’s multiple comparisons test (****p ≤ 0.0001).

https://doi.org/10.1371/journal.pgen.1008603.g001

To ensure the specificity of the Telo-3C procedure we performed the protocol without ligating or digesting the chromatin. In these conditions, we did not detect a Telo-3C product of the same intensity as compared to the complete Telo-3C protocol (Fig 1C). Similarly, when the chromatin was not crosslinked with formaldehyde, we detected only weak Telo-3C products as compared to the crosslinked sample, despite increasing amounts of chromatin input (Fig 1D). This suggests that a specific Telo-3C qPCR product could only be amplified when the loci of interest were located in close proximity. We were interested in measuring the interaction of the telomeric repeats with several loci along the subtelomere and designed primers according to the NcoI restriction sites that were available (Fig 1B). Primer P5, directly juxtaposed to the telomere repeat sequences, was used as an anchor primer in combination with P6, P7 and P9 thereby comparing the interaction of the telomeric repeats to a subtelomeric region with a distance of ~2 kb (P6) and ~9 kb (P7). With P9 we chose a primer outside the region which we would define as subtelomere with a distance of ~47 kb from the telomeric repeats. The interaction frequency was reduced to half when moving from a ~2 kb (P6) to ~ 9 kb (P7) distance from the telomeric repeats (Fig 1E), similar to what has been reported previously [45]. We also detected a Telo-3C product between primers P5 and P9 (~47 kb) which suggests that the telomere may also interact with loci of greater distance to the telomeric repeats, although with a reduced frequency (Fig 1E). The sequences of the subtelomeric Telo-3C qPCR products (P5 + P6 and P5 + P7) were confirmed by Sanger sequencing (S1A and S1B Fig). In order to understand if telomere folding occurs more frequently than a random interaction of two loci on the same chromosome, we analyzed chromosome interactions at internal NcoI restriction sites on chromosome I (Fig 1F). The respective 3C primers (P10 (anchor), P11 and P12) have similar distances as the ones used on subtelomere 1L. Although we could detect a weak interaction between these loci using the internal primer sets, the interactions between telomere 1L and its subtelomere were more robust (Fig 1G). These results demonstrate that the interactions at the end of the chromosome occur more frequently than a random encounter of two loci on the same chromosome.

In summary, we established and validated a 3C method that detects the interaction of telomeric and subtelomeric regions on a single chromosome, which provides further evidence for the presence of a folded structure at chromosome ends in budding yeast. It appears, that telomere folding occurs more frequently, or more stably, than internal interactions on the same chromosome. Moreover, the telomeric repeats might touch down at multiple regions, and the interaction frequency diminishes with increasing distance from the telomeric tract.

Telomere 1L opens during replicative senescence

To understand how telomere structure may be regulated throughout the process of replicative senescence, we passaged budding yeast lacking the RNA component of telomerase (TLC1) over multiple generations to induce gradual telomere shortening [46]. As expected, the tlc1 cells progressively experienced decreased growth potential (Fig 2A). The loss of growth potential was caused by bulk telomere shortening due to the lack of telomerase activity, as shown in a telomere restriction fragment analysis (S2A Fig, representative example of one clone). At crisis, the point of lowest growth potential, some cells eventually overcome the permanent cell cycle arrest by acquiring the capacity to elongate their telomeres based on a recombination-mediated pathway [28]. These survivors showed the amplification of TG repeats by the appearance of multiple additional bands on the Southern blot [47] (S2A Fig). We employed Telo-3C during this time course and measured the interaction of the telomere 1L with its subtelomere at the indicated time points (+ in Fig 2A). Telo-3C analysis at PD (population doubling) 9 revealed that telomere folding of tlc1 mutants was comparable to wt telomeres, despite their telomeres being much shorter (Figs 2B and S2A). However, when tlc1 cells had critically short telomeres and approached the point of telomeric crisis, telomere folding decreased significantly (PD 47, 56 and 64 in Fig 2B). Similar results were obtained when using the P5 + P7 primers (S2B Fig). In survivors, however (PD 101 in Figs 2B and S2B), telomeres re-acquired the ability to establish a fold-back structure. Using a probe specific for telomere 1L (the telomere where we measure the fold-back), we observed that at PD 9, telomeres were shorter in tlc1 cells than in wild type cells but were still fold-back proficient (Fig 2C). At PD 47 and 56, telomere 1L became even shorter and as senescent cells accumulated the fold-back structure was lost (Figs 2B, 2C and S2B). At PD 64, we observed some recombination events but the bulk of telomere 1L was still critically short (and cells remained fold-back defective) (Figs 2B, 2C and S2B). At PD 73 and 101, telomere length was recovered by HDR and this was when the fold-back began to be restored (Figs 2B, 2C and S2B). Flow cytometry analysis for DNA content revealed that the analyzed cell populations did not demonstrate drastic changes in terms of cell cycle distribution (S2C Fig). To rule out that extensive resection at short telomeres may result in loss of the NcoI restriction site, and hence loss of the Telo-3C signal, we performed the same senescence assays in an EXO1 deficient strain, where extensive telomere resection is defective [33]. In the exo1 background, we observed a similar loss of telomere folding, indicating that extensive resection via Exo1 was not responsible for the decreased Telo-3C signal (S2D and S2E Fig).

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Fig 2. Telomere 1L opens during replicative senescence.

A. The relative growth potential of 6 independent wt and tlc1 cultures was followed over ~100 PDs. Samples for Telo-3C were collected at the indicated time points (+). We defined PD 0 as the time the senescence curve was started in liquid media from the germinated spore colony. The dashed grey line indicates the clone that was used for telomere length analysis (see Figs 2C and S2A). B. Telo-3C analysis during the time course shown in Fig 2A. The relative interaction frequencies between primers P5 and P6 (Fig 1B) are shown. All interaction frequencies were normalized to a control qPCR product. Relative interaction frequencies were calculated by setting the normalized interaction frequency of the wt of the respective day to 1. Population doubling values that were used for Telo-3C correspond to those used in the senescence curve (Fig 2A) (i.e. from 24-hour saturated cultures). Mean +/- SD of 6 different clones in 3 independent experiments. Adjusted p-values were obtained from two-way ANOVA with Sidak’s multiple ikcomparisons test (*p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, n.s., not significant). C. Southern blot for telomere 1L on genomic DNA digested with SalI using a specific PCR product labeled with ³²P as a probe. One representative sample per genotype is shown (dashed grey line in Fig 2A). Population doubling values that were used for Southern blotting correspond to those used in the senescence curve (Fig 2A) (i.e. from 24-hour saturated cultures). D. Southern blot for all telomeres on XhoI-digested genomic DNA from the indicated deletion mutants using a radio-labeled (TG)n-repeat containing vector fragment as a probe. The tlc1 mutant was analyzed at ~PD 40 as calculated in Fig 2A. A representative sample for each genotype is shown. The dashed line indicates wt telomere length. E. Telomere PCR for the length of telomere 1L in the depicted mutants from the genomic DNA used in Fig 2D. The tlc1 mutant was analyzed at ~PD 40 as calculated in Fig 2A. The dashed line indicates the wt telomere length. F. Telo-3C analysis of the displayed mutants. The tlc1 mutant was analyzed at ~PD 40 as calculated in Fig 2A. The interaction frequencies between primers P5 and P6 are shown. All interaction frequencies were normalized to a control qPCR product. Relative interaction frequencies were calculated by setting the normalized interaction frequency of the wt to 1. Mean +/- SD of 3 independent experiments. Adjusted p-values were obtained from one-way ANOVA with Dunnett’s multiple comparisons test (**p ≤ 0.01, n.s., not significant). G. Telo-3C analysis of wt cells after 1 h and 2 h of MMS treatment (0.02%). The interaction frequencies between primers P5 and P6 are shown. All interaction frequencies were normalized to a control qPCR product. Relative interaction frequencies were calculated by setting the normalized interaction frequency of the untreated samples to 1. Mean +/- SD of 3 independent experiments. Adjusted p-values were obtained from two-way ANOVA with Sidak’s multiple comparisons test (**p ≤ 0.01, ****p ≤ 0.0001). PDs = population doublings.

https://doi.org/10.1371/journal.pgen.1008603.g002

The fact that telomeres remain folded at PD 9 suggests that telomere shortening alone is not responsible for the loss of the folded structure (Figs 2B and S2B). To address this further, we employed Telo-3C in several yeast mutants with very short, albeit stable, telomeres. All of the mutants analyzed harbored telomeres (both bulk telomeres and 1L specifically) that were comparable in length to telomerase negative tlc1 cells but showed neither progressive shortening nor a senescence phenotype (Fig 2D and 2E). We found that the telomere 1L of these mutants established a fold-back structure that varied in strength between mutants, but was consistently more frequent than in tlc1 mutants (Fig 2F). This suggests that short telomeres per se are still able to fold back onto the subtelomere. We cannot, however, exclude that critically short telomeres, which accumulate in telomerase negative cells, contribute to telomere unfolding during senescence. Additionally, ku70, mre11, tel1 (Fig 2F) and hrs1 (S2F Fig) mutants have all been demonstrated to be defective in telomere clustering [48,49], suggesting that clustering is not promoting the fold-back structure nor influencing the Telo-3C signal.

As replicative senescence is induced by a cell cycle arrest due to a DNA damage response at telomeres, we investigated whether other forms of DNA damage would also result in an open telomere state. Upon treatment of wild type cells with the DNA damaging agent methyl methanesulfonate (MMS), we observed decreased telomere folding, that became more pronounced over time (Figs 2G and S2G). Since both MMS treatment as well as replicative senescence lead to a G2/M delay, we addressed whether the cell cycle arrest itself may contribute to the opening of the fold-back structure. To arrest cells in G2/M in a DNA damage-independent manner, we constructed an auxin-inducible degron (AID*) version of Cdc20 (S2H Fig). This system allowed us to study the impact of a G2/M arrest on telomere folding by degrading Cdc20 upon addition of indole-3 acetic acid (IAA) to the culture media (S2H and S2I Fig). We observed reduced telomere folding in a time-dependent manner following Cdc20 depletion (S2J Fig), suggesting that the prolonged G2/M arrest of senescent cells may contribute to telomere opening.

In conclusion, we demonstrate that critically short telomeres open their folded structure during replicative senescence and prolonged exposure to MMS. The opening of the fold-back structure is not solely due to the short nature of these telomeres during senescence. Instead, the decreased telomere folding ability during crisis might rather be due to other features of the “senescence program”, including the checkpoint mediated delay at the G2/M border.

Rad51, Rad52 and Rad53 are required for telomere folding

In order to identify regulators of telomere folding, we first employed a candidate approach of factors that could potentially influence telomere fold-back formation and maintenance. First, we chose to investigate the impact of the structural maintenance of chromosomes (SMC) complexes on telomere folding. Cohesin and condensin have been demonstrated to play important roles in terms of chromosomal architecture, including chromatin loop formation and chromatin condensation, respectively [50]. We also wanted to test if the SMC5/6 complex might be involved in telomere folding due to its described roles at telomeres [51–55]. We tagged one subunit of each SMC complex with an auxin-inducible degron [56] (Fig 3A). We treated the culture with auxin for 1 h, which resulted in a nearly complete depletion of the respective proteins (Fig 3A), while not impacting the viability of the cells (S3A Fig). Telo-3C results demonstrated that telomere folding was not affected by the lack of any of the SMC complexes under these conditions (Fig 3B). Therefore, although the SMC complexes are implicated in chromosome compaction [57–59] and the establishment of chromosome domain boundaries and chromatin loops [60,61], they are not a requirement for telomere folding.

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Fig 3. Rad51, Rad52 and Rad53 are required for telomere folding.

A. SMC6, SMC1 and SMC4 were tagged with an auxin-inducible degron (AID*) and a 9MYC-tag. Western blots were performed to detect protein levels after treatment with 1 mM IAA for 1 h. B. Telo-3C analysis after depletion of SMC complex members for 1 h with 1 mM IAA. Mean +/- SD of 3 independent experiments. Adjusted p-values were obtained from two-way ANOVA with Tukey’s multiple comparisons test (*p ≤ 0.05, n.s., not significant). C. Telo-3C analysis of rif1 and rif2 mutants. Mean +/- SD of 3 independent experiments. Adjusted p-values were obtained from one-way ANOVA with Dunnett’s multiple comparisons test (n.s., not significant). D. Telo-3C analysis of rad51 and rad52 mutants. Mean +/- SD of 5 independent experiments. Adjusted p-values were obtained from one-way ANOVA with Dunnett’s multiple comparisons test (****p ≤ 0.0001). E. RAD53 was tagged with an auxin-inducible degron (AID*) and a 9MYC-tag. Western blots were performed to detect protein levels after treatment with 1 mM IAA for 2 h. F. Telo-3C analysis after depletion of Rad53 for 2 h with 1 mM IAA. Mean +/- SD of 3 independent experiments. Adjusted p-values were obtained from two-way ANOVA with Tukey’s multiple comparisons test (*p ≤ 0.05). G. Telo-3C analysis of sml1 and sml1 mec1 mutants. Mean +/- SD of 4 independent experiments. Adjusted p-values were obtained from one-way ANOVA with Tukey’s multiple comparisons test (n.s., not significant). For all Telo-3C results, the interaction frequencies between primers P5 and P6 are shown. All interaction frequencies were normalized to a control qPCR product. Relative interaction frequencies were calculated by setting the normalized interaction frequency of the respective wt sample or the untreated sample from each genotype to 1. IAA = Indole-3 acetic acid.

https://doi.org/10.1371/journal.pgen.1008603.g003

We tested the telomeric proteins Rif1 and Rif2 for their contribution to establishing telomere folding. Both Rif1 and Rif2 were previously reported to regulate telomere structure as measured by the fold-back reporter gene [15]. However, by Telo-3C we did not observe a telomere folding defect in either rif1 or rif2 deletion mutants arguing that these two telomeric proteins might not contribute to the establishment of telomere folding (Fig 3C). This discrepancy between the two methods might arise from the two different readouts employed, or due to the introduction of an artificial subtelomere with the reporter gene. Additionally, the chromatin structure at telomeres of rif1 and rif2 mutants is altered which affects natural and modified subtelomeres in different ways [4,5].

In human cells, the t-loop consists of a D-loop recombination intermediate, which is suggestive of a strand invasion event. To test whether recombination may also be involved in yeast telomere folding, we performed Telo-3C experiments in cells lacking the central HDR factors RAD51 and RAD52. We found that telomere folding was decreased in both rad51 and rad52 mutants (Figs 3D and S3B). This strongly suggests that in yeast, like in human cells, a strand invasion event contributes to the establishment of a closed telomere state. Importantly, the gene loop (between the terminator and promoter) that forms at the HEM3 locus [62] was not influenced in either rad51 or rad52 mutants (S3C Fig), suggesting that the HDR machinery may be specific for folding at chromosome ends.

As Rad51 and Rad52 promote repair in response to a DDR, we tested whether the most upstream factors of the DDR, the checkpoint kinases Mec1 (ATR) and Rad53 (CHK2), may also contribute to telomere folding. We depleted cells of Rad53 for 2 h using an auxin-inducible degron (Fig 3E), a treatment which did not result in decreased cell viability (S3D Fig). Under these conditions, we observed a significant reduction of telomere folding (Fig 3F), suggesting that an impaired DNA damage checkpoint does affect the structure of the telomere. Surprisingly, the deletion of either MEC1 (Fig 3G) or TEL1 (Fig 2F) alone did not affect the folding behavior of telomere 1L. We propose, that Mec1 and Tel1 might have overlapping functions in terms of telomere folding which could potentially compensate for each other. Testing the tel1 mec1 double mutant is complicated by the fact that it induces replicative senescence [36] and a has a high incidence of telomere fusions [38,40]. Alternatively, Rad53 may promote telomere folding in a Mec1- and Tel1-independent manner.

Loss of Sir2, Sin3 and Set2 contribute to telomere opening in senescence

In order to understand the underlying mechanisms behind the structural changes that occur at telomeres throughout senescence, we analyzed the whole proteome of tlc1 cells as they approached telomeric crisis and compared it to wt cells. Label-free quantitative proteomics analysis revealed 751 significantly downregulated and 678 upregulated proteins during senescence (Fig 4A, see S4 Fig for a comprehensive list, see S5A and S5B Fig for gene ontology analysis). To identify candidates that potentially regulate telomere folding during senescence, we compared the results from a previous screen for regulators of telomere folding in S. cerevisiae [15] with the downregulated proteins from our proteomics data set. This genetic screen by Poschke et al., identified 113 genes and we manually added sir2 to this list. Sir2 has been demonstrated to promote telomere folding, however, its deletion cannot be detected in the screening procedure due to technical reasons [12,19,63]. We found 21 overlapping candidates between the fold-back defective mutants and proteins that become downregulated during senescence (Fig 4B). Downregulation of these candidate proteins during senescence may therefore contribute to the loss of a closed telomere state. Compared to the complete set of downregulated proteins during senescence, the 21 candidates (Fig 4C) were significantly enriched for the gene ontology terms of protein and histone deacetylation (S5C Fig), suggesting an epigenetic mechanism underlying telomere folding regulation during senescence. The proteins Sir2, Sin3 and Set2 were of particular interest as they antagonize histone acetylation and were reported to be involved in telomere silencing or the regulation of telomeric chromatin [64–71]. We tested the identified chromatin modifiers in a Telo-3C assay and found that indeed the deletion mutants of SIR2, SIN3 and SET2 were telomere folding defective (Figs 4D and S6A). We further confirmed the downregulation of Sir2, Sin3 and Set2 during senescence (tlc1) as observed by mass spectrometry (Fig 4A) through western blotting (Fig 4E). In pre-senescent cells and post-senescent survivors, protein levels did not differ from those of telomerase positive wild type cells (Fig 4E). Of note, we observed a modified Sir2 species in senescent cells suggesting a specific regulation of Sir2’s activity, localization or stability during the senescence program (Fig 4E). Telomere length of these mutants is similar to wt length or in the case of sin3, only slightly shorter (Figs 4F and S6B). This further supports the notion that telomere length and the state of telomere folding are uncoupled from each other.

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Fig 4. Loss of Sir2, Sin3 and Set2 contribute to telomere opening in senescence.

A. Volcano plot for up- and downregulated proteins when comparing whole protein extracts from senescent cells (tlc1) and extracts from wt cells using label-free quantitative proteomics. Significantly changed proteins are shown in orange (p-value ≤ 0.05). B. Venn diagram of genes/proteins overlapping between downregulated proteins in senescent cells (tlc1) and mutants defective for telomere folding as described in [15]. sir2 was included in the list of fold-back defective mutants. C. Heatmap of the 21 overlapping candidates from Fig 4B and the log2 of their fold change during senescence as compared to wt. Candidates that affect histone deacetylation at telomeres are shown in bold letters. D. Telo-3C analysis in sir2, sin3, set2 and tlc1 (~PD 40) mutants. The interaction frequencies between primers P5 and P6 are shown. All interaction frequencies were normalized to a control qPCR product. Relative interaction frequencies were calculated by setting the normalized interaction frequency of the wt to 1. Mean +/- SD of 3–6 independent experiments. For sir2 and tlc1 mutants adjusted p-values were obtained from one-way ANOVA with Dunnett’s multiple comparisons test. For sin3 and set2 mutants adjusted p-values were obtained from unpaired t-tests (**p ≤ 0.01, ***p ≤ 0.001). E. Western blot for endogenously TAP-tagged Sin3 and Set2, as well as Sir2 levels in pre-senescent (~PD 30), senescent (~PD 45) and post-senescent cells (~PD 65). Sir2 and the respective actin loading control were visualized with fluorescent antibodies. All other western blots were developed using HRP-coupled antibodies. F. Southern blot for all telomeres using a (TG)n-repeat containing vector fragment labeled with ³²P as a probe. Dashed line indicates the wt telomere length. Superfluous lanes were cropped. The complete blot is shown in S6B Fig. G. TERRA levels in sir2, sin3, set2 and tlc1 (~PD 40) mutants. TERRA levels were normalized to Actin RNA levels. Relative TERRA expression levels were calculated by setting the normalized TERRA expression of wt cells to 1. Mean +/- SD of 3 independent experiments. Adjusted p-values were obtained from one-way ANOVA with Dunnett’s multiple comparisons test. No significant changes in TERRA levels were observed. H. Scheme of the PCR assay to follow the accumulation of telomere-telomere fusions (T-TFs) (Top). T-TF frequency of the indicated strains as determined by quantitative PCR from liquid cultures (~ PD 30). Mean +/- SEM of 4–6 independent samples. P-value was obtained from Mann-Whitney test (**p ≤ 0.01). PDs = population doublings.

https://doi.org/10.1371/journal.pgen.1008603.g004

We estimated the telomere silencing status by measuring the levels of the long non-coding RNA (lncRNA) TERRA from telomere 1L. TERRA is a lncRNA transcribed from subtelomeric promoters which are under the influence of the local chromatin state [72]. As expected, subtelomeres of cells lacking SIR2 were desilenced and TERRA levels were approx. 10x higher than in wt cells (Fig 4G). As previously reported, the increase in TERRA was heterogeneous in tlc1 cells, due to the stochastic appearance of critically short telomeres (Fig 4G) [73]. TERRA levels were also slightly increased in sin3 and set2 mutants (Fig 4G). Therefore, the state of telomere folding may have effects on TERRA levels in the cell.

We show here, that Sir2, Sin3 and Set2 are involved in the regulation of telomere folding. The levels of these histone modifiers are downregulated during senescence, which may contribute to the open state during this process. Interestingly, Sir2, Sin3 and Set2 protein levels were not downregulated in a folding-proficient tel1 mutant with very short telomeres (Figs 2D, 2E, 2F and S6C). This observation supports our hypothesis that telomere folding is reduced due to a feature of the senescence program, which may include chromatin alterations through downregulation of chromatin modifiers, but is not caused solely by short telomeres per se.

We were wondering what the functional consequences of reduced telomere folding are, and if the opening of the telomere structure might lead to a higher incidence of non-homologous end joining (NHEJ) between chromosome ends. We addressed how frequently telomere-telomere fusions (T-TFs) occur in sir2, sin3 and set2 mutants by quantitative PCR for T-TFs between chromosomes V and XV [40,41] (Fig 4H). As expected mec1 tel1 sml1 cells accumulated T-TFs as previously reported [38–41] (Fig 4H). In contrast, the telomeres of sir2, sin3 and set2 mutants, as well as those of telomerase negative cells (tlc1), did not present an increased incidence of T-TFs. Also the double mutants of sir2, sin3 and set2 in combination with loss of TLC1 showed similar T-TFs frequencies as wt cells (Fig 4H). Therefore, decreased folding does not render telomeres more susceptible to NHEJ.

The open state of telomeres may also influence the recombination capacity of telomeres during the senescence process. We observed that in the absence of telomerase both sir2 and set2 mutants displayed a delayed senescence phenotype (S6D Fig). This delay was completely dependent on Rad52, as its deletion led to an increased rate of senescence (S6D Fig). This suggests that an unfolded telomere state may allow increased recombination, however in the absence of Rad52, the open state telomeres may be rendered more vulnerable to becoming dysfunctional (e.g. through resection). It was not possible to make such speculations based on the sin3 mutant as it showed an enhanced senescence rate that was independent of RAD52 (S6E Fig).

Yeast strains and plasmids

Strains used in this study are listed in S1 Table. Strains were grown under standard conditions in YPD (Yeast Peptone Dextrose) at 30°C if not indicated otherwise.

Measurement of telomere length by PCR

Genomic DNA was extracted from 10 ml of exponentially growing cells with the Gentra Puregene Yeast/Bact. Kit (QIAGEN) according to the manufacturer’s protocol. For the C-tailing reaction, 100 ng of genomic DNA were mixed with 0.9 μl of NEB buffer 4 in a final volume of 9 μl. To denature the DNA, the mixture was incubated at 96°C for 10 min and cooled to 4°C. 4 units of terminal transferase (NEB) were added together with dCTP in a final concentration of 0.1 mM. The C-tailing reaction was carried out as follows: 37°C for 30 min; 65°C for 10 min; 96°C for 5 min; hold at 65°C. 30 μl of pre-heated PCR mix were added to the DNA. The PCR mix contained 4 μl of 2 mM dNTPs, 4 μl of PCR buffer (670 mM Tris-HCl pH 8.8, 160 mM (NH₄)₂SO₄, 70% glycerol, 0.1% Tween-20), 0.4 μl of Q5 Hot Start DNA Polymerase (2 u/μl, NEB) and the primers oBL358 and oBL359. The following cycling conditions were used for PCR: 95°C for 3 min; 45 cycles of 95°C for 30 s, 63°C for 15 s and 72°C for 20 s; 72°C for 5 min; hold at 4°C. PCR products were separated on an 1.8% agarose gel containing 1X SYBR Safe DNA gel stain and imaged on the Bio-Rad ChemiDoc Touch Imaging System.

Flow cytometry analysis for DNA content

0.18 OD₆₀₀ units of cells were collected and the cell pellet was washed with 1 ml of water. Cells were fixed in 70% ethanol overnight at 4°C. After fixation, cells were centrifuged at 13,000 rpm for 5 min and washed in 800 μl 50 mM Tris pH 7.5. RNA was digested in a 50 mM Tris pH 7.5 solution containing 0.25 mg/ml RNase A for 2 h at 37°C. 25 μl of 20 mg/ml proteinase K were added and incubated at 50°C for 2 h. Samples were sonicated for 10 s in a BRANSON sonifier 450 with the following settings: constant mode, 10 s, power 1. After sonication SYTOX Green was added to a final concentration of 2 μM in 50 mM Tris pH 7.5 and analyzed on a BD FACSVerse flow cytometer. 20,000 events were recorded per sample and the data was analyzed with BD FACSuite.

Flow cytometry analysis for cell viability

0.68 OD₆₀₀ units of cells were collected and the cell pellet was washed with 1 ml 50 mM Tris pH 7.5. After spinning down the cells for 1 min at 7000 rpm, the pellet was resuspended in 1 ml 50 mM Tris pH 7.5 containing 0.5 μM SYTOX Green. Cells were analyzed on a BD FACSVerse flow cytometer recording 20,000 events. The data was analyzed with FlowJo 10.6.1. As a control sample for dead cells, cells were incubated at 95°C for 15 min and subjected to the described protocol.

RNA extraction and qRT-PCR to measure TERRA levels

RNA extraction, DNase treatment and reverse transcription were performed as described in [73]. qPCR analysis was performed by the CFX384 Touch Real-Rime PCR Detection System (Bio-Rad) using DyNAmo Flash SYBR Green (Thermo Scientific).

Telomere chromosome conformation capture (Telo-3C)

25 ml of exponentially growing cells were crosslinked with 1.2% formaldehyde at OD₆₀₀ 0.4 for 20 min at room temperature. The reaction was quenched with 360 mM glycine for 5 min at RT and cells were placed on ice afterwards. The cells were washed twice with cold PBS and the pellet was resuspended in 200 μl FA-lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100). Cell lysis was performed in Lysematrix C tubes (MP Biomedicals) using the FastPrep (MP Biomedicals; 2x 30 s with 1 min on ice in between runs; 6.5 M/s). Cell extracts were recovered in FA-lysis buffer containing 0.1% sodium deoxycholate (SOD) and centrifuged for 15 min at 4°C, 13,000 rpm. The pellet was washed with FA-lysis buffer and resuspended in 200 ml of 20 mM Tris-HCl pH 7.5. The chromatin concentration of this 3C extract was estimated by determining its protein concentration by Bradford. Subsequently, the amount of chromatin corresponding to 60 μg of protein was digested with 100 units NcoI-HF (NEB) overnight at 37°C. The digestion reaction was stopped by incubating the samples at 65°C for 20 min in the presence of 1% SDS. The SDS was sequestered with 75 μl of 10% Triton X-100 and 570 μl of 20 mM Tris-HCl pH 7.5 were added. The chromatin was pelleted for 15 min at 4°C, 13,000 rpm, and resuspended in 750 μl ligation mixture containing 200 units T4 DNA ligase (NEB/IMB core facility protein production), 1X T4-Ligation buffer (NEB) and Tris-HCl pH 7.5. After ligation at RT for 5 h, RNA was digested with 20 μg RNase A and crosslinks were reversed with 7.5 μl proteinase K and 3.5 μl of 20% SDS at 65°C overnight. To isolate the DNA, the 3C reaction was mixed with 750 μl Phenol:Chloroform:Isoamylalcohol and the DNA was precipitated from the aqueous phase by adding 2 μl glycogen, 75 μl of 3 M KAc and 2 ml of 100% Ethanol. The DNA pellet was washed with 70% Ethanol and resuspended in 40 μl ddH₂O. The DNA concentration was determined with Qubit HS DNA reagent (Thermo Scientific). 1.5 ng of DNA were used per qPCR reaction, performed with CFX384 Touch Real-Rime PCR Detection System (Bio-Rad) and Dynamo Flash SYBR GREEN (Thermo Scientific). Primers for Telo-3C were used in a final concentration of 0.5 μM in the qPCR reaction. To account for the amount of input DNA that is used for the qPCR reaction, the Telo-3C-specific qPCR signal was normalized to the intensity of a control qPCR product (primers oRS39 and oRS40), which is robustly amplified from genomic DNA. The relative interaction frequencies of telomere 1L with its corresponding subtelomere in the investigated conditions were then determined relative to the respective wt or untreated sample, as follows:

The adjusted p-values were obtained from the statistical tests indicated in the figure legends. The type of post-hoc test was employed according to GraphPad Prism 8. The Tukey test was employed when comparing every mean with every other mean, such as in Fig 3B or 3G. The Dunnett’s test was used when comparing a control mean (e.g. the wt sample) to the other means, such as in Figs 2F, 3C, 3D and 4D. The Sidak method was employed when a set of means was selected to compare, such as in Fig 2B (wt and tlc1 Telo-3C interaction frequencies were compared for each time point) and Fig 2G (Telo-3C interaction frequencies +/- MMS were compared for each time point).

Protein extraction, SDS-PAGE and western blot

Proteins were extracted from 2 OD₆₀₀ units of yeast cells as described in [73]. Protein extracts were loaded on 4–15% precast SDS-PAGE gels (Bio-Rad). Proteins were blotted on a nitrocellulose membrane with the Trans-Blot Turbo Transfer System (Bio-Rad) using the high molecular weight program. The membrane was blocked for 1 h with 5% skim milk in PBST and primary antibodies were used in the following dilutions in 5% skim milk in PBST: PAP (Sigma Aldrich) at 1:3000; anti-Sir2 (Santa Cruz) at 1:1000; anti-Actin (Millipore) at 1:2000; anti-MYC-tag (Cell Signaling Technology/NEB) at 1:1000; anti-Pgk1 (Invitrogen) at 1:20,000. The membranes were incubated with the primary antibodies overnight at 4°C. After four washing steps with PBST, the respective secondary antibodies were added for 1 h at RT: goat anti-mouse-HRP conjugate (Bio-Rad) at 1:3000; goat anti-rabbit-HRP conjugate (Bio-Rad) at 1:3000; IRDye 680RD goat anti-mouse IgG (LI-COR) at 1:10,000; IRDye 800CW goat anti-rabbit IgG (LI-COR) at 1:10,000. Before detection, membranes were washed three times in PBST and once in PBS.

Membranes with HRP-coupled secondary antibodies were developed using the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific) or the Super Signal West Dura Extended Duration Substrate (Thermo Scientific) on the Bio-Rad ChemiDoc Touch Imaging System.

Membranes with secondary antibodies coupled to IRDyes were detected on the Odyssey CLx 9140 Imaging System (LI-COR) and processed via the Image studio software (LI-COR).

Southern blot

Genomic DNA (5 μg) was digested with the respective restriction enzyme for 5 h at 37°C (XhoI for all telomeres; SalI for telomere 1L). The digested DNA was separated on a 0.8% agarose gel overnight at 50 V. DNA in the gel was denatured for 1 h (0.4 M NaOH, 0.6 M NaCl) and neutralized for 1 h (1 M Trizma Base, 1.5 M NaCl, pH 7.4). DNA was transferred to a nylon membrane (Hybond NX, GE Healthcare) via capillary transfer in 10X SSC overnight and crosslinked to the membrane with UV light (auto X-link, UV Stratalinker 2400, Stratagene). The membrane was pre-hybridized for 5 h at 55°C in hybridization solution (PerfectHyb Plus Hybridization Buffer, Sigma Aldrich). Hybridization of the respective radio-labeled probe was carried out overnight at 55°C. The membrane was washed twice in 2X SSC with 0.1% SDS for 5 min and twice in 0.5X SSC with 0.1% SDS for 20 min. All washing steps were performed at 55°C. The membrane was exposed to a phosphoimager screen and the signal was detected via Typhoon FLA 9500 (GE Healthcare).

Probes for Southern blot

The telomere-specific probe was obtained by digestion of pBL423 with EcoRI followed by gel extraction using the QIAquick Gel Extraction Kit (Qiagen). Plasmid pBL423 was a kind gift from M.P. Longhese (pSP100). The probe for subtelomere 1L was generated by PCR with oligos oSM1 and oSM2 on genomic DNA of wt S. cerevisiae cells. The PCR product was separated on a 1% agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen). 60 ng of each probe were labeled with dATP [α-32P] (DECAprime kit II; Thermo Scientific) by random priming according to the manufacturer’s instructions.

Senescence curve

Liquid senescence assays were performed as described in [85]. Briefly, the OD₆₀₀ of every culture was measured every 24 h and cultures were diluted back to OD₆₀₀ 0.01 subsequently. All analysis during the senescence curve were performed on exponentially growing cultures, starting from the saturated culture after 24 h.

MS/MS

1 OD₆₀₀ unit of exponentially growing cells was resuspended in 1X LDS buffer supplemented with 100 mM DTT. Cells were heated for 10 min at 95°C and separated on a 4–12% NuPAGE Bis–Tris precasted PAGE gel (Thermo Scientific). Samples were run at 180 V for 10 min and processed by in-gel digestion [86]. Briefly, samples were reduced in reduction buffer (10 mM DTT in 50 mM ammonium bicarbonate (ABC) buffer) for 1 h at 56°C and alkylated in alkylation buffer (50 mM iodoacetamide in 50 mM ABC buffer) for 45 min in the dark. Proteins were digested with 2 μg Protease LysC (Wako Chemicals) overnight at 37°C in 50 mM ABC buffer. Digested peptides were desalted on a C18 StageTip as described [87] and analyzed by nanoflow liquid chromatography on an EASY-nLC 1000 system (Thermo Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Scientific). The peptides were separated on a self-packed reverse phase capillary (75 μm diameter, 25 cm length packed with C18 beads of 1.9 μm (Dr Maisch GmbH). The capillary was clamped on an electrospray ion source (Nanospray Flex, Thermo Scientific). A 90 min gradient starting from 2% - 60% gradient acetonitrile in 0.1% formic acid was used at a flow of 225 nl/min. Data was collected in data-dependent acquisition mode with one MS full scan followed by up to 10 MS/MS scan with HCD fragmentation.

MS data processing and bioinformatics analysis

MS raw files were processed using the MaxQuant software (version 1.5.2.8) and the ENSEMBL S. cerevisiae protein database (Saccharomyces_cerevisiae.R64-1-1.24). LFQ quantitation and match between run options were activated. MaxQuant output files were analyzed using an in-house R script. Known contaminants, reverse hits and protein groups only identified by site modification were excluded. Identified protein groups (minimum 2 peptides, 1 of them unique) were further filtered to a minimum of 2 quantification events per experiment. Missing values were imputed using a downshifted and compressed beta distribution within the 0.001 and 0.015 percentile of the measured values for each replicate individually. The LFQ intensities were log₂ transformed and a two sample Welch t-test was performed. Volcano plots were generated by plotting -log₁₀(p-values) and fold changes. The threshold line for enriched proteins is defined with p-value = 0.05. Gene ontology analysis were performed with the PantherDB.org [88] overrepresentation Test (Release 20190711) with the annotation database released on 20190703. Fisher’s exact test followed by FDR correction was applied to calculate the p-values. Heatmap for enriched proteins were generated using the “pheatmap” (version 1.0.12) or “ggplot2” (version 3.2.1) package in R. Additionally the Venn diagram was generated using the “eulerr” (version 5.1.0) package in R (version 3.5.1).

Telomere-telomere fusions analysis

Telomere-telomere fusions (T-TFs) were analyzed by quantitative PCR analyses as reported [40], with the modifications described in [41]. Briefly, ~100 ng of Sau3A-treated genomic DNA extracted by standard protocols from asynchronous cultures was PCR-amplified for quantitative analyses using a primer from the X element of chromosome XV-L and a primer from the Y’ element of chromosome V-R. A DNA fragment from HIS4 was PCR-amplified as input control. The oligonucleotides used for quantitative analyses and the PCR conditions described previously [40]. The frequency of T-TFs per genome was calculated with the formula: T-TFs/genome = 2^–N / N = Ct (T-TFs)–Ct (HIS4). Prior to applying this formula, the curves representing the increasing amounts of DNA for the two products as a function of the number of PCR cycles were confirmed to be parallel (i.e., the slope of the curve representing the log of the input amount versus ΔCt was < 0.1).

Construction of strains with auxin-inducible degrons

Strains carrying auxin-inducible degrons (AID*) for SMC complexes were created as described before [56]. Primers for tagging are listed in S2 Table.

The RAD53-AID*-9MYC construct was transferred from the strain published by Morawska and Ulrich into the S288C background [56]. For this purpose, the C-terminal sequence of RAD53 including the tag was amplified by PCR and integrated into a strain carrying leu2::AFB2::LEU2 (yKB244).

Materials

Materials such as antibodies, enzymes, chemicals and software are listed in S3 Table.

Numerical data

Underlying numerical data for all graphs are provided in S4 Table.