ERC accumulation depletes Sir2 from rDNA and induces cellular senescence by rDNA destabilization

Yeast strains and cultivation

Yeast strains and plasmids used in this study are listed in Supplementary Tables S1 and S2. YPDA medium (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose, 2% w/v agar, and 0.4% w/v adenine) or SC medium [0.67% w/v Yeast Nitrogen Base without amino acids, 2% w/v glucose (or galactose in Fig. 3B,C), 20 mg/l l-arginine HCl, 60 mg/l l-tyrosine, 80 mg/l l-isoleucine, 50 mg/l l-phenylalanine, 100 mg/l l-glutamic acid, 100 mg/l l-aspartic acid, 150 mg/l l-valine, 200 mg/l l-threonine, 400 mg/l l-serine, 20 mg/l uracil, 40 mg/l adenine sulfate, 60 mg/l l-leucine, 40 mg/l l-tryptophan, 20 mg/l l-histidine HCl, 20 mg/l l-methionine, and 120 mg/l l-lysine HCl, with the appropriate amino acid removed] was used for cultivation unless otherwise specified. For plate media, 2% agar was added. The strains were streaked out, and independent colonies were transferred into liquid media and incubated at 30°C. Before using strains NOY408-1b YEplac181 and NOY408-1b YEplac181-IGS, they were incubated for ∼30 generations in medium with the appropriate sugar. INAM treatment was performed by adding INAM (Sigma-Aldrich) to the medium at a final concentration of 25 mM.

Sorting of old cells

Old cells were sorted as described previously [41] with modifications. Logarithmic phase cells were collected by centrifugation and washed with phosphate-buffered saline (PBS). They were suspended in 100 μl of PBS (for 10⁷ cells), and 1.3 mg of sulfo-NHS-LC-biotin (Thermo Fisher Scientific) in 40 μl of PBS was added, followed by incubation at room temperature for 15 min. After incubation, the cells were collected, washed with PBS, suspended in 100 μl of PBS, transferred into 2.5% glucose YPDA medium (1% w/v yeast extract, 2% w/v peptone, 2.5% w/v glucose, 2% w/v agar, and 0.4 % w/v adenine), and incubated. After the cells were allowed to divide seven times, they were collected by centrifugation and suspended in 4 ml of PBS. A 40 μl aliquot of streptavidin-coated magnetic beads (Thermo Fisher Scientific) that were washed with PBS twice was added to the yeast suspension, and the mixture was incubated at room temperature for 15 min, followed by eight washes with PBS. Young cells were stored after the first wash. To measure bud scars, the cells were suspended in 4% (w/v) paraformaldehyde, incubated at room temperature for 5 min, washed three times with PBS, suspended in PBS, and stained with Calcofluor white (Sigma) (final 100 mg/μl) for 5 min. After two additional PBS washes, the cells were visualized by fluorescent microscopy.

Genomic DNA preparation

For pulsed-field gel electrophoresis (PFGE) and ERC assay, genomic DNA was prepared in low melting temperature agarose plugs as described previously [12, 42]. Briefly, stationary phase cells were collected and resuspended in 50 mM EDTA (pH 7.5) at a concentration of 33 μl per 5 ×ばつ 10⁷ cells. The cell suspension was incubated at 40°C and mixed with 66 μl of solution 1 [0.83% w/v low-melting-point agarose SeaPlaque GTG (Lonza), 170 mM sorbitol, 17 mM sodium citrate, 10 mM EDTA pH 7.5, 0.85% v/v β-mercaptoethanol, and 0.17 mg/ml Zymolyase 100T (Nacalai)]. The mixture was poured into a plug mold (Bio-RAD) and placed at 4°C for 20 min to solidify. The plugs were then transferred into solution 2 [450 mM EDTA (pH 7.5), 10 mM Tris–HCl (pH 7.5), 7.5% v/v β-mercaptoethanol, and 10 μg/ml RNase A (Macherey-Nagel)] and incubated at 37°C for 1 h. Subsequently, they were incubated overnight at 50°C in solution 3 [250 mM EDTA (pH 7.5), 10 mM Tris–HCl (pH 7.5), 1% w/v sodium dodecyl sulfate (SDS), and 1 mg/ml proteinase K (Nacalai)]. The plugs were washed four times for 15 min with 50 mM EDTA (pH 7.5).

Pulsed-field gel electrophoresis

PFGE was performed as described previously [12, 42]. Briefly, one-third of a plug or Hansenula wingei chromosomal DNA markers (Bio-Rad) was placed on a comb tooth. The comb was set into the gel tray, and 1% agarose solution (Pulsed Field Certified Agarose, Bio-Rad) in ×ばつ TBE [44.5 mM Tris base, 44.5 mM boric acid, and 1 mM EDTA (pH 8.0)] was poured. PFGE was run on a Bio-Rad CHEF DR-III system in 2.2 l of ×ばつ TBE under the following conditions: 3.0 V/cm for 68 h at 14°C, 120° included angle, initial switch time of 300 s, and final switch time of 900 s. After electrophoresis, DNA was stained with 0.5 μg/ml ethidium bromide (EtBr) for 30 min, and washed twice with water for 30 min each before being photographed. The stability of rDNA was assessed using Fiji (ver.2.16.0) by extracting the signal from chr. IV, measuring its width, and then extracting a segment of the same width centred on the signal peak on chr. XII. The area of the peaks was calculated for chr. XII and chr. IV in the density profile plot, using the endpoints of the extracted segment as the base. The ratio of these areas (chr. XII/chr. IV) was then determined.

Extrachromosomal rDNA circle assay

ERC assay was performed as described previously [42, 43]. Half of a plug was placed on a comb tooth. The comb was set into the gel tray (15 ×ばつ 25 cm), and 300 ml of 0.4% agarose (LABTAS + Agarose Powder, LABTAS+) in ×ばつ TAE [40 mM Tris base, 20 mM acetic acid, and 1 mM EDTA (pH 8.0)] was poured into the tray. A 500 ng aliquot of lambda HindIII DNA marker was applied to an empty lane. The electrophoresis was performed using a Sub-cell GT electrophoresis system (Bio-Rad) in 1.5 l of ×ばつ TAE at 1.0 V/cm for 48 h at 4°C with buffer circulation. The buffer was changed after 24 h. DNA was stained with 0.5 μg/ml EtBr for 30 min before being photographed.

Southern blot analysis

RNA extraction

Logarithmic phase cells were collected, and RNA was extracted using either the RNeasy Mini Kit (QIAGEN) or the NucleoSpin RNA (Macherey Nagel). For quantitative PCR (qPCR), reverse transcription and genome DNA removal were performed using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO).

DNA extraction

DNA was extracted as described previously [45] with slight modification in Fig. 3D. Stationary phase cells were collected (6.0 ×ばつ 10⁷ cells), and they were suspended in 200 μl of 0.5 M EDTA (pH 8.0), 800 μl of 1.2 M sorbitol, 10 μl of β-mercaptoethanol, and 20 μl of 50 mg/ml Zymolyase 100T. The cell extract was incubated at 37°C for 45 min, centrifuged at 4500 g for 5 min, and the supernatant was removed. The pellet was suspended in 500 μl of extraction solution [50 mM EDTA (pH 8.0), 50 mM Tris–HCl (pH 8.0), 0.5% w/v SDS] and 10 μl of 10 mg/ml proteinase K (Nacalai), incubated at 65°C for 1 h, then 200 μl of 5 M potassium acetate was added and the mixture was incubated on ice for 10 min. The extract was centrifuged for 15 min, the supernatant was transferred, and 500 μl of isopropanol was added. The mixture was centrifuged at 16 000 g for 5 min, and the supernatant was discarded. The pellet was washed with 70% ethanol, dried, then resuspended in 300 μl of TE, and 1.5 μl of 20 mg/ml RNase A (Macherey-Nagel) was added and incubated at 37°C for 1 h.

Yeast protein preparation

Yeast protein was extracted as described previously [20] with slight modification. Briefly, logarithmic phase cells were collected, and the cells were resuspended in 250 μl of water, and mixed with 37.5 μl of alkali solution (92.5% v/v 2 N NaOH, 7.5 % v/v β-mercaptoethanol), then incubated on ice for 10 min. The mixture was then combined with 50% v/v trichloroacetic acid, incubated on ice for 10 min, and centrifuged at 10 000 g for 5 min. The supernatant was removed, and the pellet was resuspended in SDS loading buffer [300 mM Tris–HCl (pH 6.8), 600 mM β-mercaptoethanol, 12% w/v SDS, 0.3% w/v Bromophenol blue, 30% v/v glycerol] to 5.0 ×ばつ 10⁶ cells/μl. The mixture was incubated at 65°C for 5 min. If cycloheximide (CHX) was used, it (water was added for the negative control) was added to the medium at a concentration of 100 μg/ml 2 h before cell collection.

Western blot analysis

Western blot was performed as described previously [20] with slight modification. The yeast protein sample was centrifuged at 21 130 g for 1 min before application. A volume of 5–10 μl of supernatant was applied to 5–20% e-PAGEL (Atto). Electrophoresis was performed in 500 ml of ×ばつ SDS–polyacrylamide gel electrophoresis (PAGE) buffer (25 mM Tris base, 192 mM glycine, and 0.1% w/v SDS) at 10–20 mA. Proteins were transferred to an Immobilon-P polyvinylidene fluoride (PVDF) membrane (Merck Millipore) in ×ばつ transfer buffer (25 mM Tris base, 192 mM glycine, and 10% v/v methanol) at 100 V for 60 min at 4°C. The membrane was soaked in 25 ml of 5% skim milk powder in ×ばつ PBS, 0.05% Tween-20 (PBS-T) for 30 min at room temperature, and then soaked in 10 ml of PBS-T with primary antibody overnight at 4°C. After that, the membrane was washed with 50 ml of PBS-T for 30 min twice. If a secondary antibody was necessary, the washed membrane was soaked in 10 ml of PBS-T with secondary antibody for 1 h at room temperature and washed with 50 ml of PBS-T for 30 min twice. Protein signals were activated with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and photographed. Stripping was performed using 20 ml of RestoreTM Western Blot Stripping Buffer (Thermo Fisher Scientific) for 15 min to detect tubulin for normalization. The membrane was washed with 25 ml of PBS-T for 5 min three times. The procedures following the blocking step were performed in the same manner as for the primary antibody. The target protein signals were normalized to tubulin signals in the quantification (target protein/tubulin). In Supplementary Fig. S3A, this value for young CHX– was normalized to 1, and degradation levels were calculated by subtracting the value for CHX+ from that for CHX– in both young and old cells.

Antibodies used for western blotting were as follows.

Sir2: goat polyclonal anti-Sir2 (yN-19) (Santa Cruz Biotechnology)

Goat IgG-HRP: donkey polyclonal anti-goat IgG-HRP (Santa Cruz Biotechnology)

Tubulin: rat monoclonal anti-tubulin alpha:HRP (clone YL1/2) (Bio-Rad)

HA-HRP: mouse monoclonal anti-HA-tag (F-7) HRP (Santa Cruz Biotechnology

These antibodies are also listed in Supplementary Table S3.

Chromatin immunoprecipitation

Logarithmic phase cells were collected, suspended in 10 ml of PBS, and 278 μl of 37% formaldehyde solution (FUJIFILM Wako) was added. The mixture was incubated for 15 min at room temperature, mixed with 500 μl of 2.5 M glycine, and incubated for 5 min at room temperature. The cells (almost 1–3 ×ばつ 10⁷ cells) were washed with 500 μl of PBS and lysed with 300 μl of buffer 1 [140 mM NaCl, 0.1% w/v sodium deoxycholate, 50 mM HEPES-KOH (pH 7.5), 1 mM EDTA (pH 8.0), 1% v/v Triton X-100, and Complete EDTA-free Protease Inhibitor Cocktail (Roche)] by zirconia grinding beads (YTZ-ball) with a diameter of 0.5 mm (Nikkato) and a Multi Beads Shocker (Yasui Kikai) for 30 s five times. The lysate was sonicated using a Bioruptor (Cosmo Bio) for 30 s five times at high speed and centrifuged at 9391 g for 5 min. The supernatant was mixed with 20 μl of Dynabeads Protein G (Thermo Fisher Scientific), 1 μl of antibody, and buffer 1 (up to a total volume of 321 μl). The mixture was incubated at 4°C overnight, washed with 1 ml of buffer 1 twice, 1 ml of buffer 1′ [500 mM NaCl, 0.1% w/v sodium deoxycholate, 50 mM HEPES-KOH (pH 7.5), 1 mM EDTA (pH 8.0), and 1% v/v Triton X-100] twice, 1 ml of wash buffer [250 mM NaCl, 0.5% w/v sodium deoxycholate, 10 mM Tris–HCl (pH 8.0), 0.5% Nonidet P-40] twice, and 1 ml of ×ばつ TE (pH 8.0) once. The pellet was resuspended in 50 μl of ×ばつ TE (pH 8.0), mixed with 1 μl of 20 mg/ml RNase A (Macherey-Nagel), and incubated at 37°C for 10 min. After that, 50 μl of 1% w/v SDS and 1 mg/ml proteinase K (Nakalai) was added and incubated at 42°C for 30 min, then at 65°C for 3 h. The DNA was extracted by NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel).

Antibodies used for ChIP were as follows.

Sir2: goat polyclonal anti-Sir2 (yN-19) (Santa Cruz Biotechnology)

Histone H3: rabbit polyclonal anti-histone H3 (Abcam)

Histone H3K9Ac: rabbit polyclonal anti-acetyl-histone H3 (Lys9) (Millipore)

Histone H3K14Ac: rabbit polyclonal anti-acetyl-histone H3 (Lys14) (Millipore)

Histone H4K16 Ac: rabbit polyclonal anti-histone H4K16 Ac (Active Motif)

RNA polymerase II: rat monoclonal RNA pol II antibody (mAb) (Active Motif)

These antibodies are also listed in Supplementary Table S3.

Polysome profiling

Quantitative PCR analysis

qPCR was performed using a Thermal Cycler Dice® Real Time System II (TaKaRa) or a QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific) with THUNDERBIRD SYBR qPCR Mix (TOYOBO) or THUNDERBIRD Next SYBR qPCR Mix (TOYOBO) under the following conditions: denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Primer sequences used for qPCR were as follows :

E-pro transcription (Figs 2F, 3B, 4C, and 5A; Supplementary Fig. S4C) (5′-CCCATAACTAACCTACCATTCGA and 5′-TCAAGTAGTAGCAACCCAATGAG)

ACT1 mRNA and ACT1 region (Figs 2F, 3B, D, 4C, and 5A–C; Supplementary Figs S3B, D, S4A, C, and S5D) (5′-CGAATTGAGAGTTGCCCCAG and 5′-CAAGGACAAAACGGCTTGGA)

E-pro region (Figs 3D, 4B, and 5B–D; Supplementary Figs S4A and S5C) (5′-GCGGTATGCGGAGTTGTAAG and 5′-CGGTTTTGTTCTCTTCCCTCC)

SIR2 mRNA (Supplementary Fig. S3B, D) (5′-CCGAGGATTTGAACTCGTTATAC and 5′-CCAAATCTTGAACACGCTCTTGC)

GCN5 mRNA (Supplementary Fig. S5D) (5′-CATCAGATTGAAGAGGATCACTTG and 5′-GGTGCCCTCTTGTTTATTGGTCTC)

TEL07L (Fig. 5B) (5′-AACCACCATCCATCTCTCTACTTACTACTA and 5′-AGAACAACAGTACAGTGAGTAGGACATG).

These primers are also listed in Supplementary Table S4. For quantification except Fig. 5B, we calculated ΔCt (target Ct – standard Ct) and determined relative expression using the 2^(–ΔCt) method. The average of these values in the control group was normalized to 1. In Fig. 5B, after calculating 2^(–ΔCt), the percentage of input DNA (%IN) was derived.

Changes in rDNA instability and E-pro activation with aging

To investigate rDNA instability in old cells, we harvested them using a biotin–streptavidin strategy [41] (Fig. 2A). Briefly, yeast cell surfaces are labeled with biotin, which remains only on mother cells after budding. Thus, labeled old cells were harvested by capturing with streptavidin after cultivation, while the remaining cells were used as young cells. Using this strategy, old cells were accurately separated (∼7–8 divisions) (Fig. 2B, C), and subsequently used for analysis.

First, we compared rDNA instability between young and old cells using PFGE to detect rDNA copy number fluctuations. Southern blotting was performed to identify chr. XII, employing two probes. Probe 1 (rDNA probe) detects the rDNA sequence, identifying both chr. XII and ERCs, whereas Probe 2 (non-rDNA probe) detects the short arm region of chr. XII (Fig. 2D), identifying chr. XII only.

We also utilized the rARS-Δ3 strain, in which a non-essential element of the replication origin (ACS-III) is deleted, significantly reducing replication initiation efficiency, particularly in ERCs [40]. As a result, replication of ERCs is rare, and their numbers are considerably lower than in the wild-type strain. In the rARS-Δ3 strain, the status of genomic rDNA is more clearly observed, as the effect of ERCs is minimized. In the Southern blotting experiment (Fig. 2E), sharp signals were detected only in old cells when using Probe 1 as well as the EtBr-stained gel. These signals were absent with Probe 2 and were weaker in the rARS-Δ3 strain compared with the wild-type strain, suggesting that these signals represent ERCs. Furthermore, to confirm this, exonuclease V treatment was performed before PFGE to digest linear DNA (Supplementary Fig. S1). The sharp signals persisted while other chromosomal signals almost disappeared, suggesting that they consist of circular DNA. This result further supports the idea that these signals represent ERCs. However, the apparent size of these circular DNAs was comparable with that of chromosomes, leading us to consider that their exact size could not be accurately measured under the current PFGE conditions. This behavior is consistent with previous studies on PFGE, which demonstrated that the mobility of supercoiled circular DNA is largely independent of pulse time, causing it to migrate differently from large linear chromosomes [48, 49]. Finally, referring to Southern blotting analysis, rDNA instability was observed in old cells in both strains, as indicated by Probe 2. These results are consistent with those of previous studies [50, 51].

Since previous research has shown that rDNA instability is caused by the activation of E-pro transcription [16, 17], we measured E-pro transcript levels using quantitative reverse transcription–PCR (RT–qPCR). The results showed that E-pro transcript levels increased with aging. In old cells, E-pro transcripts were ∼30-fold higher in the wild-type strain and 20-fold higher in the rARS-Δ3 strain compared with young cells (Fig. 2F), consistent with earlier findings [50]. These results indicate that in old cells, E-pro on the chromosome is activated, leading to increased rDNA instability.

The effect of increased transcripts from the IGS region and the change of Sir2 levels with aging

As shown above, E-pro transcript levels increased with aging, which is suggested to contribute to rDNA instability. However, it remains unclear whether the transcripts themselves or the transcriptional process cause rDNA instability. To address this question, we introduced plasmids containing the IGS region of rDNA, where the E-pro was replaced by the Gal 1/10 promoter (Gal-pro), into yeast (Fig. 3A), while the genomic E-pro remains unchanged. Gal-pro transcript levels from the IGS region were controlled by the sugar in the medium. We used glucose medium as a negative control alongside galactose medium. Additionally, a strain introduced with only vector plasmids was used as another negative control. These two strains were incubated for ∼30 generations in the presence of the appropriate sugar after their construction to observe changes in rDNA before being used for experiments.

RT–qPCR results showed that Gal-pro transcript levels increased in the galactose medium (Fig. 3B). These strains were then analyzed using PFGE. No differences in the sharpness of chr. XII signals were observed. Moreover, the signal intensity per unit area was calculated for quantification (a simplified explanation is provided here), but no significant difference was detected (Fig. 3C). Details of the method are provided in the Materials and methods. Previous studies showed that transcription activation by replacing E-pro with Gal-pro in the genome causes rDNA instability [16, 17]. However, our experiment showed that the increase in transcript levels from the plasmids does not affect rDNA stability. These results suggest that either E-pro transcripts act solely in cis, or that rDNA instability is caused by the act of E-pro transcription itself rather than by the resulting transcripts, a perspective that aligns with the model proposed in the previous study [17].

We then investigated the cause of the increase in E-pro transcript levels. It is known that the number of ERCs, which include the E-pro region, is higher in old cells than in young cells [36], raising the possibility that the increase in E-pro transcript levels may be caused by an increase in ERCs. To test this, we determined the rDNA copy number (genomic rDNA + ERC) by qPCR. This analysis revealed that rDNA copy number in old cells was approximately twice as high as in young cells (Fig. 3D), a ratio much lower than that of E-pro transcript levels (>30-fold) (Fig. 2F, left). Furthermore, E-pro transcript levels increased with aging in the rARS-Δ3 strain, which has lower ERC levels than the wild-type strain (Fig. 2F, right). The findings suggest that the increase in E-pro transcript levels is caused by transcriptional activation, not ERC accumulation.

To explore the cause of E-pro transcription activation, we focused on Sir2. Previous studies reported that Sir2 maintains rDNA stability by regulating E-pro transcription [17, 18] and decreases with aging [32]. In contract, our experiment showed that Sir2 levels did not decrease with aging (Fig. 3E; Supplementary Fig. S2A–D), as determined by western blotting. Furthermore, Sir2 protein stability (Supplementary Fig. S3A), SIR2 mRNA levels (Supplementary Fig. S3B), and SIR2 translation efficiency (Supplementary Fig. S3C, D) were analyzed using western blotting after treatment with CHX, which inhibits protein synthesis, RT–qPCR, and polysome profiling followed by RT–qPCR, respectively. None of these analyses provided data supporting a decrease in Sir2 levels with aging. In fact, SIR2 mRNA levels were slightly increased with aging (Supplementary Fig. S3B). These findings align with the previous report [20] suggesting that increased rDNA copies enhance SIR2 transcription. This occurs because UAF, which represses SIR2 expression, is titrated by the rDNA copies (see Introduction). Furthermore, Rrn5—the least abundant protein in UAF [52]—shows decreased levels with aging (Supplementary Fig. S3E). Moreover, to further confirm our results, we normalized Sir2 protein levels to the nucleolar protein Rrn5 and found that the Sir2/Rrn5 ratio did not decrease with aging (Supplementary Fig. S3E).

The relationship between histone acetylation in the E-pro region and rDNA instability

Under our experimental conditions, Sir2 levels were not affected by aging, suggesting that factors other than Sir2 contribute to E-pro transcription activation. Based on previous studies [27, 28], we examined histone modifications by chromatin immunoprecipitation (ChIP)-qPCR. The results showed that histone H3K14 and H4K16 acetylation levels increased with aging (Fig. 4A, B). Furthermore, we performed ChIP-qPCR to analyze RNA polymerase II (Pol2) localization in E-pro. We used input DNA of ACT1 for normalization to remove the effect of increased rDNA copy number with aging. We revealed that the amount of Pol2 which combines with E-pro increases with aging (Supplementary Fig. S4A).

Histone H4K16 acetylation is known to affect E-pro transcription and rDNA instability [31]. In contrast, the role of H3K14 acetylation has not been reported previously. In budding yeast, Sas3 and Gcn5 function as histone H3K14 acetylases [53–55]. To investigate the effects of histone H3K14 acetylation on rDNA stability, we established sas3 and gcn5 deletion mutants (sas3Δ and gcn5Δ, respectively). PFGE analysis showed no significant differences in rDNA stability in either young or old sas3Δ or gcn5Δ cells (Supplementary Fig. S4B). In sas3Δ, no significant changes in the fold change of E-pro transcript levels were observed. Thus, further analyses of sas3Δ were not performed (Supplementary Fig. S4C).

In contrast, the fold change in E-pro transcript levels with aging was ∼3.5-fold in the gcn5Δ strain compared with 18-fold in the wild-type strain (Fig. 4C). We speculated that rDNA in old cells might be too unstable to detect the difference using PFGE. To address this, we performed an ERC assay. ERC levels, which reflect rDNA instability, were lower in the gcn5Δ strain compared with the wild-type strain in old cells but not in young cells (Fig. 4D; Supplementary Fig. S5B).

ERC levels are influenced not only by rDNA stability but also by the replicative initiation activity, as observed in the rARS-Δ3 strain. Interestingly, ERC levels in young gcn5Δ cells were not significantly reduced compared with wild-type cells, a phenotype that differs from the rARS-Δ3 strain, in which ERC levels are reduced in young cells (Supplementary Fig. S5A, B), as reported in a previous study [51]. These findings suggest that rDNA in the gcn5Δ strain is more stable than in the wild-type strain in old cells.

Given that Gcn5 acetylates not only H3K14 but also H3K9 [53, 56], we proceeded to investigate H3K9 acetylation. While H3K9 acetylation levels showed an increasing trend, they did not reach statistical significance (Supplementary Fig. S5C).

Additionally, H3K14R and H3K14Q mutants, which are non-acetylatable and mimic acetylation, respectively, were analyzed using PFGE. The chr. XII signals in the H3K14R strain were sharper than those in the wild-type strain. Moreover, quantification of signal intensity per unit area (simplified explanation provided here) indicated that rDNA in the H3K14R strain was more stable than in the wild-type strain (Fig. 4E). Details of the method are provided in the Materials and methods. In contrast, no difference in rDNA instability was observed between the H3K14Q strain and the wild-type strain.

GCN5 expression levels did not change with aging (Supplementary Fig. S5D, E). While rDNA stability improved in the gcn5Δ mutant, it remained unstable in old cells compared with young cells. This suggests the involvement of additional factors that influence rDNA instability in old cells.

The changes in Sir2 activity and localization with aging

In summary, Sir2 protein levels do not decrease with aging, but the acetylation levels of H3K14 and H4K16 in the E-pro region increase. Since these histone residues are deacetylated by Sir2, we next examined the enzymatic activity of Sir2. First, INAM treatment was performed to enhance Sir2 activity by increasing NAD⁺ levels and counteracting the inhibitory effect of NAM on Sir2 activation [34, 35]. However, this treatment did not affect the fold change in E-pro transcript levels with aging (Fig. 5A). These results suggest that Sir2 activity is sufficient for deacetylation in both young and old cells.

Next, we analyzed Sir2 localization using ChIP-qPCR. Sir2 association with E-pro in old cells was approximately half that in young cells when normalized to the input DNA of E-pro. However, no significant difference was observed when normalized to the input DNA of the ACT1 region (Fig. 5B, C). We interpreted these results as being caused by an increase in rDNA copy number with aging (Fig. 3D). Specifically, the amount of Sir2 on a rDNA copy does not change with aging. However, as the number of rDNA copies increases due to ERC accumulation, the relative amount of Sir2 bound to genomic rDNA decreases, leading to histone acetylation.

Supporting this hypothesis, plasmids containing the IGS region but modified with a galactose-inducible promoter (Gal-pro) instead of E-pro increased transcript levels from the IGS region, even though the Gal-pro in these plasmids was inactive in glucose (Fig. 3B), suggesting that E-pro transcription in genomic rDNA is activated. As the Gal-pro plasmid includes the RFB which is associated with Sir2 [30], Sir2 may be sequestered. In line with this, the localization levels of Sir2 at E-pro decreased when the IGS region was transformed by these plasmids (Fig. 5D). Moreover, in the rARS-Δ3 strain, which exhibits lower ERC proliferation than the wild-type strain, the fold change in E-pro transcript levels with aging was lower than in the wild-type strain (Fig. 2F). The reason for this may be that the lower rDNA copy number, resulting from reduced ERC proliferation, affects not only the amount of template DNA but also the extent to which Sir2 decreases on genomic rDNA. Taken together, these findings suggest that Sir2 activity does not decrease, at least by the midpoint of the yeast lifespan when rDNA instability occurs. Instead, its activity within genomic rDNA is reduced due to ERCs sequestering Sir2.

• 1. López-Otín C, Blasco MA, Partridge L et al. The hallmarks of aging. Cell. 2013; 153:1194–217. 10.1016/j.cell.202211001.. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 2. Moskalev AA, Shaposhnikov MV, Plyusnina EN et al. The role of DNA damage and repair in aging through the prism of Koch-like criteria. Ageing Res Rev. 2013; 12:661–84. 10.1016/j.arr.201202001. [DOI] [PubMed] [Google Scholar]
• 3. Kobayashi T Ribosomal RNA gene repeats, their stability and cellular senescence. Proc Jpn Acad, Ser B. 2014; 90:119–29. 10.2183/pjab.90.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 4. Sasaki M, Kobayashi T Regulatory processes that maintain or alter ribosomal DNA stability during the repair of programmed DNA double-strand breaks. Genes Genet Syst. 2023; 98:103–19. 10.1266/ggs.22-00046. [DOI] [PubMed] [Google Scholar]
• 5. Hori Y, Engel C, Kobayashi T Regulation of ribosomal RNA gene copy number, transcription and nucleolus organization in eukaryotes. Nat Rev Mol Cell Biol. 2023; 24:414–29. 10.1038/s41580-022-00573-9. [DOI] [PubMed] [Google Scholar]
• 6. Kobayashi T Regulation of ribosomal RNA gene copy number and its role in modulating genome integrity and evolutionary adaptability in yeast. Cell Mol Life Sci. 2011; 68:1395–403. 10.1007/s00018-010-0613-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 7. Petes TD Yeast ribosomal DNA genes are located on chromosome XII. Proc Natl Acad Sci USA. 1979; 76:410–4. 10.1073/pnas.76.1.410. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 8. Kobayashi T, Heck DJ, Nomura M et al. Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I. Genes Dev. 1998; 12:3821–30. 10.1101/gad.12.24.3821. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 9. Brewer BJ, Fangman WL A replication fork barrier at the 3′ end of yeast ribosomal RNA genes. Cell. 1988; 55:637–43. 10.1016/0092-8674(88)90222-X. [DOI] [PubMed] [Google Scholar]
• 10. Brewer BJ, Lockshon D, Fangman WL The arrest of replication forks in the rDNA of yeast occurs independently of transcription. Cell. 1992; 71:267–76. 10.1016/0092-8674(92)90355-G. [DOI] [PubMed] [Google Scholar]
• 11. Kobayashi T, Hidaka M, Nishizawa M et al. Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae. Mol Gen Genet. 1992; 233:355–62. 10.1007/BF00265431. [DOI] [PubMed] [Google Scholar]
• 12. Sasaki M, Kobayashi T Ctf4 prevents genome rearrangements by suppressing DNA double-strand break formation and its end resection at arrested replication forks. Mol Cell. 2017; 66:533–45. 10.1016/j.molcel.2017年04月02日0. [DOI] [PubMed] [Google Scholar]
• 13. Kobayashi T A new role of the rDNA and nucleolus in the nucleus—rDNA instability maintains genome integrity. Bioessays. 2008; 30:267–72. 10.1002/bies.20723. [DOI] [PubMed] [Google Scholar]
• 14. Ganley ARD, Hayashi K, Horiuchi T et al. Identifying gene-independent noncoding functional elements in the yeast ribosomal DNA by phylogenetic footprinting. Proc Natl Acad Sci USA. 2005; 102:11787–92. 10.1073/pnas.0504905102. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 15. Hosoyamada S, Sasaki M, Kobayashi T The CCR4–NOT complex maintains stability and transcription of rRNA genes by repressing antisense transcripts. Mol Cell Biol. 2019; 40:e00320-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 16. Saka K, Ide S, Ganley ARD et al. Cellular senescence in yeast is regulated by rDNA noncoding transcription. Curr Biol. 2013; 23:1794–8. 10.1016/j.cub.2013年07月04日8. [DOI] [PubMed] [Google Scholar]
• 17. Kobayashi T, Ganley ARD Recombination regulation by transcription-induced cohesin dissociation in rDNA repeats. Science. 2005; 309:1581–4. 10.1126/science.1116102. [DOI] [PubMed] [Google Scholar]
• 18. Kobayashi T, Horiuchi T, Tongaonkar P et al. SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast. Cell. 2004; 117:441–53. 10.1016/S0092-8674(04)00414-3. [DOI] [PubMed] [Google Scholar]
• 19. Kaeberlein M, McVey M, Guarente L The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev. 1999; 13:2570–80. 10.1101/gad.13.19.2570. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 20. Iida T, Kobayashi T RNA polymerase I activators count and adjust ribosomal RNA gene copy number. Mol Cell. 2019; 73:645–54. 10.1016/j.molcel.2018年11月02日9. [DOI] [PubMed] [Google Scholar]
• 21. Imai S, Armstrong CM, Kaeberlein M et al. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature. 2000; 403:795–800. 10.1038/35001622. [DOI] [PubMed] [Google Scholar]
• 22. Landry J, Slama JT, Sternglanz R Role of NAD⁺ in the deacetylase activity of the SIR2-like proteins. Biochem Biophys Res Commun. 2000; 278:685–90. 10.1006/bbrc.2000.3854. [DOI] [PubMed] [Google Scholar]
• 23. Kanfi Y, Naiman S, Amir G et al. The sirtuin SIRT6 regulates lifespan in male mice. Nature. 2012; 483:218–21. 10.1038/nature10815. [DOI] [PubMed] [Google Scholar]
• 24. Rine J, Herskowitz I Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae. Genetics. 1987; 116:9–22. 10.1093/genetics/116.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 25. Gottschling DE, Aparicio OM, Billington BL et al. Position effect at S. cerevisiaetelomeres: reversible repression of pol II transcription. Cell. 1990; 63:751–62. 10.1016/0092-8674(90)90141-Z. [DOI] [PubMed] [Google Scholar]
• 26. Strahl-Bolsinger S, Hecht A, Luo K et al. SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev. 1997; 11:83–93. 10.1101/gad.11.1.83. [DOI] [PubMed] [Google Scholar]
• 27. Braunstein M, Sobel RE, Allis CD et al. Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern. Mol Cell Biol. 1996; 16:4349–56. 10.1128/MCB.16.8.4349. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 28. Thompson JS, Ling X, Grunstein M Histone H3 amino terminus is required for telomeric and silent mating locus repression in yeast. Nature. 1994; 369:245–7. 10.1038/369245a0. [DOI] [PubMed] [Google Scholar]
• 29. Buck SW, Sandmeier JJ, Smith JS RNA polymerase I propagates unidirectional spreading of rDNA silent chromatin. Cell. 2002; 111:1003–14. 10.1016/S0092-8674(02)01193-5. [DOI] [PubMed] [Google Scholar]
• 30. Huang J, Moazed D Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing. Genes Dev. 2003; 17:2162–76. 10.1101/gad.1108403. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 31. Cesarini E, D’Alfonso A, Camilloni G H4K16 acetylation affects recombination and ncRNA transcription at rDNA in Saccharomyces cerevisiae. Mol Biol Cell. 2012; 23:2770–81. 10.1091/mbc.e12-02-0095. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 32. Dang W, Steffen KK, Perry R et al. Histone H4 lysine 16 acetylation regulates cellular lifespan. Nature. 2009; 459:802–7. 10.1038/nature08085. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 33. Lin SS, Manchester JK, Gordon JI Enhanced gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae. J Biol Chem. 2001; 276:36000–7. 10.1074/jbc.M103509200. [DOI] [PubMed] [Google Scholar]
• 34. McClure JM, Wierman MB, Maqani N et al. Isonicotinamide enhances Sir2 protein-mediated silencing and longevity in yeast by raising intracellular NAD+ concentration. J Biol Chem. 2012; 287:20957–66. 10.1074/jbc.M112.367524. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 35. Sauve AA, Moir RD, Schramm VL et al. Chemical activation of Sir2-dependent silencing by relief of nicotinamide inhibition. Mol Cell. 2005; 17:595–601. 10.1016/j.molcel.2004年12月03日2. [DOI] [PubMed] [Google Scholar]
• 36. Sinclair DA, Guarente L Extrachromosomal rDNA circles—a cause of aging in yeast. Cell. 1997; 91:1033–42. 10.1016/S0092-8674(00)80493-6. [DOI] [PubMed] [Google Scholar]
• 37. Russell AE, Schafer BJ et al. Dynamics of age-related catastrophic mitotic failures and recovery in yeast. bioRxiv9 November 2018, preprint: not peer reviewed 10.1101/466797v2. [DOI] [Google Scholar]
• 38. Morlot S, Song J, Léger-Silvestre I et al. Excessive rDNA transcription drives the disruption in nuclear homeostasis during entry into senescence in budding yeast. Cell Rep. 2019; 28:408–422. 10.1016/j.celrep.2019年06月03日2. [DOI] [PubMed] [Google Scholar]
• 39. Fehrmann S, Paoletti C, Goulev Y et al. Aging yeast cells undergo a sharp entry into senescence unrelated to the loss of mitochondrial membrane potential. Cell Rep. 2013; 5:1589–99. 10.1016/j.celrep.2013年11月01日3. [DOI] [PubMed] [Google Scholar]
• 40. Ganley ARD, Ide S, Saka K et al. The effect of replication initiation on gene amplification in the rDNA and its relationship to aging. Mol Cell. 2009; 35:683–93. 10.1016/j.molcel.2009年07月01日2. [DOI] [PubMed] [Google Scholar]
• 41. Park PU, McVey M, Guarente L Separation of mother and daughter cells. Methods Enzymol. 2002; 351:468–77. [DOI] [PubMed] [Google Scholar]
• 42. Sasaki M, Kobayashi T Gel electrophoresis analysis of rDNA instability in Saccharomyces cerevisiae. Methods Mol Biol. 2021; 2153:403–25. [DOI] [PubMed] [Google Scholar]
• 43. Goto M, Sasaki M, Kobayashi T The S-phase cyclin Clb5 promotes rRNA gene (rDNA) stability by maintaining replication initiation efficiency in rDNA. Mol Cell Biol. 2021; 41:1–19. 10.1128/MCB.00324-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 44. Yokoyama M, Sasaki M, Kobayashi T Spt4 promotes cellular senescence by activating non-coding RNA transcription in ribosomal RNA gene clusters. Cell Rep. 2023; 42:111944. 10.1016/j.celrep.2022.111944. [DOI] [PubMed] [Google Scholar]
• 45. Murakami H, Borde V, Nicolas A et al. Gel electrophoresis assays for analyzing DNA double-strand breaks in Saccharomyces cerevisiae at various spatial resolutions. Methods Mol Biol. 2009; 557:117–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 46. Schindelin J, Arganda-Carreras I, Frise E et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9:676–82. 10.1038/nmeth.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 47. Ohgane K, Yoshioka H Quantification of Gel Bands by an Image J Macro, Band/Peak Quantification Tool. protocols.io. 2019; v1: 10.17504/protocols.io.7vghn3w. [DOI] [Google Scholar]
• 48. Simske JS, Scherer S Pulsed-field gel electrophoresis of circular DNA. Nucleic Acids Res. 1989; 17:4359–65. 10.1093/nar/17.11.4359. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 49. Beverley SM Characterization of the "unusual" mobility of large circular DNAs in pulsed field-gradient electrophoresis. Nucleic Acids Res. 1988; 16:925–39. 10.1093/nar/16.3.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 50. Pal S, Postnikoff SD, Chavez M et al. Impaired cohesion and homologous recombination during replicative aging in budding yeast. Sci Adv. 2018; 4:eaaq0236. 10.1126/sciadv.aaq0236. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 51. Hattori M, Horigome C, Aspert T et al. Changed life course upon defective replication of ribosomal RNA genes. Genes Genet Syst. 2022; 97:285–95. 10.1266/ggs.22-00100. [DOI] [PubMed] [Google Scholar]
• 52. Kulak NA, Pichler G, Paron I et al. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods. 2014; 11:319–24. 10.1038/nmeth.2834. [DOI] [PubMed] [Google Scholar]
• 53. Kuo MH, Brownell JE, Sobel RE et al. Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines. Nature. 1996; 383:269–72. 10.1038/383269a0. [DOI] [PubMed] [Google Scholar]
• 54. Jaiswal D, Turniansky R, Green EM Choose your own adventure: the role of histone modifications in yeast cell fate. J Mol Biol. 2017; 429:1946–57. 10.1016/j.jmb.2016年10月01日8. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 55. Howe L, Auston D, Grant P et al. Histone H3 specific acetyltransferases are essential for cell cycle progression. Genes Dev. 2001; 15:3144–54. 10.1101/gad.931401. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 56. Bonnet J, Wang C-Y, Baptista T et al. The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription. Genes Dev. 2014; 28:1999–2012. 10.1101/gad.250225.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 57. Huynh MT, Yadav SP, Reese JC et al. Nucleosome dynamics during transcription elongation. ACS Chem Biol. 2020; 15:3133–42. 10.1021/acschembio.0c00617. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 58. Zippo A, Serafini R, Rocchigiani M et al. Histone crosstalk between H3S10ph and H4K16ac generates a histone code that mediates transcription elongation. Cell. 2009; 138:1122–36. 10.1016/j.cell.2009年07月03日1. [DOI] [PubMed] [Google Scholar]
• 59. Houseley J, Kotovic K, El Hage A et al. Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control. EMBO J. 2007; 26:4996–5006. 10.1038/sj.emboj.7601921. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 60. Gnügge R, Rudolf F Saccharomyces cerevisiae shuttle vectors. Yeast. 2017; 34:205–21. [DOI] [PubMed] [Google Scholar]
• 61. Michel AH, Kornmann B, Dubrana K et al. Spontaneous rDNA copy number variation modulates Sir2 levels and epigenetic gene silencing. Genes Dev. 2005; 19:1199–210. 10.1101/gad.340205. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 62. Moazed D Common themes in mechanisms of gene silencing. Mol Cell. 2001; 8:489–98. 10.1016/S1097-2765(01)00340-9. [DOI] [PubMed] [Google Scholar]
• 63. Smeal T, Claus J, Kennedy B et al. Loss of transcriptional silencing causes sterility in old mother cells of S. cerevisiae. Cell. 1996; 84:633–42. 10.1016/S0092-8674(00)81038-7. [DOI] [PubMed] [Google Scholar]
• 64. Kim S, Ohkuni K, Couplan E et al. The histone acetyltransferase GCN5 modulates the retrograde response and genome stability determining yeast longevity. Biogerontology. 2004; 5:305–16. 10.1007/s10522-004-2568-x. [DOI] [PubMed] [Google Scholar]
• 65. John S, Howe L, Tafrov ST et al. The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)–FACT complex. Genes Dev. 2000; 14:1196–208. 10.1101/gad.14.10.1196. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 66. Eberharter A, Sterner DE, Schieltz D et al. The ADA complex is a distinct histone acetyltransferase complex in Saccharomyces cerevisiae. Mol Cell Biol. 1999; 19:6621–31. 10.1128/MCB.19.10.6621. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 67. McCormick MA, Mason AG, Guyenet SJ et al. The SAGA histone deubiquitinase module controls yeast replicative lifespan via Sir2 interaction. Cell Rep. 2014; 8:477–86. 10.1016/j.celrep.2014年06月03日7. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 68. Lim S, Ahn H, Duan R et al. The Spt7 subunit of the SAGA complex is required for the regulation of lifespan in both dividing and nondividing yeast cells. Mech Ageing Dev. 2021; 196:111480. 10.1016/j.mad.2021.111480. [DOI] [PubMed] [Google Scholar]
• 69. Fine RD, Maqani N, Li M et al. Depletion of limiting rDNA structural complexes triggers chromosomal instability and replicative aging of Saccharomyces cerevisiae. Genetics. 2019; 212:75–91. 10.1534/genetics.119.302047. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 70. Sun Y, Yu R, Guo H-B et al. A quantitative yeast aging proteomics analysis reveals novel aging regulators. GeroScience. 2021; 43:2573–93. 10.1007/s11357-021-00412-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 71. Maltby VE, Martin BJE, Brind’Amour J et al. Histone H3K4 demethylation is negatively regulated by histone H3 acetylation in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 2012; 109:18505–10. 10.1073/pnas.1202070109. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 72. Nakanishi S, Sanderson BW, Delventhal KM et al. A comprehensive library of histone mutants identifies nucleosomal residues required for H3K4 methylation. Nat Struct Mol Biol. 2008; 15:881–8. 10.1038/nsmb.1454. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 73. Turner KM, Deshpande V, Beyter D et al. Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity. Nature. 2017; 543:122–5. 10.1038/nature21356. [DOI] [PMC free article] [PubMed] [Google Scholar]

Supplementary Materials

Data Availability Statement

Original data reported in this paper will be shared at: https://data.mendeley.com/preview/3jj7gf3ttf?a=8c9ac88c-cfb6-4350-ba4d-cd300ff6ef33.