A subtelomeric region affects telomerase-negative replicative senescence in Saccharomyces cerevisiae - PubMed (original) (raw)

A subtelomeric region affects telomerase-negative replicative senescence in Saccharomyces cerevisiae

Pascale Jolivet et al. Sci Rep. 2019.

Abstract

In eukaryotes, telomeres determine cell proliferation potential by triggering replicative senescence in the absence of telomerase. In Saccharomyces cerevisiae, senescence is mainly dictated by the first telomere that reaches a critically short length, activating a DNA-damage-like response. How the corresponding signaling is modulated by the telomeric structure and context is largely unknown. Here we investigated how subtelomeric elements of the shortest telomere in a telomerase-negative cell influence the onset of senescence. We found that a 15 kb truncation of the 7L subtelomere widely used in studies of telomere biology affects cell growth when combined with telomerase inactivation. This effect is likely not explained by (i) elimination of sequence homology at chromosome ends that would compromise homology-directed DNA repair mechanisms; (ii) elimination of the conserved subtelomeric X-element; (iii) elimination of a gene that would become essential in the absence of telomerase; and (iv) heterochromatinization of inner genes, causing the silencing of an essential gene in replicative senescent cells. This works contributes to better delineate subtelomere functions and their impact on telomere biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Figure 1

Experimental system to shorten a single telomere in the cell. The chromosome end containing the 7L telomere (a) is modified in two ways. In control (CTL) cells (b), the last 15 kb of the telomere end are replaced by a construct in which a URA3 marker is flanked by two Flippase Recognition Target (FRT) sites and followed by a wild-type length telomeric tract. (c) In cells able to generate a very short telomere (VST), the URA3 marker is followed by extratelomeric repeats that inhibit the action of telomerase on telomeric repeats in cis. This results in a short terminal telomeric tract. (b,c) Upon telomerase removal and induction of flippase (encoded by FLP1), sequences between the FRT sites are excised in a circle that is diluted out upon successive cell divisions. Remaining telomeres are of wild-type length (b) or very short (c). (d) The wild type 6R chromosome end is modified to generate 6R-CTL (e) and 6R-VST (f) in a manner similar to 7L (a–c). Chromosome maps and annotations are from the Saccharomyces Genome Database and Centre for Genetic Architecture of Complex Traits website (

https://www.le.ac.uk/colleges/medbiopsych/research/gact/resources/yeast-telomeres

) using the sequence of strain S288C as reference. Red and orange colors correspond to genes encoding minimally characterized proteins or dubious/putative proteins, respectively. Note that subtelomeric sequences of strains W303 – used in this work - and S288C – used as reference - 7L diverge in a region spanning Ty5 to YGL262W.

Figure 2

Figure 2

Effect of the subtelomeric region on replicative senescence. 16 telomerase-negative individual spores carrying the telomere 7L-CTL (blue), 7L-VST (red), 6R-CTL (black) or 6R-VST (purple) (see Fig. 1b,c, e,f) were germinated for two days on selective media. Colonies grown on selective plates for 2 days were then resuspended to equal concentrations and 10-fold dilutions were spotted on solid media, grown at 30 °C for 2 days (passage 1). This procedure was repeated twice (passage 2 and 3). (a) Cells from passage 1 were used to prepare DNA and telomere length measurements were performed by telomere-PCR using specific primers amplifying either the 7L or the 6R-derived telomeres. Median telomere length is shown. Error bars correspond to SD. Adjusted p-values were obtained by the Wilcoxon rank-sum test with a false discovery rate correction **p < 0.01 (n = 14, 14, 16 and 9, respectively). Plates were scanned at high resolution (b) and analyzed to obtain a numerical value for each serial dilution set that is related to the intensity of the spots (c). Adjusted p-values were obtained by the Wilcoxon rank-sum test with a false discovery rate correction *p-value < 0.05, **p-value < 0.01 and ***p-value < 0,001. n = 16 for 7L-CTL, 6R-CTL and 6R-VST, n = 15 for 7L-VST. See Supplementary Table 3 for detailed p-values.

Figure 3

Figure 3

The homologous recombination machinery counteracts senescence both in the presence and absence of subtelomeric elements at the VST. (a) The absence of Rad51 accelerates replicative senescence in the presence or absence of subtelomeric elements at the shortest telomere. Quantitative analysis of serial spot assays of senescence for cells carrying 7L-VST or 6R-VST as in Fig. 2c. Adjusted p-values were obtained by the Wilcoxon rank-sum test with a false discovery rate correction *p-value < 0.05, **p-value < 0.01 and ***p-value < 0,001. n = 7, 6, 5, 5 for each strain set respectively. See Supplementary Table 3 for detailed p-values. (b) Rad51 and Rad52 preferentially associate with short telomeres in the presence and absence of subtelomere. For each indicated strain, 7L-CTL, 7L-VST, 6R-CTL or 6R-VST, a mixed population of hundreds of independent telomerase-negative clones was grown for ~30 population doublings after sporulation. Chromatin was immunoprecipitated using primary antibodies against either Rad52 (left panel) or Rad51 (right panel). The association of each protein to 7L, 6R or Y’ telomeres or to the ARO1 locus was quantified by qPCR and the fold increase of telomere enrichment over ARO1 is represented. Two biologically independent experiments are shown.

Figure 4

Figure 4

Subtelomeres affect senescence independently of the X-element. (a) Scheme of the deletion of the15 kb, the terminal 8 kb, and the internal 8–14 kb of the sub-telomere 7L. Annotations are as in Fig. 1a. (b,d,f) Spores of the indicated genotype were spotted in parallel for three serial passages and analyzed as in Fig. 2b,c (c,e,g). Adjusted P-values were obtained by the Wilcoxon rank-sum test with a false discovery rate correction *p-value < 0.05, **p-value < 0.01 and ***p-value < 0,001. n = 8 for each strain set. See Supplementary Table 3 for detailed p-values.

Figure 5

Figure 5

Chromatin is likely unchanged in strains lacking terminal 15 kb of 7L. (a) Genetic elements overexpressed upon methyl methanesulfonate (MMS) treatment, hydroxyurea (HU) treatment, or in tlc1- Δ are indicated with “+” signs, according to,. (b,c) mRNA levels of indicated coding sequences in indicated strains were assessed by qPCR, normalized by ACT1 mRNA and by corresponding levels in TLC1 7L-WT strains. Error bars correspond to SD. *p-value < 0.05 obtained with one-tailed Student T-test. n ≥ 3. (d) Spores of the indicated genotype were spotted in parallel for three serial passages on YPD or YPD complemented with 5 mM Nicotinamide (NAM) and analyzed as in Fig. 2b,c (e). Adjusted P-values were obtained by the Wilcoxon rank-sum test with a false discovery rate correction *p-value < 0.05 and **p-value < 0.01. n = 8 for each strain set. See Supplementary Table 3 for detailed p-values.

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References

    1. Lingner J, Cooper JP, Cech TR. Telomerase and DNA end replication: no longer a lagging strand problem? Science. 1995;269:1533–1534. doi: 10.1126/science.7545310. - DOI - PubMed
    1. Soudet J, Jolivet P, Teixeira MT. Elucidation of the DNA end-replication problem in Saccharomyces cerevisiae. Mol. Cell. 2014;53:954–964. doi: 10.1016/j.molcel.2014.02.030. - DOI - PubMed
    1. Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985;43:405–413. doi: 10.1016/0092-8674(85)90170-9. - DOI - PubMed
    1. Lingner J, et al. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science. 1997;276:561–567. doi: 10.1126/science.276.5312.561. - DOI - PubMed
    1. Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature. 1990;345:458–460. doi: 10.1038/345458a0. - DOI - PubMed

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