Histone depletion prevents telomere fusions in pre-senescent cells

RESEARCH ARTICLE Histone depletion prevents telomere fusions in pre-senescent cells Marta Barrientos-Moreno, Marina Murillo-Pineda, Ana M. Muñoz-Cabello, Fe ´lix Prado* Department of Genome Biology, Andalusian Molecular Biology and Regenerative Medicine Center (CABIMER), CSIC-University of Seville-University Pablo de Olavide, Seville, Spain *[email protected] Abstract Upon telomerase inactivation, telomeres gradually shorten with each cell division until cells enter replicative senescence. In Saccharomyces cerevisiae, the kinases Mec1/ATR and Tel1/ATM protect the genome during pre-senescence by preventing telomere-telomere fusions (T-TFs) and the subsequent genetic instability associated with fusion-bridge-break- age cycles. Here we report that T-TFs in mec1Δtel1Δcells can be suppressed by reducing the pool of available histones. This protection associates neither with changes in bulk telomere length nor with major changes in the structure of subtelomeric chromatin. We show that the absence of Mec1 and Tel1 strongly augments double-strand break (DSB) repair by non-homologous end joining (NHEJ), which might contribute to the high frequency of T-TFs in mec1Δtel1Δcells. However, histone depletion does not prevent telomere fusions by inhibiting NHEJ, which is actually increased in histone-depleted cells. Rather, histone depletion protects telomeres from fusions by homologous recombination (HR), even though HR is proficient in maintaining the proliferative state of pre-senescent mec1Δtel1Δcells. Therefore, HR during pre-senescence not only helps stalled replication forks but also prevents T- TFs by a mechanism that, in contrast to the previous one, is promoted by a reduction in the histone pool and can occur in the absence of Rad51. Our results further suggest that the Mec1-dependent depletion of histones that occurs during pre-senescence in cells without telomerase (tlc1Δ) prevents T-TFs by favoring the processing of unprotected telomeres by Rad51-independent HR. Author summary Telomere shortening upon telomerase inactivation leads to an irreversible cell division arrest known as replicative senescence, which is considered as a tumor suppressor mechanism. Since pre-senescence is critical for tissue homeostasis, cells are endowed with recombination mechanisms that facilitate the replication of short telomeres and prevent premature entry into senescence. Consequently, pre-senescent cells divide with critically short telomeres, which have lost most of their shelterin proteins. The tumor suppressor genes ATR and ATM, as well as their yeast homologs Mec1 and Tel1, prevent telomere fusions during pre-senescence by unknown mechanisms. Here we show that the absence PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 1 / 22 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Barrientos-Moreno M, Murillo-Pineda M, Muñoz-Cabello AM, Prado F (2018) Histone depletion prevents telomere fusions in presenescent cells. PLoS Genet 14(6): e1007407. https://doi.org/10.1371/journal.pgen.1007407 Editor: Jin-Qiu Zhou, Chinese Academy of Sciences, CHINA Received: November 8, 2017 Accepted: May 9, 2018 Published: June 7, 2018 Copyright: ©2018 Barrientos-Moreno et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Funding: MM-P and AMM-C were recipient of a pre-doctoral training grant (FPU) and a Juan de la Cierva post-doctoral grant from the Spanish Ministry of Education. Research was funded by the Andalusian Government (P12-CTS-2270) and the Spanish Ministry of Economy and Competitivity (BFU2012-38171 and BFU2015-63698-P). The funders had no role in study design, data collection of Mec1 and Tel1 strongly augments DSB repair by non-homologous end joining, which might explain the high rate of telomere fusions in mec1Δtel1Δcells. Moreover, we show that a reduction in the pool of available histones prevents telomere fusions in mec1Δtel1Δ cells by stimulating Rad51-independent homologous recombination. Our results suggest that the Mec1-dependent process of histone depletion that accompanies pre-senescence in cells lacking telomerase activity is required to prevent telomere fusions by promoting the processing of unprotected telomeres by recombination instead of non-homologous end joining. Introduction Telomeres are highly specialized nucleoprotein structures that hide the ends of chromosomes from double-strand break (DSB) repair and DNA damage checkpoint activities. In this way, telomeres protect the chromosome ends from fusions and degradations and from eliciting an erroneous DNA damage response. Accordingly, defects in telomere maintenance are linked to cancer and aging [1]. Telomere DNA consists of repeated DNA sequences that end in a 30sin- gle-stranded G-rich tail. To compensate for the natural shortening that telomeres undergo every cell cycle during DNA replication, many cells express a reverse transcriptase, the telomerase, which adds telomere repeats. However, telomerase expression is repressed in many tissues of multicellular organisms, leading to continuous telomere erosion that eventually activates replicative senescence [2]. Premature senescence entry can affect tissue homeostasis [3] and accordingly, cells are endowed with mechanisms to maintain the proliferative state of cells with short telomeres. As a major risk for genome integrity during this time is the instability of critically short telomeres, an essential task during pre-senescence is to protect these telomeres. In sharp contrast to dysfunctional telomeres caused by mutations in the telomeric shelterin proteins, eroded telomeres by physiological shortening are able to repress nonhomologous end joining (NHEJ), thus preventing telomere-telomere fusions (T-TFs) and the subsequent genetic instability associated with fusion-bridge-breakage cycles [4,5]. In yeast, telomeres consist of ~300 bp of TG 1-3 repeats that are covered by 15–20 molecules of Rap1 (repressor activator protein 1) and its partners Rif1 and Rif2, and ~12–15 bases of a G tail that are covered by the Cdc13/Stn1/Ten1 complex. Similar to most organisms, yeast also contains two classes of subtelomeric elements: X elements, which are present at virtually all telomeres, and Y0-elements, which are present in zero to four tandem copies immediately internal to the telomere repeats [6]. In yeast cells that lack telomerase, telomeres progressively shorten with each cell cycle until cells enter replicative senescence with critically short but protected telomeres [7–10]. Mec1 and Tel1 (yeast homologs of the tumor suppressor genes ATR and ATM, respectively) are master checkpoint kinases with specific and redundant roles in many processes related to genome integrity, such as DSB signaling [11,12]. Specifically, Mec1 transduces the signal that activates senescence in cells lacking telomerase when telomeres reach a critical length [13,14]. Telomeres regulate the binding and activity of many DNA repair and checkpoint factors that are essential for telomere maintenance but prevent that these factors process the chromosome ends as DSBs. Thus, Mec1/ATR and Tel1/ATM binding to telomeres is regulated in order to facilitate their role in promoting the recruitment of the telomerase to short telomeres [15–19] without leading to an inadvertent activation of the DNA damage checkpoint signaling [20–22]. The telomerase recruitment function is carried out mainly by Tel1 in wildtype cells, although Mec1 can partially complement this function in the absence of Tel1 [17]. Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 2 / 22 and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Consistently, telomeres are barely affected in mec1Δcells, are very short but stable in tel1Δ cells, and only the lack of both Mec1 and Tel1 leads to short and unstable telomeres and to the activation of replicative senescence [7,8]. However, and in contrast to cells lacking telomerase, mec1Δtel1Δcells accumulate T-TFs, indicating that Mec1 and Tel1 have additional functions in protecting telomeres [9,23,24]. These functions seem to be conserved as inferred from the accumulation of telomere fusions observed in yeast, fly and mammalian cells lacking ATM and ATR [25–28]. Yeast and mammalian cells also share a reduction in the synthesis of histones during presenescence [29,30]. The mechanism of histone reduction has been elucidated in yeast cells lacking the telomerase RNA coding gene (TLC1) [30]. In particular, it has been reported that telomere shortening is accompanied by a relocalization of Rap1 from eroded telomeres to the promoter of hundreds of new genes. A subset of these genes includes the core histone–encoding genes, which are repressed by Rap1 leading to a reduction in the pool of available histones and a loss of histones at Rap1-targeted promoters. Importantly, Rap1 relocalization and histone depletion require Mec1 [30]. In this study, we asked what defects in mec1Δtel1Δcells are due to their inability to reduce the level of histones as compared to tlc1Δcells. We show that an induced reduction in the pool of available histones in mec1Δtel1Δdoes not affect the length of telomeres or the entry into senescence, but prevents T-TFs. This histone depletion–induced protection require a HR mechanism that, in contrast to the one that maintains the proliferative state of mec1Δtel1Δ cells during pre-senescence, can occur in the absence of Rad51. Likewise, cells lacking Tlc1 requires Rad51-independent HR to prevent telomere fusions, opening the possibility that the Mec1-dependent depletion of histones that occurs during pre-senescence protects telomeres from fusions by favoring the repair of unprotected ends by HR rather than NHEJ. Results Histone depletion induction prevents T-TFs in mec1Δtel1Δpre-senescent cells To address the genetic consequences of the inability to reduce the amount of histones during pre-senescence of cells lacking Mec1 and Tel1, we induced a partial depletion of histones in mec1Δtel1Δcells using a previously reported genetic system, in which the sole source of histone H4 is under the control of the doxycycline-inducible tet promoter (t::HHF2) [31]. For this, MEC1/mec1ΔTEL1/tel1ΔHHF1/hhf1ΔHHF2/hhf2Δdiploids transformed with the plasmid p413TARtetH4 were dissected on plates containing rich medium with 5 μg/mL doxycycline and the colonies were streaked on the same medium; a smear of cells from this streak was then restreaked after 3 days, and this step was repeated for several times (each streak involved ~20 generations). Mec1 is essential to maintain the levels of dNTPs during replication and DNA damage, but can be eliminated without affecting viability in cells lacking Sml1, a Mec1-regulated inhibitor of the dNTPs synthesis [32]. Since the intracellular pool of dNTPs has a direct impact on telomere biology [33–35], all analyses were performed in sml1Δstrains except for tlc1Δ, which was compared with its isogenic wild-type strain. Wild-type and mec1Δ tel1Δcells from streak 1 displayed similar levels of histone H4, which dropped two-fold in mec1Δtel1Δt::HHF2 cells (Fig 1A). However, histone levels in mec1Δtel1Δt::HHF2 cells were still higher than those displayed by cells lacking telomerase. This difference was confirmed by chromatin fractionation and western blot (Fig 1B and S1A Fig). To follow senescence, we analyzed cell growth during several streaks from different spores of each genotype. As previously reported, cells lacking Mec1 and Tel1 senesced after 3 to 5 streaks but a small fraction of them survived senescence (S1B and S1C Fig) [7]. This loss of cell Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 3 / 22 Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 4 / 22 viability was less clear in some clones, likely due to the high cell-to-cell heterogeneity in the entry into senescence [36]. Moreover, viability was much lower in mec1Δtel1Δcells than in tlc1Δcells (S1C Fig), consistent with the major functions of Mec1 and Tel1 in genome integrity. Parallel cultures from mec1Δtel1Δt::HHF2 spores from the same diploids also displayed high growth clonal variation with a similar pattern of senescence (S1B Fig) and viability (S1C Fig). However, a more detailed analysis showed that histone depletion slightly rescued the growth defects of mec1Δtel1Δpre-senescent cells (S1D Fig), and this suppression was not associated with an increase in cell viability but with a slight reduction in the doubling time (S1E and S1F Fig). To address whether histone depletion prevents T-TFs in cells lacking Mec1 and Tel1, DNA from liquid cultures from streak 1 was isolated and amplified by semiquantitative PCR to specifically detect T-TFs between chromosomes V and XV as previously reported [9]. Histone depletion largely suppressed T-TFs in mec1Δtel1Δt::HHF2 pre-senescent cells (Fig 1C). To calculate more precisely this effect, the frequency of T-TFs was determined by quantitative PCR (Fig 1D). The frequency of telomere fusions between the right arm of chromosome V and the left arm of chromosome XV was 3.2 ×10 −3 /genome in mec1Δtel1Δcells. Histone depletion suppressed T-TFs in mec1Δtel1Δt::HHF2 an average of ~73 times. Finally, a detailed analysis in pre-senescent (streak S1), senescent (streak S4) and surviving cells (streaks S7 and S10) showed that T-TFs accumulated in mec1Δtel1Δduring pre-senescence but disappeared in the surviving cells, and that histone depletion prevented T-TFs during the whole process (Fig 1E). Indeed, T-TF accumulation in mec1Δtel1Δand protection by histone depletion was observed as early as in cultures inoculated directly from the spore (~25–30 generations) (S2A Fig). Many of the phenotypes associated with partial depletion of histone H4 can be mimicked in cac1Δrtt106Δreplication-coupled chromatin assembly mutants: loss of chromatin integrity and negative supercoiling, replication fork instability, hyper-recombination and chromosome missegregation [37–41]. Thus, we analyzed T-TFs in mec1Δtel1Δcac1Δrtt106Δcells from streak S1 to test whether telomere protection in mec1Δtel1Δt::HHF2 was due to global defects in chromatin assembly. Notably, the absence of Cac1 and Rtt106 did not reduce the amount of T-TFs induced by mec1Δtel1Δ(Fig 1D and 1F), suggesting that the effects of histone depletion in preventing T-TFs are not due to global defects in replication-coupled chromatin assembly. In Drosophila melanogaster, the accumulation of T-TFs in the absence of ATR and ATM can be suppressed by depleting the histone variant H2A.Z, which restores the loading of the HOAP capping protein [42]. Given that histone depletion is likely to reduce the amount of H2A.Z (Htz1 in yeast)-containing nucleosomes, we addressed the effect of the lack of Htz1 in the formation of T-TFs in mec1Δtel1Δcells. The absence of Htz1 did not prevent the formation of T-TFs in mec1Δtel1Δhtz1Δ(Fig 1D and S2B Fig). Interestingly, both mec1Δtel1Δ cac1Δrtt106Δand mec1Δtel1Δhtz1Δcells senesced much earlier (streaks S2-S3) than mec1Δ Fig 1. Histone depletion prevents T-TFs in mec1Δtel1Δpre-senescent cells. (A) Histone H4 levels in mec1Δtel1Δand mec1Δtel1Δt::HHF2cells (sml1Δ background), and wild type and tlc1Δcells from streak 1-derived cultures as determined by western blot. The amount of histone H4 was normalized to the total amount of protein. The average and SEM of the indicated number of independent strains are shown. (B) Histone H4 and Pgk1 levels at chromatin and soluble fractions of the indicated strains from S1-derived cultures. (C) T-TFs in wild type, t::HHF2,mec1Δtel1Δand mec1Δtel1Δt::HHF2(sml1Δbackground) from liquid cultures from streak 1 as determined by semiquantitative PCR. A PCR control without DNA was included (-). A DNA fragment from HIS4 was PCR-amplified as input control. A scheme of the PCR assay to follow the accumulation of T-TFs is shown. (D) T-TF frequency of the indicated strains as determined by quantitative PCR from S1 streaks. The average and SEM are shown, except for mec1Δtel1Δt::HHF2GAL1::HA-RAD52in which the range is indicated, as n = 2. The number of DNA samples from independent strains, which include those used for semiquantitative PCR and telomere length analyses, is shown in parenthesis for each genotype. One (P-values <0.01) and three (P-values <0.0001) asterisks indicate statistically significant difference according to a Student’s t-test (Mann-Whitney). (E) Accumulation of T-TFs during pre-senescence (streak S1), senescence (S4), and post-senescence (S7 and S10). (F) T-TFs in mec1Δtel1Δ,cac1Δrtt106Δand mec1Δ tel1Δ,cac1Δrtt106Δ(sml1Δbackground) from streak 1 biomass as determined by semiquantitative PCR. All analyses were performed with haploid strains (indicated below each genotype) obtained from heterozygous diploids and streaked for several times on rich medium plates. https://doi.org/10.1371/journal.pgen.1007407.g001 Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 5 / 22 tel1Δcells, and surviving cells took longer to appear (S2C and S2D Fig), suggesting that Cac1 and Rtt106, as well as Htz1, are required to delay the entry into senescence. Telomere protection against fusions by histone depletion in mec1Δtel1Δt:: HHF2 is not associated with bulk telomere elongation T-TFs in mec1Δtel1Δcells can be suppressed if telomeres are artificially elongated by expressing a Cdc13-Est2 fusion protein [9]. This might explain the disappearance of T-TFs in postsenescent cells (Fig 1E), in which survival is associated with telomere lengthening [7]. Thus, we asked whether histone depletion increases telomere length in mec1Δtel1Δcells. For a direct comparison, telomere length was analyzed with the same DNA samples used for T-TF analyses. Total DNA was digested with XhoI, run into a gel and hybridized to a Y0-specific probe. This assay generates a broad band (~1.25 kb in wild-type cells) encompassing the telomere fragments from Y0-containing telomeres, and two upper bands (~6.7 and ~5.2 kb) that represent the two sizes of tandemly arranged Y0subtelomeric repeats (Fig 2A) [7,43,44]. Telomere length in t::HHF2 cells was not apparently affected (Fig 2B, top, and S3A Fig), although the analysis of individual chromosomes showed shorter telomeres than those in wild-type cells in some cases (e.g., Fig 3A). As reported, tel1Δbut not mec1Δcells displayed short telomeres, whereas mec1Δtel1Δcells rapidly shortened their telomeres; the mec1Δtel1Δmutant displayed short telomeres as early as ~25–30 generations after dissection of heterozygous diploids (S3B Fig) [7], which showed wild-type telomeres (S3B Fig) and did not accumulate T-TFs (S2A Fig). Telomere length analysis during pre-senescence, senescence and post-senescence showed a subpopulation of cells in some clones with longer Y0-containing telomeres, which were reduced and disappeared as cells entered into senescence; we do not have an explanation for these events, although they do not seem to be related to the accumulation of T-TFs as they appeared both in mec1Δtel1Δand mec1Δtel1Δt::HHF2 cells (see asterisks in Fig 2B, and S3A and S3B Fig). Indeed, this subpopulation was also detected in tel1Δ(S3B Fig). Importantly, histone depletion did not affect bulk telomere length in mec1Δtel1Δt::HHF2 cells as compared to mec1Δtel1Δcells, which were similar from S1 to S10 (Fig 2B, top gel, and S3A and S3B Fig). A similar result was obtained using a TG 1–3 telomere-specific probe, which also detected telomeres that only contain X subtelomeric elements (Fig 2A and 2B, bottom gel, and S3C Fig). These results suggest that telomere protection against fusions by histone depletion in mec1Δ tel1Δt::HHF2 cells is not due to bulk telomere elongation. The telomere-specific probe also showed that the amount of X-only telomeres is reduced in mec1Δtel1Δcells (see arrows in Fig 2B, bottom gel, and S3C Fig). This is a characteristic of type I survivors, which extend telomeres by Y0-element acquisition through Rad51-dependent HR mechanisms [45], although Rad51-independent, Rad59-dependent Y0-acquisition events can also be detected during presenescence in cells lacking telomerase [44]. Accordingly, surviving mec1Δtel1Δcells amplified the Y0elements (5.2 and 6.7 kb bands), whereas telomere length remained as short as in S1 (Fig 2B, top gel) [7]. The acquisition of Y0subtelomeric elements might explain the reduction in T-TFs observed in mec1Δtel1Δsurvivors (Fig 1E) because the T-TF assay is based on a X- only telomere. Histone depletion alters telomeric chromatin in mec1Δtel1Δt::HHF2 but not in tlc1Δpre-senescent cells Next, we wondered whether the accumulation of T-TFs in mec1Δtel1Δcells, and its suppression by histone depletion are associated with specific changes in the structure of the subtelomeric chromatin. To address this, we analyzed nucleosome positioning at the subtelomeric X element of the left telomere of chromosome III (TEL03L) by indirect–end labeling of MNaseI– Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 6 / 22 Fig 2. Histone depletion does not affect bulk telomere length in mec1Δtel1Δcells. (A) Schematic structure of yeast telomeres. The position of the Y0and TG 1-3 probes used in (B) is shown. (B) Telomere length of the strains analyzed in Fig 1E (S1, pre-senescent cells; S4, senescent cells; S7 and S10, surviving cells) as determined by Southern blot of the Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 7 / 22 treated cells from streak 2–derived cultures (Fig 3A). We also analyzed the chromatin structure of the subtelomeric Y0elements because X elements are characterized by low nucleosome density [46]. For this, and due to the lack of specific DNA sequences to follow nucleosome positioning at a single Y0element, global chromatin accessibility was followed with a probe that hybridizes with the long Y0subtelomeric repeats (Fig 3B). Signal profiles from lanes displaying similar MNase digestion were plotted to compare the patterns of MNaseI accessibility. The DNA samples used for T-TFs with either a telomere-proximal Y0(top gel) or a TG 1-3 (bottom gel) probe. Subpopulations of long telomeres and X-only telomeres are indicated with asterisks and arrows, respectively. https://doi.org/10.1371/journal.pgen.1007407.g002 Fig 3. Chromatin analyses of subtelomeric elements in mec1Δtel1Δand tlc1Δpre-senescent cells. Nucleosome positioning at the left telomere of chromosome III (TEL03L) (A) and global nucleosome pattern at the long subtelomeric Y0elements (B) from streak 2-derived cultures of the indicated strains. The profiles of MNaseI accessibility of the indicated lanes (vertical arrow), which display similar MNase digestion, are shown on the right. Subtle (asterisks) and major (arrows) changes in chromatin structure are marked in the profiles. Schemes with the position of nucleosomes from the BamHI site at the Ty5-1 element to the end of TEL03L (A), and the bulk nucleosome pattern of the ~2.3 Kb ClaI-ClaI fragment from the long Y0elements (B) are shown on the left. Note that the analysis in (A) shows nucleosome positioning from a specific site at a single telomere, whereas the analysis in (B) shows the pattern of nucleosomes at and around the probe from all long Y0elements. Ovals indicate the putative nucleosomes inferred from the MNaseI digestion analysis. https://doi.org/10.1371/journal.pgen.1007407.g003 Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 8 / 22 absence of Mec1 and Tel1 did not lead to major changes in the structure of either the subtelomeric elements or the Ty5-1 proximal transposon (Fig 3A and 3B; compare mec1Δtel1Δwith the wild type). Remarkably, chromatin structure was also basically unaltered in tlc1Δcells as compared to the wild type (Fig 3A and 3B; only a subtle modification in the Y0element that was shared by all mutants, and therefore was not associated with T-TFs), despite having reduced histone levels [30]. In line with this result, histone loss seems to be specific for Rap1- targeted promoters in tlc1Δcells [30], suggesting that chromatin assembly is properly regulated under conditions of programmed histone depletion during pre-senescence. In contrast to mec1Δtel1Δand tlc1Δcells, histone depletion caused major changes in the chromatin structure of the Ty5-1 transposon (gain or loss of DNA accessibility sites; marked with arrows) and subtle changes in the Y0and X elements (high background signal and small changes in DNA accessibility sites; marked with asterisks) in both t::HHF2 and mec1Δtel1Δt:: HHF2 cells (Fig 3A and 3B; compare t::HHF2 and mec1Δtel1Δt::HHF2 with the wild type). Finally, we analyzed the same regions in cac1Δrtt106Δand mec1Δtel1Δcac1Δrtt106Δcells. These mutants displayed similar chromatin changes at the Ty5-1 transposon as t::HHF2 and mec1Δtel1Δt::HHF2 cells (Fig 3A; compare the changes marked with arrows in cac1Δrtt106Δ and mec1Δtel1Δcac1Δrtt106Δwith those in t::HHF2 and mec1Δtel1Δt::HHF2), as expected for mutants affected in replication-coupled chromatin assembly. However, subtelomeric Y0 chromatin was much less affected in cac1Δrtt106Δcells than in t::HHF2 cells (Fig 3B; compare the Y0element MNaseI profiles in t::HHF2 and mec1Δtel1Δt::HHF2 with those in cac1Δ rtt106Δand mec1Δtel1Δcac1Δrtt106Δ). Therefore, subtelomeric chromatin changes seem to occur specifically in response to induced histone depletion. Histone depletion and mec1Δtel1Δincrease NHEJ T-TFs in mec1Δtel1Δcells are NHEJ events [9]. To explore the possibility that histone depletion impairs NHEJ, we used an in vivo plasmid-recircularization assay in which the repair of an induced DSB can only occur by NHEJ [47]. For this, S2 pre-senescent cells were transformed with a plasmid linearized at a region with no homology in the yeast genome. Accordingly, cells lacking the NHEJ protein Ku70, but not the recombination protein Rad52 were defective in plasmid recircularization (Fig 4A). Interestingly, whereas the absence of telomerase activity in tlc1Δcells did not affect NHEJ efficiency as compared to wild-type cells, the lack of Mec1 and Tel1 –but not of Mec1 or Tel1 –increased the efficiency of NHEJ repair ~7-fold (Fig 4A), which might explain the high levels of T-TFs in mec1Δtel1Δcells. Remarkably, histone depletion also increased NHEJ efficiency in t::HHF2 (~9-fold), suggesting that not only the absence of Mec1 and Tel1 but also histone depletion inhibit DNA resection, thus increasing NHEJ frequency. The triple mutant mec1Δtel1Δt::HHF2 led to an additive increase (~20-fold) (Fig 4A), suggesting that mec1Δtel1Δand histone-depleted cells affect DNA resection by different mechanisms. Importantly, the fact that mec1Δtel1Δt::HHF2 cells, which have protected telomeres, displayed even higher NHEJ levels than mec1Δtel1Δcells rules out the possibility that histone depletion prevents T-TFs in mec1Δtel1Δt::HHF2 cells by impairing NHEJ. Protection against mec1Δtel1Δ-induced telomere fusions by histone depletion requires homologous recombination A putative protection mechanism against T-TFs might be HR, which competes with NHEJ for DSB processing [48]. In yeast, HR is required to delay senescence early after telomerase inactivation, likely through template switching mechanisms that seem to facilitate the restart of stalled replication forks with the sister chromatid [43–45,49–55]. Thus, a regulated activity of Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 9 / 22 Telomere-telomere fusions analysis Telomere-telomere fusions (T-TFs) were analyzed by semiquantitative and quantitative PCR analyses as reported [9]. Briefly, ~100 ng of Sau3A-treated genomic DNA extracted by standard protocols from asynchronous cultures was PCR-amplified for semiquantitative analyses using a primer from the X element of chromosome XV-L and a primer from the Y0element of chromosome V-R (coordinates 183–207 and 576759–576783, respectively; Stanford Genome Database). A DNA fragment from HIS4 was PCR-amplified as input control. T-TFs and HIS4 were PCR-amplified using 35 or 20 cycles, respectively, under the conditions previously reported. Oligonucleotides for PCR amplification are listed in S2 Table. Quantitative analyses were performed by real-time PCR by using the same amount of Sau3A-treated genomic DNA, the oligonucleotides described above for semiquantitative analyses and the PCR conditions described previously [9]. 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). Telomere length analysis Total DNA from asynchronous cultures was extracted by standard protocols. DNA samples were digested with XhoI and run in 1.2% TBE 1×agarose gels for 15 hours at 2 V/cm. Gels were blotted onto Hybond-XL membranes and hybridized either at 65˚C with a 32 P-labeled PCR fragment containing 600 bp from XhoI to the centromere-distal end of the subtelomeric Y’ element (probe Y0) or at 37˚C with a TG 1–3 oligo labeled at the 50terminus with ATP (γ- 32 P) and T4 polynucleotide kinase (TG 1–3 probe). Oligonucleotides are listed in S2 Table. All signals were quantified in a Fuji FLA5100 with the ImageGauge analysis program. Chromatin analysis by MNaseI digestion Nucleosome positioning at TEL03L was determined by micrococcal nuclease (MNaseI) digestion and indirect end-labelling [31]. MNaseI–treated DNA was digested with BamHI, resolved in a 1.5% agarose gel, blotted onto a HybondTM-XL membrane and probed with a 230-bp 32 P-labeled PCR fragment located at 60 bp from the BamHI site that reveals nucleosome positioning from this restriction site to TEL03L. Chromatin structure of the long subtelomeric Y0 elements was determined by the same method, except that MNaseI–treated DNA was digested with ClaI, and probed with a 517-bp 32 P-labeled fragment located at 412 bp from the centromere-proximal ClaI site that reveals bulk chromatin accessibility (mono-, di-, tri- . . . nucleosomes) at and around the probed region. Oligonucleotides for PCR amplification are listed in S2 Table. MNaseI profiles were generated with the ImageGauge analysis program. Western blot Yeast protein extracts were prepared using the TCA protocol as described [31] and run on a 15% sodium dodecyl sulfate-polyacrylamide gel. Histone H4 and Pgk1 were detected with the rabbit polyclonal ab10158 (Abcam) and the mouse polyclonal 22C5D8 (Invitrogen) primary antibodies, respectively, and either fluorophore-conjugate or peroxidase-conjugate secondary antibodies. Total protein was determined by using the TFX Stain-Free FastCast Acrylamide kit (Biorad) [77]. Bands were visualized and quantified using either the Odyssey infrared Imaging System (Licor) or the ChemiDoc MP image system (Biorad). Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 16 / 22 Chromatin fractionation Chromatin fractionation was performed as described for young yeast cells [71] with some modifications. Samples (30 ml) from mid-log phase cultures were collected by centrifugation, washed with cold 0.1mM Tris pH 9.4, 10mM DTT, and incubated for 15 min in 1 ml of the same buffer on ice. Cells were then washed with cold spheroplasting buffer (20mM Hepes pH 7.4, 1.2mM sorbitol, Roche Complete EDTA free protease inhibitor cocktail) and incubated with 1 ml of the same buffer with 210 μg zymoliase 20T for 1 h at 30˚C. The spheroplasts were collected, washed twice with cold washing buffer (20mM Tris pH 7.4, 20mM KCl, 1M sorbitol, 0.1μM spermine, 0.25μM spermidine, protease inhibitors), and resuspended in 1 ml lysis buffer (20mM Tris pH 7.4, 20mM KCl, 1M sorbitol, 0.1μM spermine, 0.25μM spermidine, 1% Triton X-100, protease inhibitors) for 5 min on ice. An aliquot (80 μl) was removed for the total sample, and the remaining sample was centrifuged for 15 min at 13000 g at 4˚C to separate soluble (supernatant) and chromatin-enriched (pellet) fractions. Each pellet was washed with 0.5 ml cold lysis buffer and resuspended in 80 μl of water, and chromatin, soluble and total samples were mixed with SDS buffer for western blot analyses. Similar volumes were loaded for each sample, and similar cell equivalents of the chromatin and soluble fractions were loaded for the fractionation controls. Statistical analyses Statistical analyses were performed using the Prism software (Graphpad). Numerical data that underlies graphs and statistical analyses are provided in S3 Table. Supporting information S1 Fig. Induced histone depletion hardly affects mec1Δtel1Δcell growth. (A) Histone H4 and Pgk1 levels at chromatin and soluble fractions of the indicated strains from S1-derived cultures. The average and range from two independent experiments is shown on the bottom. (B) Cell growth analysis of wild type, t::HHF2,mec1Δtel1Δand mec1Δtel1Δt::HHF2 cells (sml1Δ background). Strains are indicated in parenthesis. See text for details. (C) Cell viability from S1, S3 and S6-derived cultures of sml1Δmec1Δtel1Δ(strains 43D, 5D and 53D), sml1Δmec1Δ tel1Δt::HHF2 (strains 7D, 8C and 40A) and tlc1Δ(strains 3C, 4B and 5A) cells. (D–F) Cell growth (D), viability (E), and doubling time (F) of wild type, t::HHF2,mec1Δtel1Δand mec1Δ tel1Δt::HHF2 cells (sml1Δbackground) from streak 2-derived cultures. Cell growth analysis in (D) was performed by plating ten-fold serial dilutions from the same number of mid-log phase cells. Cell viability was determined as the frequency of cells from an asynchronous liquid culture able to form colonies. The total amount of cells was counted in a Burker chamber. The average and SEM of three independent strains are plotted. (TIF) S2 Fig. Histone depletion-mediated protection of telomeres in mec1Δtel1Δt::HHF2 is independent of Htz1. (A) T-TFs in mec1Δtel1Δand mec1Δtel1Δt::HHF2 cells (sml1Δback- ground) from spore-inoculated cultures and diploids heterozygous for the indicated markers. The result from four (spores) and two (diploids) independent strains (indicated below each genotype) is shown. (B) T-TFs accumulation in sml1Δmec1Δtel1Δhtz1Δcells from streak 1 biomass from the indicated strains. T-TFs from sml1Δmec1Δtel1Δand sml1Δmec1Δtel1Δt:: HHF2 cells from streak 1 biomass was included as control. (C, D) Cell growth analysis of mec1Δtel1Δhtz1Δcells (C) and mec1Δtel1Δcac1Δrtt106Δcells (D) (sml1Δbackground) from the indicated strains. Diploids heterozygous for those markers were dissected on rich-medium Telomere protection by histone depletion PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007407 June 7, 2018 17 / 22 plates, and cells were streaked for several times on the same medium (S1 to S7). (TIF) S3 Fig. Telomere length analyses in mec1Δtel1Δand mec1Δtel1Δt::HHF2. (A, B) Telomere length of the indicated strains as determined by southern blot using either a telomere-proximal Y0probe (A, B) or a TG 1-3 probe (C). See legend to Fig 2 for more details. (TIF) S4 Fig. Role of HR on telomere fusions and telomere length in mec1Δtel1Δcells. (A) T-TF accumulation in sml1Δmec1Δtel1Δand sml1Δmec1Δtel1Δrad52Δstrains (indicated below each genotype) from streak S1 biomass, as determined by semi-quantitative PCR. (B) HA-Rad52 is not functional. Cell growth was determined for wild-type and GAL1::HA-RAD52 strains in glucose and galactose medium in the absence or presence of MMS at the indicated concentrations. (C) T-TF accumulation in mec1Δtel1Δcells is not associated with changes in bulk telomere length. Telomere length of the indicated strains from streak S1 biomass was determined by probing DNA samples from Fig 4C with a telomere-proximal Y0probe. All samples were run in the same gel. (TIF) S5 Fig. Histone overexpression in tlc1Δcells. (A) Histone H4 levels in mec1Δtel1Δand tlc1Δ cells, and in tlc1Δcells transformed with either p426-H3.4.2A.2B (histone overexpression) or pRS426 (empty vector) from streak 1-derived cultures as determined by western blot. The amount of histone H4 was normalized to the amount of Pgk1. The average and range of 2 independent strains are shown, as well as the image of one the blots. (B) T-TFs in tlc1Δcells transformed with either p426-H3.4.2A.2B (histone overexpression) or pRS426 (empty vector) from streak 1-derived cultures. Similar results were obtained with 8 more spores. tlc1Δstrains were obtained from TLC1/tlc1Δdiploids transformed with the corresponding plasmid. (TIF) S6 Fig. T-TF variability in tlc1Δrad52Δcells is not associated with differences in bulk telomere length. (A, B) T-TF accumulation (A) and telomere length (B) of the indicated strains from S1 biomass, as determined by semiquantitative PCR and southern blot (using a Y0-spe- cific probe), respectively. Total DNA was split into two samples for T-TF and telomere length analyses. Asterisks in (B) indicate subpopulations of long telomeres. (TIF) S1 Table. Saccharomyces cerevisiaestrains used in this study. (DOCX) S2 Table. Oligonucleotides used in this study. (DOCX) S3 Table. Numerical data underlying graphs. 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