Yaniv Harari

Environmental Stresses Disrupt Telomere Length Homeostasis

Telomeres protect the chromosome ends from degradation and play crucial roles in cellular aging and disease. Recent studies have additionally found a correlation between psychological stress, telomere length, and health outcome in humans. However, studies have not yet explored the causal relationship between stress and telomere length, or the molecular mechanisms underlying that relationship. Using yeast as a model organism, we show that stresses may have very different outcomes: alcohol and acetic acid elongate telomeres, whereas caffeine and high temperatures shorten telomeres. Additional treatments, such as oxidative stress, show no effect. By combining genome-wide expression measurements with a systematic genetic screen, we identify the Rap1/Rif1 pathway as the central mediator of the telomeric response to environmental signals. These results demonstrate that telomere length can be manipulated, and that a carefully regulated homeostasis may become markedly deregulated in opposing directions in response to different environmental cues.

The role of Holliday junction resolvases in the repair of spontaneous and induced DNA damage

DNA double-strand breaks (DSBs) and other lesions occur frequently during cell growth and in meiosis. These are often repaired by homologous recombination (HR). HR may result in the formation of DNA structures called Holliday junctions (HJs), which need to be resolved to allow chromosome segregation. Whereas HJs are present in most HR events in meiosis, it has been proposed that in vegetative cells most HR events occur through intermediates lacking HJs. A recent screen in yeast has shown HJ resolution activity for a protein called Yen1, in addition to the previously known Mus81/Mms4 complex. Yeast strains deleted for both YEN1 and MMS4 show a reduction in growth rate, and are very sensitive to DNA-damaging agents. In addition, we investigate the genetic interaction of yen1 and mms4 with mutants defective in different repair pathways. We find that in the absence of Yen1 and Mms4 deletion of RAD1 or RAD52 have no further effect, whereas additional sensitivity is seen if RAD51 is deleted. Finally, we show that yeast cells are unable to carry out meiosis in the absence of both resolvases. Our results show that both Yen1 and Mms4/Mus81 play important (although not identical) roles during vegetative growth and in meiosis.

Tor Complex 1 Controls Telomere Length by Affecting the Level of Ku

Telomeres are specialized DNA-protein structures at the ends of eukaryotic chromosomes. Telomeric DNA is synthesized by telomerase, which is expressed only at the early stages of development [1, 2]. To become malignant, any cell has to be able to replenish telomeres [3]. Thus, understanding how telomere length is monitored has significant medical implications, especially in the fields of aging and cancer. In yeast, telomerase is constitutively active. A large network of genes participates in controlling telomere length [4-8]. Tor1 and Tor2 (targets of rapamycin [9]) are two similar kinases that regulate cell growth [10]. Both can be found as part of the TOR complex 1 (TORC1 [11]), which coordinates the response to nutrient starvation and is sensitive to rapamycin [12]. The rapamycin-insensitive TOR complex 2 (TORC2) contains only Tor2 and regulates actin cytoskeleton polarization [13]. Here we provide evidence for a role of TORC1 in telomere shortening upon starvation in yeast cells. The TORC1 signal is transduced by the Gln3/Gat1/Ure2 pathway, which controls the levels of the Ku heterodimer, a telomere regulator. We discuss the potential implications for the usage of rapamycin as a therapeutic agent against cancer and the effect that calorie restriction may have on telomere length.

Nature vs nurture: interplay between the genetic control of telomere length and environmental factors.

Telomeres are nucleoprotein structures that cap the ends of the linear eukaryotic chromosomes, thus protecting their stability and integrity. They play important roles in DNA replication and repair and are central to our understanding of aging and cancer development. In rapidly dividing cells, telomere length is maintained by the activity of telomerase. About 400 TLM (telomere length maintenance) genes have been identified in yeast, as participants of an intricate homeostasis network that keeps telomere length constant. Two papers have recently shown that despite this extremely complex control, telomere length can be manipulated by external stimuli. These results have profound implications for our understanding of cellular homeostatic systems in general and of telomere length maintenance in particular. In addition, they point to the possibility of developing aging and cancer therapies based on telomere length manipulation.

Telomere length kinetics assay (TELKA) sorts the telomere length maintenance (tlm) mutants into functional groups

Genome-wide systematic screens in yeast have uncovered a large gene network (the telomere length maintenance network or TLM), encompassing more than 400 genes, which acts coordinatively to maintain telomere length. Identifying the genes was an important first stage; the next challenge is to decipher their mechanism of action and to organize then into functional groups or pathways. Here we present a new telomere-length measuring program, TelQuant, and a novel assay, telomere length kinetics assay, and use them to organize tlm mutants into functional classes. Our results show that a mutant defective for the relatively unknown MET7 gene has the same telomeric kinetics as mutants defective for the ribonucleotide reductase subunit Rnr1, in charge of the limiting step in dNTP synthesis, or for the Ku heterodimer, a well-established telomere complex. We confirm the epistatic relationship between the mutants and show that physical interactions exist between Rnr1 and Met7. We also show that Met7 and the Ku heterodimer affect dNTP formation, and play a role in non-homologous end joining. Thus, our telomere kinetics assay uncovers new functional groups, as well as complex genetic interactions between tlm mutants.

Regulation of Elg1 activity by phosphorylation

ELG1 is a conserved gene with important roles in the maintenance of genome stability. Elg1s activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its Fanconi Anemia-related mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice and acts as a tumor suppressor in mice and humans. Elg1 encodes a protein that forms an RFC-like complex that unloads the replicative clamp, PCNA, from DNA, mainly in its SUMOylated form. We have identified 2 different regions in yeast Elg1 that undergo phosphorylation. Phosphorylation of one of them, S112, is dependent on the ATR yeast ortholog, Mec1, and probably is a direct target of this kinase. We show that phosphorylation of Elg1 is important for its role at telomeres. Mutants unable to undergo phosphorylation suppress the DNA damage sensitivity of Δrad5 mutants, defective for an error-free post-replicational bypass pathway. This indicates a role of phosphorylation in the regulation of DNA repair. Our results open the way to investigate the mechanisms by which the activity of Elg1 is regulated during DNA replication and in response to DNA damage.

Do long telomeres affect cellular fitness

Telomeres protect the chromosome ends and maintain the genome stability; they, therefore, play important roles in aging and cancer. Despite the wide variability in telomere length among eukaryotes, in all telomerase-expressing cells telomere length is strictly controlled within a very narrow range. In humans, telomeres shorten with age, and it has been proposed that telomere shortening may play a causal role in aging. Using yeast strains with genetically or physiologically generated differences in telomere length, we have explored the question of whether having long telomeres affects telomere function and fitness or cellular lifespan. We found no effect of long telomeres on vegetative cell division, meiosis, or in cellular lifespan. No positive or negative effect on fitness was observed either under stressful conditions.

Genome-wide studies of telomere biology in budding yeast

Telomeres are specialized DNA-protein structures at the ends of eukaryotic chromosomes. Telomeres are essential for chromosomal stability and integrity, as they prevent chromosome ends from being recognized as double strand breaks. In rapidly proliferating cells, telomeric DNA is synthesized by the enzyme telomerase, which copies a short template sequence within its own RNA moiety, thus helping to solve the “end-replication problem”, in which information is lost at the ends of chromosomes with each DNA replication cycle. The basic mechanisms of telomere length, structure and function maintenance are conserved among eukaryotes. Studies in the yeast Saccharomyces cerevisiae have been instrumental in deciphering the basic aspects of telomere biology. In the last decade, technical advances, such as the availability of mutant collections, have allowed carrying out systematic genome-wide screens for mutants affecting various aspects of telomere biology. In this review we summarize these efforts, and the insights that this Systems Biology approach has produced so far.

An anti-checkpoint activity for rif1.

Chromosomal double-strand breaks (DSBs) are among the most severe lesions a cell has to deal with: if left unrepaired, they may lead to cell death or cancer. Thus, efficient mechanisms have evolved that respond to the presence of DSBs. These are collectively called the “DNA damage response” (DDR), or the “DNA damage checkpoint”. As a result of intensive studies by many research groups in several model organisms, the basic mechanisms that respond to DNA damage have been delineated: following the formation of DSBs, the broken ends are resected, exposing single-stranded DNA (ssDNA) which gets covered by Replication Protein A (RPA), eliciting cell cycle arrest through a complex cascade of protein recruitment and phosphorylation in which several kinases take part (reviewed in [1]). The ends of linear eukaryotic chromosomes, called telomeres, resemble DSBs; however, they do not normally elicit the checkpoint: the DNA ends are somehow “hidden” from the checkpoint-activating mechanisms. This is a very important feature, as it prevents continuous cell cycle arrests or inappropriate (and undesirable) repair of the natural chromosome ends. However, the precise mechanism(s) by which telomeres avoid checkpoint activation have remained elusive. In the accompanying paper, Xue et al. [2] identify Saccharomyces cerevisiae Rif1 as an important telomeric factor with an anti-checkpoint role. Yeast telomeres maintain their integrity by the action of three different protein complexes: the CST (Cdc13-Stn1-Ten1) complex, which resembles RPA and binds to the telomeric G-rich single-stranded 3’ end; the Yku70/80 heterodimer, which blocks single-stranded DNA formation specifically in G1 [3]; and the Rap1 protein, which binds the TG-rich telomeric dsDNA and recruits two additional proteins, Rif1 and Rif2, via its C-terminus [4]. The Rif1 and Rif2 proteins seem to have important, yet different, roles in determining the integrity and length of telomeres [4]–[6]. Xue and co-workers [2] have studied the recruitment of several proteins to the telomeres in a strain carrying the temperature-sensitive cdc13-1 allele. In such strains, upon transfer of the cells to the restrictive temperature (e.g., 36°C) telomeres become uncapped and DNA resecting factors such as Sgs1 and Exo1 are recruited, generating ssDNA [7]. The authors followed the recruitment of the various factors, as well as the binding of checkpoint proteins, by chromatin immuno-precipitation (ChIP) at telomeric, subtelomeric, and unrelated sequences after transfer of the cells to the restrictive temperature. As expected, once resection by Sgs1 and Exo1 started, the amount of Rap1 bound to the telomeric sequences diminished (as Rap1 binds dsDNA); however, surprisingly, Rif1 accumulated with the same pattern as that of the DNA processing enzymes. This was true even in strains in which the C-terminus of Rif1 (thought to be essential for its recruitment) was deleted. Thus, Rif1 can associate to resected telomeres independently of Rap1. The presence of Rif1 had a negative effect on the recruitment of the checkpoint sensors RPA, Ddc2ATRIP, Ddc1RAD9, and Rad953BP1: a much higher recruitment of these proteins was seen in strains lacking Rif1 than in the wild type. Moreover, with time after temperature shift, the negative effect of Rif1 was stronger at proximal sites than at the subtelomeric sequences, suggesting that the Rif1 protein itself moves; these effects were not caused by increased ssDNA levels or by changes in the dynamics of resection. Thus, it appears that Rif1 travels with the resection machinery at telomeres, preventing the local activation of the checkpoint by interfering with the recruitment of RPA and checkpoint sensors (Figure 1). Rif1 seems to act by de-sensitizing cells to the presence of ssDNA: whereas cdc13-1 RIF1+/RIF1-CΔ cells respond to the presence of ssDNA when its level reaches 6%–10% (at 27°C) but not at low ssDNA levels (e.g., at 25°C), cdc13-1 rif1Δ cells already arrest in the cell cycle in the presence of only 2% ssDNA (at 25°C). Figure 1 Rif1 works as an anti-checkpoint protein. If Rif1 sets the threshold for the DDR, then overexpression of the protein might elevate the threshold: indeed, cdc13-1 cells overexpressing Rif1 were able to grow at 29°C, an effect similar to the one obtained by deleting checkpoint components such as RAD24RAD17 and RAD17RAD9 [8]. Thus, Rif1 over-expression has the same effect as a checkpoint knockout, abrogating cell cycle arrest. Moreover, increasing expression of Rif1 in cdc13-1 cells already arrested at the restrictive temperature allowed them to exit the cell cycle arrest, demonstrating that Rif1 can out-compete the checkpoint proteins already present at the telomeres and extinguish an ongoing checkpoint response. Interestingly, this effect was telomere specific, as no anti-checkpoint effect could be seen associated with non-telomeric-induced DSBs. Some time ago Weinert and colleagues [9] showed that the presence of a telomeric tract close to an artificial DSB gradually turned off the DDR elicited by the DSB. The molecular nature of this anti-checkpoint effect was not clear at the time, but the Rif1 protein seems to fit all the requirements for such an anti-checkpoint factor: it is specific for telomeres, acts in cis, and does not affect the resection or the repair of the broken ends. The identification of Rif1 as an anti-checkpoint factor is a huge step forward; however, many questions remain: If Rif1 activity is independent of Rap1, what is the mechanism of its recruitment? Unlike its vertebrate ortholog, the yeast Rif1 lacks a C-terminal DNA-binding domain [10]. Does yeast Rif1 require an additional factor for binding? Does it move with the DNA-resection machinery by being somehow linked to it? Interestingly, the vertebrate Rif1 protein was shown to interact with DNA and with the BLM protein (the ortholog of yeast’s Sgs1) [10]. An intriguing hypothesis is that Rif1 may be bound to Rap1 at normal telomeres; when telomeres become uncapped, the resection machinery may advance along the chromosome, dislodging Rap1 and concomitantly recruiting Rif1. What then is the role of Rif2? Genetic analysis has shown that its role is independent of Rif1 in determining telomere length [4]. Finally, what is the mechanism by which Rif1 can turn off an ongoing checkpoint response? An attractive idea proposed by Xue et al. [2] is that Rif1 may help recruit phosphatases to de-phosphorylate the central checkpoint kinases. Interestingly, mammalian Rif1 was thought to function differently from yeast Rif1, as it can be found at non-telomeric locations and does not co-localize with Rap1 at normal telomeres [11]. The data presented here, however, suggest that Rif1 activity in yeast is independent of Rap1 and that yeast and mammalian proteins may share more features than originally thought. Remarkably, Rif1 expression is elevated in human breast tumors, and its expression status is also positively correlated with differentiation degrees of invasive ductal carcinoma of the breast [12]. If the anti-checkpoint role of Rif1 is conserved in mammalian cells, the increased levels of Rif1 may artificially increase the threshold for ssDNA recognition, allowing cells to continue their proliferation in the presence of unrepaired DNA damage without eliciting the DDR.

Role of the ESCRT complexes in telomere biology

ABSTRACT Eukaryotic chromosomal ends are protected by telomeres from fusion, degradation, and unwanted double-strand break repair events. Therefore, telomeres preserve genome stability and integrity. Telomere length can be maintained by telomerase, which is expressed in most human primary tumors but is not expressed in the majority of somatic cells. Thus, telomerase may be a highly relevant anticancer drug target. Genome-wide studies in the yeast Saccharomyces cerevisiae identified a set of genes associated with telomere length maintenance (TLM genes). Among the tlm mutants with short telomeres, we found a strong enrichment for those affecting vacuolar and endosomal traffic (particularly the endosomal sorting complex required for transport [ESCRT] pathway). Here, we present our results from investigating the surprising link between telomere shortening and the ESCRT machinery. Our data show that the whole ESCRT system is required to safeguard proper telomere length maintenance. We propose a model of impaired end resection resulting in too little telomeric overhang, such that Cdc13 binding is prevented, precluding either telomerase recruitment or telomeric overhang protection. IMPORTANCE Telomeres are the ends of eukaryotic chromosomes. They are necessary for the proper replication of the genome and protect the chromosomes from degradation. In a large-scale systematic screen for mutants that affect telomere length in yeast, we found that mutations in any of the genes encoding the ESCRT complexes, required for the formation of transport vesicles within the cell, cause telomere shortening. We carried out an analysis of the mechanisms disrupted in these mutants and found that they are defective for the ability to elongate short telomeres, probably due to faulty end processing. We discuss the significance of these findings and how they could be relevant to anticancer therapies. Telomeres are the ends of eukaryotic chromosomes. They are necessary for the proper replication of the genome and protect the chromosomes from degradation. In a large-scale systematic screen for mutants that affect telomere length in yeast, we found that mutations in any of the genes encoding the ESCRT complexes, required for the formation of transport vesicles within the cell, cause telomere shortening. We carried out an analysis of the mechanisms disrupted in these mutants and found that they are defective for the ability to elongate short telomeres, probably due to faulty end processing. We discuss the significance of these findings and how they could be relevant to anticancer therapies.