The PIAS homologue Siz2 regulates perinuclear telomere position and telomerase activity in budding yeast

Budding yeast telomeres are reversibly bound at the nuclear envelope through two partially redundant pathways that involve the Sir2/3/4 silencing complex and the Yku70/80 heterodimer. To better understand how this is regulated, we studied the role of SUMOylation in telomere anchoring. We find that the PIAS-like SUMO E3 ligase Siz2 sumoylates both Yku70/80 and Sir4 in vivo and promotes telomere anchoring to the nuclear envelope. Remarkably, loss of Siz2 also provokes telomere extension in a telomerase-dependent manner that is epistatic with loss of the helicase Pif1. Consistent with our previously documented role for telomerase in anchorage, normal telomere anchoring in siz2 Δ is restored by PIF1 deletion. By live-cell imaging of a critically short telomere, we show that telomeres shift away from the nuclear envelope when elongating. We propose that SUMO-dependent association with the nuclear periphery restrains bound telomerase, whereas active elongation correlates with telomere release.     

Cdc13 telomere capping decreases Mec1 association but does not affect Tel1 association with DNA ends

Chromosome ends, known as telomeres, have to be distinguished from DNA breaks that activate DNA damage checkpoint. Two large protein kinases, ataxia-teleangiectasia mutated (ATM) and ATM-Rad3-related (ATR), control not only checkpoint activation but also telomere length. In budding yeast, Mec1 and Tel1 correspond to ATR and ATM, respectively. Here, we show that Cdc13-dependent telomere capping attenuates Mec1 association with DNA ends. The telomeric TG repeat sequence inhibits DNA degradation and decreases Mec1 accumulation at the DNA end. The TG-mediated degradation block requires binding of multiple Cdc13 proteins. The Mre11-Rad50-Xrs2 complex and Exo1 contribute to DNA degradation at DNA ends. Although the TG sequence impedes Exo1 association with DNA ends, it allows Mre11 association. Moreover, the TG sequence does not affect Tel1 association with the DNA end. Our results suggest that the Cdc13 telomere cap coordinates Mec1 and Tel1 accumulation rather than simply covering the DNA ends at telomeres.     

Chromosome looping in yeast: telomere pairing and coordinated movement reflect anchoring efficiency and territorial organization

Long-range chromosome organization is known to influence nuclear function. Budding yeast centromeres cluster near the spindle pole body, whereas telomeres are grouped in five to eight perinuclear foci. Using live microscopy, we examine the relative positions of right and left telomeres of several yeast chromosomes. Integrated lac and tet operator arrays are visualized by their respective repressor fused to CFP and YFP in interphase yeast cells. The two ends of chromosomes 3 and 6 interact significantly but transiently, forming whole chromosome loops. For chromosomes 5 and 14, end-to-end interaction is less frequent, yet telomeres are closer to each other than to the centromere, suggesting that yeast chromosomes fold in a Rabl-like conformation. Disruption of telomere anchoring by deletions of YKU70 or SIR4 significantly compromises contact between two linked telomeres. These mutations do not, however, eliminate coordinated movement of telomere (Tel) 6R and Tel6L, which we propose stems from the territorial organization of yeast chromosomes.     

Reciprocal association of the budding yeast ATM-related proteins Tel1 and Mec1 with telomeres in vivo

The phosphoinositide (PI)-3-kinase-related kinase (PIKK) family proteins Tel1p and Mec1p have been implicated in the telomere integrity of Saccharomyces cerevisiae. However, the mechanism of PIKK-mediated telomere length control remains unclear. Here, we show that Tel1p and Mec1p are recruited to the telomeres at specific times in the cell cycle in a mutually exclusive manner. In particular, Mec1p interacts with the telomeres during late S phase and is associated preferentially with shortened telomeres. We propose a model in which telomere integrity is maintained by the reciprocal association of PIKKs, and Mec1p acts as a sensor for structural abnormalities in the telomeres. Our study suggests a mechanistic similarity between telomere length regulation and DNA double-strand break repair, both of which are achieved by the direct association of PIKKs.     

Mammalian telomere biology

The review considers the function of the important chromosome regions telomeres in normal and immortal cells. Telomeres are dynamic nucleoprotein structures that cap the ends of eukaryotic chromosomes, protecting them from degradation and end-to-end fusion. The functional state of telomeres depends on many interrelated parameters such as telomerase activity, the status of the telomere safety complex shelterin, and telomere-associated proteins (replication, recombination, DNA break repair factors, etc.). Special attention is paid to the mechanisms that control the telomere length in normal and immortal cells as well as in cells containing or lacking active telomerase. The features attributed to an alternative telomere length control are analyzed, in particular, in view of a recently discovered additional mechanism of telomere shortening by t-cycle trimming. The possibility of expressing both telomerase-dependent and recombinational pathways of telomere length control in normal mammalian cells is considered, as well as the role of shelterin proteins in choosing one of them to be dominant. The review additionally discusses the role of telomeres in the spatial organization of the nucleus during mitosis and meiosis and specific telomere organizations in mammals, including Iberian shrews with their unusual or rare chromosome structures.     

Sudden telomere lengthening triggers a Rad53-dependent checkpoint in Saccharomyces cerevisiae

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.     

Inositol diphosphate signaling regulates telomere length

Activation of phospholipase C-dependent inositol polyphosphate signaling pathways generates distinct messengers derived from inositol 1,4,5-trisphosphate that control gene expression and mRNA export. Here we report the regulation of telomere length by production of a diphosphorylinositol tetrakisphosphate, PP-IP4, synthesized by the KCS1 gene product. Loss of PP-IP4 production results in lengthening of telomeres, whereas overproduction leads to their shortening. This effect requires the presence of Tel1, the yeast homologue of ATM, the protein mutated in the human disease ataxia telangiectasia. Our data provide in vivo evidence of a regulatory link between inositol polyphosphate signaling and the checkpoint kinase family and describe a third nuclear process modulated by phospholipase C activation.     

Tel1p preferentially associates with short telomeres to stimulate their elongation

In many organisms, telomeric DNA consists of long tracts of short repeats. Shorter tracts are preferentially lengthened by telomerase, suggesting a conserved mechanism that recognizes and elongates short telomeres. Tel1p, an ATM family checkpoint kinase, plays an important role in telomere elongation, as cells lacking Tel1p have short telomeres and show reduced recruitment of telomerase components to telomeres. We show that Tel1p association increased as telomeres shortened in vivo in the presence or absence of telomerase and that Tel1p preferentially associated with the shortest telomeres. Tel1p association was independent of Tel1p kinase activity and enhanced by Mre11p. Tel1p overexpression simultaneously stimulated telomerase-mediated elongation and Tel1p association with all telomeres. Thus, Tel1p preferentially associates with the shortest telomeres and stimulates their elongation by telomerase.     

Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition

The budding yeast Cdc13, Stn1 and Ten1 (CST) proteins are proposed to function as an RPA-like complex at telomeres that protects (‘caps'') chromosome ends and regulates their elongation by telomerase. We show that Stn1 has a critical function in both processes through the deployment of two separable domains. The N terminus of Stn1 interacts with Ten1 and carries out its essential capping function. The C terminus of Stn1 binds both Cdc13 and Pol12, and we present genetic data indicating that the Stn1–Cdc13 interaction is required to limit continuous telomerase action. Stn1 telomere association, similar to that of Cdc13, peaks during S phase. Significantly, the magnitude of Stn1 telomere binding is independent of telomere TG tract length, suggesting that the negative effect of Stn1 on telomerase action might be regulated by a modification of CST activity or structure in cis at individual telomeres. Genetic analysis suggests that the Tel1 kinase exerts an effect in parallel with the Stn1 C terminus to counteract its inhibition of telomerase. These data provide new insights into the coordination of telomere capping and telomerase regulation.     

Telomerase can inhibit the recombination-based pathway of telomere maintenance in human cells

Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.     

S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association

In diverse organisms, the Mre11 complex and phosphoinositide 3-kinase-related kinases (PIKKs), such as Tel1p and Mec1p from S. cerevisiae, are key mediators of DNA repair and DNA damage checkpoints that also function at telomeres. Here, we use chromatin immunoprecipitation (ChIP) to determine if Mre11p, Tel1p, or Mec1p affects telomere maintenance by promoting recruitment of telomerase subunits to S. cerevisiae telomeres. We find that recruitment of Est2p, the catalytic subunit of telomerase, and Est1p, a telomerase accessory protein, was severely reduced in mre11Delta and tel1Delta cells. In contrast, the levels of Est2p and Est1p binding in late S/G2 phase, the period in the cell cycle when yeast telomerase lengthens telomeres, were indistinguishable in wild-type (WT) and mec1Delta cells. These data argue that Mre11p and Tel1p affect telomere length by promoting telomerase recruitment to telomeres, whereas Mec1p has only a minor role in telomerase recruitment in a TEL1 cell.     

Dependence of the regulation of telomere length on the type of subtelomeric repeat in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, chromosomes terminate with approximately 400 bp of a simple repeat poly(TG(1-3)). Based on the arrangement of subtelomeric X and Y' repeats, two types of yeast telomeres exist, those with both X and Y' (Y' telomeres) and those with only X (X telomeres). Mutations that result in abnormally short or abnormally long poly(TG(1-3)) tracts have been previously identified. In this study, we investigated telomere length in strains with two classes of mutations, one that resulted in short poly(TG(1-3)) tracts (tel1) and one that resulted in elongated tracts (pif1, rap1-17, rif1, or rif2). In the tel1 pif1 strain, Y' telomeres had about the same length as those in tel1 strains and X telomeres had lengths intermediate between those in tel1 and pif1 strains. Strains with either the tel1 rap1-17 or tel1 rif2 genotypes had short tracts for all chromosome ends examined, demonstrating that the telomere elongation characteristic of rap1-17 and rif2 strains is Tel1p-dependent. In strains of the tel1 rif1 or tel1 rif1 rif2 genotypes, telomeres with Y' repeats had short terminal tracts, whereas most of the X telomeres had long terminal tracts. These results demonstrate that the regulation of telomere length is different for X and Y' telomeres.     

Importance of TRF1 for functional telomere structure

Telomeres are comprised of telomeric DNA sequences and associated binding molecules. Their structure functions to protect the ends of linear chromosomes and ensure chromosomal stability. One of the mammalian telomere-binding factors, TRF1, localizes telomeres by binding to double-stranded telomeric DNA arrays. Because the overexpression of wild-type and dominant-negative TRF1 induces progressive telomere shortening and elongation in human cells, respectively, a proposed major role of TRF1 is that of a negative regulator of telomere length. Here we report another crucial function of TRF1 in telomeres. In conditional mouse TRF1 null mutant embryonic stem cells, TRF1 deletion induced growth defect and chromosomal instability. Although no clear telomere shortening or elongation was observed in short term cultured TRF1-deficient cells, abnormal telomere signals were observed, and TRF1-interacting telomere-binding factor, TIN2, lost telomeric association. Furthermore, another double-stranded telomeric DNA-binding factor, TRF2, also showed decreased telomeric association. Importantly, end-to-end fusions with detectable telomere signals at fusion points accumulated in TRF1-deficient cells. These results strongly suggest that TRF1 interacts with other telomere-binding molecules and integrates into the functional telomere structure.     

Tel1 kinase and subtelomere-bound Tbf1 mediate preferential elongation of short telomeres by telomerase in yeast

Telomerase enables telomere length homeostasis, exhibiting increasing preference for telomeres as their lengths decline. This regulation involves telomere repeat-bound Rap1, which provides a length-dependent negative feedback mechanism, and the Tel1 and Mec1 kinases, which are positive regulators of telomere length. By analysing telomere elongation of wild-type chromosome ends at single-molecule resolution, we show that in tel1Delta cells the overall frequency of elongation decreases considerably, explaining their short telomere phenotype. At an artificial telomere lacking a subtelomeric region, telomere elongation no longer increases with telomere shortening in tel1Delta cells. By contrast, a natural telomere, containing subtelomeric sequence, retains a preference for the elongation of short telomeres. Tethering of the subtelomere binding protein Tbf1 to the artificial telomere in tel1Delta cells restored preferential telomerase action at short telomeres; thus, Tbf1 might function in parallel to Tel1, which has a crucial role in a TG-repeat-controlled pathway for the activation of telomerase at short telomeres.     

Tel1 and Rif2 oppositely regulate telomere protection at uncapped telomeres in Saccharomyces cerevisiae

It has been well documented that Tel1 positively regulates telomere-end resection by promoting Mre11-Rad50-Xrs2(MRX) activity, while Rif2 negatively regulates telomere-end resection by inhibiting MRX activity. At uncapped telomeres, whether Tel1 or Rif2 plays any role remains largely unknown. In this work, we examined the roles of Tel1 and Rif2 at uncapped telomeres in yku70△ and/or cdc13-1 mutant cells cultured at non-permissive temperature. We found that deletion of TEL1 exacerbates the temperature sensitivity of both yku70△ and cdc13-1 cells. Further epistasis analysis indicated that MRX and Tel1 function in the same pathway in telomere protection. Consistently, TEL1 deletion increases accumulation of Exo1-dependent telomeric single-stranded DNA(ssDNA) at uncapped telomeres, which stimulates checkpoint-dependent cell cycle arrest. Moreover, TEL1 deletion in yku70△ cells facilitates Rad51-dependent Y0 recombination. In contrast, RIF2 deletion in yku70△ cells decreases the accumulation of telomeric ssDNA after 8 h of incubation at the non-permissive temperature of 37℃ and suppresses the temperature sensitivity of yku70△ cells, likely due to the increase of Mre11 association at telomeres.Collectively, our findings indicate that Tel1 and Rif2 regulate telomere protection at uncapped telomeres via their roles in balancing MRX activity in telomere resection.     

Age-related telomere attrition in the human putamen

Age is a major risk factor for neurodegenerative diseases. Shortening of leucocyte telomeres with advancing age, arguably a measure of “biological” age, is a known phenomenon and epidemiologically correlated with age-related disease. The main mechanism of telomere shortening is cell division, rendering telomere length in post-mitotic cells presumably stable. Longitudinal measurement of human brain telomere length is not feasible, and cross-sectional cortical brain samples so far indicated no attrition with age. Hence, age-related changes in telomere length in the brain and the association between telomere length and neurodegenerative diseases remain unknown. Here, we demonstrate that mean telomere length in the putamen, a part of the basal ganglia, physiologically shortens with age, like leukocyte telomeres. This was achieved by using matched brain and leukocyte-rich spleen samples from 98 post-mortem healthy human donors. Using spleen telomeres as a reference, we further found that mean telomere length was brain region-specific, as telomeres in the putamen were significantly shorter than in the cerebellum. Expression analyses of genes involved in telomere length regulation and oxidative phosphorylation revealed that both region- and age-dependent expression pattern corresponded with region-dependent telomere length dynamics. Collectively, our results indicate that mean telomere length in the human putamen physiologically shortens with advancing age and that both local and temporal gene expression dynamics correlate with this, pointing at a potential mechanism for the selective, age-related vulnerability of the nigro-striatal network.     

Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres

Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.