Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres

In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3' of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3' terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.     

The Rap1p-telomere complex does not determine the replicative capacity of telomerase-deficient yeast

Telomeres are nucleoprotein structures that cap the ends of chromosomes and thereby protect their stability and integrity. In the presence of telomerase, the enzyme that synthesizes telomeric repeats, telomere length is controlled primarily by Rap1p, the budding yeast telomeric DNA binding protein which, through its C-terminal domain, nucleates a protein complex that limits telomere lengthening. In the absence of telomerase, telomeres shorten with every cell division, and eventually, cells enter replicative senescence. We have set out to identify the telomeric property that determines the replicative capacity of telomerase-deficient budding yeast. We show that in cells deficient for both telomerase and homologous recombination, replicative capacity is dependent on telomere length but not on the binding of Rap1p to the telomeric repeats. Strikingly, inhibition of Rap1p binding or truncation of the C-terminal tail of Rap1p in Kluyveromyces lactis and deletion of the Rap1p-recruited complex in Saccharomyces cerevisiae lead to a dramatic increase in replicative capacity. The study of the role of telomere binding proteins and telomere length on replicative capacity in yeast may have significant implications for our understanding of cellular senescence in higher organisms.     

Yeast Est2p affects telomere length by influencing association of Rap1p with telomeric chromatin

In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.     

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.     

Mutant Telomeric Repeats in Yeast Can Disrupt the Negative Regulation of Recombination-Mediated Telomere Maintenance and Create an Alternative Lengthening of Telomeres-Like Phenotype

Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent of human ALT cells. In this study, we generated telomeres composed of either of two types of mutant telomeric repeats, Acc and SnaB, that each alter the binding site for the telomeric protein Rap1. We show here that arrays of both types of mutant repeats present basally on a telomere were defective in negatively regulating telomere length in the presence of telomerase. Similarly, when each type of mutant repeat was spread to all chromosome ends in cells lacking telomerase, they led to the formation of telomeres produced by RTE that were much longer than those seen in cells with only wild-type telomeric repeats. The Acc repeats produced the more severe defect in both types of telomere maintenance, consistent with their more severe Rap1 binding defect. Curiously, although telomerase deletion mutants with telomeres composed of Acc repeats invariably showed extreme telomere elongation, they often also initially showed persistent very short telomeres with few or no Acc repeats. We suggest that these result from futile cycles of recombinational elongation and truncation of the Acc repeats from the telomeres. The presence of extensive 3′ overhangs at mutant telomeres suggests that Rap1 may normally be involved in controlling 5′ end degradation.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication

Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.     

Rap1 affects the length and heterogeneity of human telomeres

Telomere length is controlled in part by cis-acting negative regulators that limit telomere extension by telomerase. In budding yeast, the major telomere length regulator scRap1 binds to telomeric DNA and acts to inhibit telomere elongation in cis. Because the human Rap1 ortholog hRap1 does not bind to telomeric DNA directly but is recruited to telomeres by TRF2, we examined its role in telomere length control. The data are consistent with hRap1 being a negative regulator of telomere length, indicating functional conservation. Deletion mapping confirmed that hRap1 is tethered to telomeres through interaction of its C terminus with TRF2. The telomere length phenotypes of hRap1 deletion mutants implicated both the BRCT and Myb domain as protein interaction domains involved in telomere length regulation. By contrast, scRap1 binds to telomeres with its Myb domains and uses its C terminus to recruit the telomere length regulators Rif1 and Rif2. Together, our data show that although the role of Rap1 at telomeres has been largely conserved, the domains of Rap1 have undergone extensive functional changes during eukaryotic evolution. Surprisingly, hRap1 alleles lacking the BRCT domain diminished the heterogeneity of human telomeres, indicating that hRap1 also plays a role in the regulation of telomere length distribution.     

Telomerase enzymatic component hTERT shortens long telomeres in human cells

Telomere lengths are tightly regulated within a narrow range in normal human cells. Previous studies have extensively focused on how short telomeres are extended and have demonstrated that telomerase plays a central role in elongating short telomeres. However, much about the molecular mechanisms of regulating excessively long telomeres is unknown. In this report, we demonstrated that the telomerase enzymatic component, hTERT, plays a dual role in the regulation of telomere length. It shortens excessively long telomeres and elongates short telomeres simultaneously in one cell, maintaining the optimal telomere length at each chromosomal end for efficient protection. This novel hTERT-mediated telomere-shortening mechanism not only exists in cancer cells, but also in primary human cells. The hTERT-mediated telomere shortening requires hTERT’s enzymatic activity, but the telomerase RNA component, hTR, is not involved in that process. We found that expression of hTERT increases telomeric circular DNA formation, suggesting that telomere homologous recombination is involved in the telomere-shortening process. We further demonstrated that shelterin protein TPP1 interacts with hTERT and recruits hTERT onto the telomeres, suggesting that TPP1 might be involved in regulation of telomere shortening. This study reveals a novel function of hTERT in telomere length regulation and adds a new element to the current molecular model of telomere length maintenance.     

Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.     

铅和硒对端粒长度、端粒酶及端粒结合蛋白的影响

以酿酒酵母细胞为实验材料 ,在分子水平上研究铅 (Pb)和硒 (Se)对端粒长度、端粒酶及端粒结合蛋白的影响。结果发现 :与对照组相比 ,添加 1mg/LPb的培养基中培养 10 0代后的酿酒酵母细胞中端粒DNA的平均长度缩短 ,端粒结合蛋白Rap1p含量减少 ,而且Rap1p蛋白的二级结构受到扰动、端粒酶活性降低、43%的细胞死亡。加 1mg/LSe培养 10 0代后的酿酒酵母细胞与对照组相比 ,细胞中端粒平均长度增加 ,Rap1p蛋白浓度和二级结构保持稳定 ,端粒酶活性增加 ,细胞正常存活。以上结果表明 ,Pb对酿酒酵母细胞端粒有损伤 ,而且其损伤在子代细胞中有累积效应 ;而Se对Pb损伤具有一定程度的修复保护作用 ,适量给机体补Se对抑制细胞损伤和衰老有一定作用。由于端粒的特殊结构特征 ,推断Pb和Se是通过作用于端粒酶及端粒结合蛋白而间接影响端粒长度的     

HOT1 is a mammalian direct telomere repeat‐binding protein contributing to telomerase recruitment

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase–telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC‐based DNA–protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere‐binding protein 1 (HOT1). HOT1 directly and specifically binds double‐stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase‐dependent telomere elongation.     

Telomere Length Dynamics in Telomerase-Positive Immortal Human Cell Populations

It has been proposed that the progressive shortening of telomeres in somatic cells eventually results in senescence. Previous experiments have demonstrated that many immortal cell lines have acquired telomerase activity leading to stabilization of telomere length. Telomere dynamics and telomerase activity were examined in the telomerase-positive immortal cell lines HeLa and 293 and subclones derived from them. A mass culture of HeLa cells had a stable mean telomere length over 60 population doublings (PD)in vitro.Subclones of this culture, however, had a range of mean telomere lengths indicating that telomeric heterogeneity exists within a population with a stable mean telomere length. Some of the subclones lacked detectable telomerase activity soon after isolation but regained it by PD 18, suggesting that at least some of the variation in telomere length can be attributed to variations in telomerase activity levels. 293 subclones also varied in telomere length and telomerase activity. Some telomerase-positive 293 subclones contained long telomeres that gradually shortened, demonstrating that factors other than telomerase also act to modulate telomere length. Fluctuations in telomere length in telomerase-positive immortalized cells may contribute to chromosomal instability and clonal evolution.     

Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro

Although vertebrate telomeres are highly conserved, telomere dynamics and telomerase profiles vary among species. The objective of the present study was to examine telomerase activity and telomere length profiles of transformed and non-transformed avian cells in vitro. Non-transformed chicken embryo fibroblasts (CEFs) showed little or no telomerase activity from the earliest passages through senescence. Unexpectedly, a single culture of particularly long-lived senescent CEFs showed telomerase activity after over 250 days in culture. Transformed avian lines (six chicken, two quail and one turkey) and tumor samples (two chicken) exhibited telomerase activity. Telomere length profiles of non-transformed CEF cultures derived from individual embryos of an inbred line (UCD 003) exhibited cycles of shortening and lengthening with a substantial net loss of telomeric DNA by senescence. The telomere length profiles of several transformed cell lines resembled telomere length profiles of senescent CEFs in that they exhibited little of the typical smear of terminal restriction fragments (TRFs) suggesting that these transformed cells may possess a reduced amount of telomeric DNA. These results show that avian telomerase activity profiles are consistent with the telomerase activity profiles of human primary and transformed cells. Further, monitoring of telomere lengths of primary cells provides evidence for a dynamic series of changes over the lifespan of any specific cell culture ultimately resulting in net telomeric DNA loss by senescence.