Rad59-Facilitated Acquisition of Y′ Elements by Short Telomeres Delays the Onset of Senescence

Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG_1–3-like tracts present between subtelomeric X and Y′ elements, which is followed by BIR-mediated non-reciprocal translocation of Y′ element and terminal TG_1–3 repeats from the donor end onto the shortened telomere. We found that choice of the Y′ donor was not random, since both engineered telomere VII-L and native VI-R acquired Y′ elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y′ translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG_1–3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y′ translocation events taking place during presenescence are genetically separable from Rad51-dependent Y′ amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y′ translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation.     

Impairment of cytotype regulation of P-element activity in Drosophila melanogaster by mutations in the Su(var)205 gene

Cytotype regulation of transposable P elements in the germ line of Drosophila melanogaster is associated with maternal transmission of P elements inserted at the left telomere of the X chromosome. This regulation is impaired in long-term stocks heterozygous for mutations in Suppressor of variegation 205 [Su(var)205], a gene implicated in the control of telomere length. Regulation by TP5, a structurally incomplete P element at the X telomere, is more profoundly impaired than regulation by TP6, a different incomplete P element inserted at the same site in a TAS repeat at the X telomere. Genetic analysis with the TP5 element indicates that its regulatory ability is not impaired in flies whose fathers came directly from a stock heterozygous for a Su(var)205 mutation, even when the flies themselves carry this mutation. However, it is impaired in flies whose grandfathers came from such a stock. Furthermore, this impairment occurs even when the Su(var)205 mutation is not present in the flies themselves or in their mothers. The impaired regulatory ability of TP5 persists for at least several generations after TP5 X chromosomes extracted from a long-term mutant Su(var)205 stock are made homozygous in the absence of the Su(var)205 mutation. Impairment of TP5-mediated regulation is therefore not directly dependent on the Su(var)205 mutation. However, it is characteristic of the six mutant Su(var)205 stocks that were tested and may be related to the elongated telomeres that develop in these stocks. Impairment of regulation by TP5 is also seen in a stock derived from Gaiano, a wild-type strain that has elongated telomeres due to a dominant mutation in the Telomere elongation (Tel) gene. Regulation by TP6 is not impaired in the Gaiano genetic background. The regulatory abilities of the TP5 and TP6 elements are therefore not equally susceptible to the effects of elongated telomeres in the mutant Su(var)205 and Gaiano stocks.     

Effect of Telomere Proximity on Telomere Position Effect,Chromosome Healing,and Sensitivity to DNA Double-Strand Breaks in a Human Tumor Cell Line

The ends of chromosomes, called telomeres, are composed of a DNA repeat sequence and associated proteins, which prevent DNA degradation and chromosome fusion. We have previously used plasmid sequences integrated adjacent to a telomere to demonstrate that mammalian telomeres suppress gene expression, called telomere position effect (TPE). We have also shown that subtelomeric regions are highly sensitive to double-strand breaks, leading to chromosome instability, and that this instability can be prevented by the addition of a new telomere to the break, a process called chromosome healing. We have now targeted the same plasmid sequences to a site 100 kb from a telomere in a human carcinoma cell line to address the effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to double-strand breaks. The results demonstrate a substantial decrease in TPE 100 kb from the telomere, demonstrating that TPE is very limited in range. Chromosome healing was also diminished 100 kb from the telomere, consistent with our model that chromosome healing serves as a repair process for restoring lost telomeres. Conversely, the region 100 kb from the telomere was highly sensitive to double-strand breaks, demonstrating that the sensitive region is a relatively large target for ionizing radiation-induced chromosome instability.Telomeres are composed of a six-base pair repeat sequence and associated proteins that together form a cap to protect the ends of chromosomes and prevent chromosome fusion (6). Telomeres are actively maintained by the enzyme telomerase in human germ line cells but shorten with age in most somatic cells due to the low level of expression of telomerase (12). When a telomere shortens to the point that it is recognized as a double-strand break (DSB), it serves as a signal for replicative cell senescence (13). Human cells that lose the ability to senesce continue to show telomere shortening and eventually enter crisis, which involves increased chromosome fusion, aneuploidy, and cell death (11, 15). An important step that is required for continued division of cancer cells is therefore that they possess the ability to maintain telomeres, not only to avoid senescence but also to avoid chromosome fusion brought on by crisis (11, 25).In addition to their role in protecting the ends of chromosomes, telomeres can also inhibit the expression of nearby genes, called telomere position effect (TPE). TPE has been proposed to have a role in the cellular response to changes in telomere length (26); however, the function of TPE remains unknown. TPE has been extensively studied in Saccharomyces cerevisiae using transgenes integrated near telomeres on truncated chromosomes (1, 2, 22, 47). These studies demonstrated that TPE involves changes in chromatin conformation and is dependent upon both the distance from the telomere and telomere length (55). Subsequent studies of endogenous yeast genes, however, revealed that the influence of TPE on gene expression varies depending on the presence of insulator sequences (18, 45). TPE also occurs in mammalian cells and has been implicated in the loss of expression of genes relocated near telomeres in a variety of human syndromes (9, 16, 28, 58, 59). As in yeast, transgenes located near telomeres have been used to study TPE in the C33-A (32) and HeLa (4) human cervical carcinoma cell lines. We have also studied TPE using transgenes located adjacent to telomeres in mouse embryonic stem (ES) cells, mouse embryo fibroblasts, and transgenic mice (43). However, none of the studies of TPE in mammalian cells has addressed the distance over which TPE extends from the telomere, and so the number of genes whose expression is likely to be affected is not known.The presence of a telomere can also influence the sensitivity of subtelomeric regions to DSBs. We previously demonstrated the sensitivity of subtelomeric regions to DSBs using selectable transgenes and a recognition site for the I-SceI endonuclease that are integrated immediately adjacent to a telomere. Unlike I-SceI-induced DSBs at most locations, which primarily result in small deletions (27, 34, 46, 50), I-SceI-induced DSBs near telomeres commonly result in large deletions, gross chromosome rearrangements (GCRs), and chromosome instability in both mouse ES cells (37) and human tumor cells (65). Therefore, depending on the size of the sensitive region, the combined targets of the subtelomeric regions on all telomeres could contribute significantly to the genomic instability caused by ionizing radiation or other agents that produce DSBs (35). This sensitivity to DSBs may result from a deficiency in DSB repair since regions near telomeres in yeast are deficient in nonhomologous end joining, resulting in an increase in GCRs (48). One possible reason for a deficiency in DSB repair near telomeres is the role of the telomere in preventing chromosome fusion. Telomeric repeat sequences in yeast have been shown to suppress the activation of cell cycle checkpoints in response to DSBs (39). Similarly, the human TRF2 protein, which is required to prevent chromosome fusion, has been demonstrated to inhibit ATM (31), whose activation is instrumental in the repair of DSBs in heterochromatin (20).One mechanism for avoiding the consequences of DSBs near telomeres is through the addition of a new telomere to the site of a DSB, termed chromosome healing (44). Studies in yeast have shown that chromosome healing occurs through the de novo addition of telomeric repeat sequences by telomerase (14, 33, 38). Chromosome healing in S. cerevisiae is inhibited by the 5′-3′ helicase, Pif1 (52), with Pif1-deficient cells showing up to a 1,000-fold increase in chromosome healing (33, 38). The ability of Pif1 to inhibit chromosome healing has been proposed to serve as a mechanism to prevent chromosome healing from interfering with DSB repair (63). Mammalian cells that express telomerase are also capable of performing chromosome healing. We have shown that chromosome healing can also occur following spontaneous telomere loss (17, 49) or DSBs near telomeres in a human cancer cell line (65) or mouse ES cells (19, 54). We have also shown that chromosome healing can prevent the chromosome instability resulting from DSBs near telomeres (19). Because the de novo addition of telomeric repeat sequences has not been observed in mammalian cells at I-SceI-induced DSBs at interstitial sites (27, 34, 46, 50), we have proposed that chromosome healing is inhibited at most locations but serves as an important mechanism for dealing with DSBs near telomeres that would otherwise result in chromosome instability. However, an alternative possibility that has not been ruled out is that chromosome healing also occurs at interstitial sites but that the large terminal deletions that it causes at these sites results in cell death.In the present study, we address several key questions regarding the importance of telomere proximity on TPE, chromosome healing, and sensitivity to DSBs by investigating how telomere proximity affects these processes. The first of these questions involves establishing the distance over which TPE extends from the telomere to gain insights into the numbers of genes that would be affected by changes in TPE. Second, we will investigate whether chromosome healing can occur at a site that is distant from a telomere but in which terminal deletions are known not to be lethal. This will determine for the first time whether chromosome healing is limited to regions near telomeres. Finally, we will investigate the size of the region near a telomere that is sensitive to DSBs, which will address the potential importance of the subtelomeric region as a target for ionizing radiation-induced genomic instability (35). The distance over which a telomere can exert its effects was investigated by comparing TPE, chromosome healing, and the sensitivity to DSBs at a site 100 kb from a telomere with a site immediately adjacent to the same telomere. As a control for the efficiency of generating DSBs at these sites, we have also analyzed the frequency of small deletions, the most common type of I-SceI-induced DNA rearrangement at interstitial sites in mammalian cells (27, 60). Small deletions serve as an excellent internal control for comparing the frequency of other types of rearrangements since we have previously observed a similar frequency of small deletions at telomeric and interstitial sites (65). The results provide important information on the distance over which a telomere can influence TPE, chromosome healing, and the sensitivity to DSBs.     

端粒位置效应与人类衰老

端粒随细胞分裂进行性缩短不但防止了人类肿瘤的发展,而且与人类的衰老密切相关。另外,端粒中存在一种特殊的现象：端粒位置效应,它首先在酵母中发现,表现为靠近端粒序列附近的基因表达因端粒的位置效应而沉默。在人类细胞中也存在端粒位置效应,并且有多种因子参与此效应,它可能对细胞生长停止、肿瘤以及衰老发生时等许多随端粒缩短密切相关基因的程序性表达产生重要作用。     

Construction and behavior of circularly permuted and telocentric chromosomes in Saccharomyces cerevisiae.

We developed techniques that allow us to construct novel variants of Saccharomyces cerevisiae chromosomes. These modified chromosomes have precisely determined structures. A metacentric derivative of chromosome III which lacks the telomere-associated X and Y' elements, which are found at the telomeres of most yeast chromosomes, behaves normally in both mitosis and meiosis. We made a circularly permuted telocentric version of yeast chromosome III whose closest telomere was 33 kilobases from the centromere. This telocentric chromosome was lost at a frequency of 1.6 X 10(-5) per cell compared with a frequency of 4.0 X 10(-6) for the natural metacentric version of chromosome III. An extremely telocentric chromosome whose closet telomere was only 3.5 kilobases from the centromere was lost at a frequency of 6.0 X 10(-5). The mitotic stability of telocentric chromosomes shows that the very high frequency of nondisjunction observed for short linear artificial chromosomes is not due to inadequate centromere-telomere separation.     

spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast.

Telomeres are essential for genome integrity. scRap1 (S. cerevisiae Rap1) directly binds to telomeric DNA and regulates telomere length and telomere position effect (TPE) by recruiting two different groups of proteins to its RCT (Rap1 C-terminal) domain. The first group, Rif1 and Rif2, regulates telomere length. The second group, Sir3 and Sir4, is involved in heterochromatin formation. On the other hand, human TRF1 and TRF2, as well as their fission yeast homolog, Taz1, directly bind to telomeric DNA and negatively regulate telomere length. Taz1 also plays important roles in TPE and meiosis. Human Rap1, the ortholog of scRap1, negatively regulates telomere length and appears to be recruited to telomeres by interacting with TRF2. Here, we describe two novel fission yeast proteins, spRap1 (S. pombe Rap1) and spRif1 (S. pombe Rif1), which are orthologous to scRap1 and scRif1, respectively. spRap1 and spRif1 are independently recruited to telomeres by interacting with Taz1. The rap1 mutant is severely defective in telomere length control, TPE, and telomere clustering toward the spindle pole body (SPB) at the premeiotic horsetail stage, indicating that spRap1 has critical roles in these telomere functions. The rif1 mutant also shows some defects in telomere length control and meiosis. Our results indicate that Taz1 provides binding sites for telomere regulators, spRap1 and spRif1, which perform the essential telomere functions. This study establishes the similarity of telomere organization in fission yeast and humans.     

Limitations of silencing at native yeast telomeres

Silencing at native yeast telomeres, in which the subtelomeric elements are intact, is different from silencing at terminal truncations. The repression of URA3 inserted in different subtelomeric positions at several chromosome ends was investigated. Many ends exhibit very little silencing close to the telomere, while others exhibit substantial repression in limited domains. Silencing at native ends is discontinuous, with maximal repression found adjacent to the ARS consensus sequence in the subtelomeric core X element. The level of repression declines precipitously towards the centromere. Mutation of the ARS sequence or an adjacent Abf1p-binding site significantly reduces silencing. The subtelomeric Y' elements are resistant to silencing along their whole length, yet silencing can be re-established at the proximal X element. Deletion of PPR1, the transactivator of URA3, and SIR3 overexpression do not increase repression or extend spreading of silencing to the same extent as with terminally truncated ends. sir1Delta causes partial derepression at X-ACS, in contrast to the lack of effect seen at terminal truncations. orc2-1 and orc5-1 have no effect on natural silencing yet cause derepression at truncated ends. X-ACS silencing requires the proximity of the telomere and is dependent on SIR2, SIR3, SIR4 and HDF1. The structures found at native yeast telomeres appear to limit the potential of repressive chromatin.     

Chromatin modifiers and recombination factors promote a telomere fold-back structure,that is lost during replicative senescence

Telomeres have the ability to adopt a lariat conformation and hence, engage in long and short distance intra-chromosome interactions. Budding yeast telomeres were proposed to fold back into subtelomeric regions, but a robust assay to quantitatively characterize this structure has been lacking. Therefore, it is not well understood how the interactions between telomeres and non-telomeric regions are established and regulated. We employ a telomere chromosome conformation capture (Telo-3C) approach to directly analyze telomere folding and its maintenance in S. cerevisiae. We identify the histone modifiers Sir2, Sin3 and Set2 as critical regulators for telomere folding, which suggests that a distinct telomeric chromatin environment is a major requirement for the folding of yeast telomeres. We demonstrate that telomeres are not folded when cells enter replicative senescence, which occurs independently of short telomere length. Indeed, Sir2, Sin3 and Set2 protein levels are decreased during senescence and their absence may thereby prevent telomere folding. Additionally, we show that the homologous recombination machinery, including the Rad51 and Rad52 proteins, as well as the checkpoint component Rad53 are essential for establishing the telomere fold-back structure. This study outlines a method to interrogate telomere-subtelomere interactions at a single unmodified yeast telomere. Using this method, we provide insights into how the spatial arrangement of the chromosome end structure is established and demonstrate that telomere folding is compromised throughout replicative senescence.     

Telomere length, chromatin structure and chromosome fusigenic potential

Telomeres are specialized structures at chromosome ends that are thought to function as buffers against chromosome fusion. Several studies suggest that telomere shortening may render chromosomes fusigenic. We used a novel quantitative fluorescence in situ hybridization procedure to estimate telomere length in individual mammalian chromosomes, and G-banding and chromosome painting techniques to determine chromosome fusigenic potential. All analysed Chinese hamster and mouse cell lines exhibited shorter telomeres at short chromosome arms than at long chromosome arms. However, no clear link between short telomeres and chromosome fusigenic potential was observed, i.e. frequencies of telomeric associations were higher in cell lines exhibiting longer telomeres. We speculate that chromosome fusigenic potential in mammalian cell lines may be determined not only by telomere length but also by the status of telomere chromatin structure. This is supported by the observed presence of chromatin filaments linking telomeres in Chinese hamster chromosomes and of multibranched chromosomes oriented end-to-end in the murine severe combined immunodeficient (SCID) cell line. Multibranched chromosomes are the hallmark of the human ICF (Immune deficiency, Centromeric instability, Facial abnormalities) syndrome, characterized by alterations in heterochromatin structure. Received: 13 June 1997; in revised form: 3 August 1997 / Accepted: 4 August 1997     

Telomere dynamics and genome stability in the human pancreatic tumor cell line MIAPaCa-2

Telomeres are specialized structures found at the ends of eukaryotic chromosomes serving as guardians of genome stability. In normal cells telomeres shorten with each cell division, but immortal cells undergoing multiple divisions constantly have to maintain telomere lengths above a critical level. This is accomplished either through expression of telomerase or the alternative recombination pathway (ALT). In the present study, we analyzed telomere dynamics of the telomerase positive human pancreatic tumor cell line MIAPaCa-2. The cells demonstrated genomic instability with a high frequency of chromosomal aberrations resulting in differences between individual karyotypes within the same cell population. The telomeres were short when compared with normal human fibroblasts, and about 39% of the chromosome ends did not have detectable telomere repeats as demonstrated by PNA-FISH. In many cases telomere signals were missing even when sister chromatids were strongly labeled. In addition, we used an internal PNA probe specific for the X chromosome, present in a single copy in these cells, in order to follow telomere dynamics on individual chromatids. High heterogeneity in telomere signals among individual X chromosomes as well as between their sister chromatids suggested sudden and stochastic loss or gain of telomere repeats. Such constant genomic instability often results in apoptosis and death of a fraction of cells present in the culture at all times. We discuss possible molecular mechanisms that may explain this observed telomere heterogeneity and possible adaptive repair mechanisms by which these cells maintain their chromosomes in order to survive such extreme and permanent genomic instability.     

Telomere dynamics in an immortal human cell line.

The integration of transfected plasmid DNA at the telomere of chromosome 13 in an immortalized simian virus 40-transformed human cell line provided the first opportunity to study polymorphism in the number of telomeric repeat sequences on the end of a single chromosome. Three subclones of this cell line were selected for analysis: one with a long telomere on chromosome 13, one with a short telomere, and one with such extreme polymorphism that no distinct band was discernible. Further subcloning demonstrated that telomere polymorphism resulted from both gradual changes and rapid changes that sometimes involved many kilobases. The gradual changes were due to the shortening of telomeres at a rate similar to that reported for telomeres of somatic cells without telomerase, eventually resulting in the loss of nearly all of the telomere. However, telomeres were not generally lost completely, as shown by the absence of polymorphism in the subtelomeric plasmid sequences. Instead, telomeres that were less than a few hundred base pairs in length showed a rapid, highly heterogeneous increase in size. Rapid changes in telomere length also occurred on longer telomeres. The frequency of this type of change in telomere length varied among the subclones and correlated with chromosome fusion. Therefore, the rapid changes in telomere length appeared occasionally to result in the complete loss of telomeric repeat sequences. Rapid changes in telomere length have been associated with telomere loss and chromosome instability in yeast and could be responsible for the high rate of chromosome fusion observed in many human tumor cell lines.     

Taming the tiger by the tail: modulation of DNA damage responses by telomeres

Telomeres are by definition stable and inert chromosome ends, whereas internal chromosome breaks are potent stimulators of the DNA damage response (DDR). Telomeres do not, as might be expected, exclude DDR proteins from chromosome ends but instead engage with many DDR proteins. However, the most powerful DDRs, those that might induce chromosome fusion or cell‐cycle arrest, are inhibited at telomeres. In budding yeast, many DDR proteins that accumulate most rapidly at double strand breaks (DSBs), have important functions in physiological telomere maintenance, whereas DDR proteins that arrive later tend to have less important functions. Considerable diversity in telomere structure has evolved in different organisms and, perhaps reflecting this diversity, different DDR proteins seem to have distinct roles in telomere physiology in different organisms. Drawing principally on studies in simple model organisms such as budding yeast, in which many fundamental aspects of the DDR and telomere biology have been established; current views on how telomeres harness aspects of DDR pathways to maintain telomere stability and permit cell‐cycle division are discussed.     

Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing

The preconditions and early steps of meiotic chromosome pairing were studied by fluorescence in situ hybridization (FISH) with chromosome- specific DNA probes to mouse and human testis tissue sections. Premeiotic pairing of homologous chromosomes was not detected in spermatogonia of the two species. FISH with centromere- and telomere- specific DNA probes in combination with immunostaining (IS) of synaptonemal complex (SC) proteins to testis sections of prepuberal mice at days 4-12 post partum was performed to study sequentially the meiotic pairing process. Movements of centromeres and then telomeres to the nuclear envelope, and of telomeres along the nuclear envelope leading to the formation of a chromosomal bouquet were detected during mouse prophase. At the bouquet stage, pairing of a mouse chromosome-8- specific probe was observed. SC-IS and simultaneous telomere FISH revealed that axial element proteins appear as large aggregates in mouse meiocytes when telomeres are attached to the nuclear envelope. Axial element formation initiates during tight telomere clustering and transverse filament-IS indicated the initiation of synapsis during this stage. Comparison of telomere and centromere distribution patterns of mouse and human meiocytes revealed movements of centromeres and then telomeres to the nuclear envelope and subsequent bouquet formation as conserved motifs of the pairing process. Chromosome painting in human spermatogonia revealed compacted, largely mutually exclusive chromosome territories. The territories developed into long, thin threads at the onset of meiotic prophase. Based on these results a unified model of the pairing process is proposed.