A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus

Coats plus (CP) can be caused by mutations in the CTC1 component of CST, which promotes polymerase α (polα)/primase-dependent fill-in throughout the genome and at telomeres. The cellular pathology relating to CP has not been established. We identified a homozygous POT1 S322L substitution (POT1^CP) in two siblings with CP. POT1^CP induced a proliferative arrest that could be bypassed by telomerase. POT1^CP was expressed at normal levels, bound TPP1 and telomeres, and blocked ATR signaling. POT1^CP was defective in regulating telomerase, leading to telomere elongation rather than the telomere shortening observed in other telomeropathies. POT1^CP was also defective in the maintenance of the telomeric C strand, causing extended 3′ overhangs and stochastic telomere truncations that could be healed by telomerase. Consistent with shortening of the telomeric C strand, metaphase chromosomes showed loss of telomeres synthesized by leading strand DNA synthesis. We propose that CP is caused by a defect in POT1/CST-dependent telomere fill-in. We further propose that deficiency in the fill-in step generates truncated telomeres that halt proliferation in cells lacking telomerase, whereas, in tissues expressing telomerase (e.g., bone marrow), the truncations are healed. The proposed etiology can explain why CP presents with features distinct from those associated with telomerase defects (e.g., dyskeratosis congenita).     

Human XPF controls TRF2 and telomere length maintenance through distinctive mechanisms

XPF-ERCC1, a structure-specific endonuclease, is involved in nucleotide excision repair, crosslink repair and homologous recombination. XPF-ERCC1 is also found to interact with TRF2, a duplex telomeric DNA binding protein. We have previously shown that XPF-ERCC1 is required for TRF2-promoted telomere shortening. However, whether XPF-ERCC1 by itself has a role in telomere length maintenance has not been determined. Here we report that overexpression of XPF induces telomere shortening in XPF-proficient cells whereas XPF complementation suppresses telomere lengthening in XPF-deficient cells. These results suggest that XPF-ERCC1 can function as a negative mediator of telomere length maintenance. In addition, we find that introduction of wild type XPF into XPF-deficient cells leads to over 40% reduction in TRF2 association with telomeric DNA, indicating that XPF-ERCC1 negatively regulates TRF2 binding to telomeric DNA. Furthermore, we show that XPF carrying mutations in the conserved nuclease domain fails to control TRF2 association with telomeric DNA but it is competent for modulating telomere length maintenance. These results imply that XPF-ERCC1 controls TRF2 and telomere length maintenance through two distinctive mechanisms, with the former requiring its nuclease activity. Our results further imply that TRF2 association with telomeres may be deregulated in cells derived from XPF patients.     

Massive telomere loss and telomerase RNA expression in dexamethasone-induced apoptosis in mouse thymocytes

Apoptosis is a well-recognized process of cell death occurring under several physiological and pathological conditions and represents the principal mechanism involved in cell selection in the thymus. Glucocorticoids are well known to stimulate apoptosis in rat thymocytes. However, it is unclear whether the same changes occur after in vivo glucocorticoid treatment in mice. Chromosomal stability and cell viability require a proficient telomeric end-capping function. Cells with critical telomere shortening and telomerase dysfunction undergo increased apoptosis. In turn, the change in telomere function in cells undergoing apoptosis is not fully characterized. In order to investigate this, we studied the changes in thymocytes after dexamethasone administration in BALB/c mice. The loss of normal thymocytes coincided with the appearance of small dense cells with characteristic features of apoptosis including condensed chromatin, internucleosomal DNA cleavage, and a "hypodiploid" peak on flow cytometry, which suggested that dexamethasone-induced thymocyte apoptosis in BALB/c mice could be considered a well-defined experimental model for studying apoptotic processes. Dexamethasone-treated thymocytes exhibited rapid and dynamic loss of telomeric sequences and up-regulation of telomerase RNA as an early event in the apoptotic process. Telomerase activity was unchanged in this event. Thereafter, telomere gain associated with an increase in telomerase activity occurred in the regenerative process of the thymus. These results suggest a role of telomere loss and up-regulation of telomerase RNA as key apoptosis sensors.     

Role of oxidative stress in telomere shortening in cultured fibroblasts from normal individuals and patients with ataxia-telangiectasia

Cells from patients with the autosomal recessive disorder ataxia-telangiectasia (A-T) display accelerated telomere shortening upon culture in vitro. It has been suggested that A-T cells are in a chronic state of oxidative stress, which could contribute to their enhanced telomere shortening. In order to examine this hypothesis, we monitored the changes in telomere length in A-T homozygous, heterozygous and control fibroblasts cultured in vitro under various conditions of oxidative stress using quantitative fluorescent in situ hybridization. Compared with normal cells, the rate of telomere shortening was 1.5-fold increased under 'normal' levels of oxidative stress in A-T heterozygous cells and 2.4-3.2-fold in A-T homozygous cells. Mild chronic oxidative stress induced by hydrogen peroxide increased the rate of telomere shortening in A-T cells but not in normal fibroblasts and the telomere shortening rate decreased in both normal and A-T fibroblasts if cultures were supplemented with the anti-oxidant phenyl-butyl-nitrone. Increased telomere shortening upon oxidative stress in A-T cells was associated with a significant increase in the number of extra-chromosomal fragments of telomeric DNA and chromosome ends without detectable telomere repeats. We propose that the ATM (A-T mutated) protein has a role in the prevention or repair of oxidative damage to telomeric DNA and that enhanced sensitivity of telomeric DNA to oxidative damage in A-T cells results in accelerated telomere shortening and chromosomal instability.     

Telomere-independent reduction of human B lymphocyte: proliferation during long-term culture

Telomeres and telomerase, the telomere lengthening enzyme, have been shown to play a central role in the long-term ability of cells to proliferate and maintain viability. In opposition to transformed cells, normal somatic cells express a low level of telomerase, which results in the gradual shortening of their telomeres after each division and in cell senescence once a critical telomere length is reached. We have tested the hypothesis that shortening of telomeres could limit the expansion of normal human B lymphocytes maintained in long-term culture using a CD40/CD154 system. Measurement of temolerase activity in cell lysates showed a rapid up-regulation of telomerase following the initiation of the culture that was dependent on the CD40 signaling. The high level of telomerase activity and the corresponding long telomere structures remained constant for the 35 day culture period in which a gradual reduction of the cell expansion rate is observed. We conclude that the gradual in vitro senescence of cultured B cells does not correlate with a corresponding loss of telomerase activity and of telomere length. Rather the phenomenon may be related to an intrinsic property of the proliferating B cells to differentiate into Ig-secreting cells.     

Telomere length and telomerase activity: effect of ageing on human NK cells

Telomeres are repeats of TTAGGG sequences located at the end of eukaryotic chromosomes. They are essential for stabilisation and protection of chromosomal ends and for the regulation of cell replicative capacity. Due to the end-replication defect of DNA polymerase, telomeres shorten progressively with each cell division and telomere length may be an indicator of the replicative history of a cell. Compensatory mechanisms for the telomere loss have been identified. The most widely studied one is mediated by telomerase a ribonuclear protein-enzyme complex that synthesise telomeric repeats. In this study we have investigated whether NK cells, derived from a group of old healthy subjects, underwent the modifications of telomere length and telomerase activity observed in other sub-populations of lymphocytes with advancing age. We demonstrated that: (a) telomere shortening occurred and telomerase activity decreased in human NK cells with ageing; (b) the rate of telomere loss was different under and over 80 years of age; (c) similarly to telomere shortening, the modification of telomerase activity was particularly evident in octogenarians; (d) subjects with the most evident modifications of telomeres and telomerase were the oldest and those with increased NK cell numbers.     

Analyses and comparisons of telomerase activity and telomere length in human T and B cells: Insights for epidemiology of telomere maintenance

Telomeres are the DNA–protein complexes that protect the ends of eukaryotic chromosomes. The cellular enzyme telomerase counteracts telomere shortening by adding telomeric DNA. A growing body of literature links shorter telomere length and lower telomerase activity with various age-related diseases and earlier mortality. Thus, leukocyte telomere length (LTL) and telomerase activity are emerging both as biomarkers and contributing factors for age-related diseases. However, no clinical study has directly examined telomerase activity and telomere length in different lymphocyte subtypes isolated from the same donors, which could offer insight into the summary measure of leukocyte telomere maintenance.We report the first quantitative data in humans examining both levels of telomerase activity and telomere length in four lymphocyte subpopulations from the same donors—CD4+, CD8+CD28+ and CD8+CD28? T cells and B cells, as well as total PBMCs—in a cohort of healthy women. We found that B cells had the highest telomerase activity and longest telomere length; CD4+ T cells had slightly higher telomerase activity than CD8+CD28+ T cells, and similar telomere length. Consistent with earlier reports that CD8+CD28? T cells are replicatively senescent cells, they had the lowest telomerase activity and shortest telomere length. In addition, a higher percentage of CD8+CD28? T cells correlated with shorter total PBMC TL (r = ? 0.26, p = 0.05). Interestingly, telomerase activities of CD4+ and CD8+CD28+ T cells from the same individual were strongly correlated (r = 0.55, r < 0.001), indicating possible common mechanisms for telomerase activity regulation in these two cell subtypes. These data will facilitate the understanding of leukocyte aging and its relationship to human health.     

Exploiting the telomere machinery to put the brakes on inflamm-aging

Telomere shortening accompanies mammalian aging in vivo, and the burden of senescent cells with short telomeres and a senescence-associated secretory phenotype (SASP) increases with aging. The release into the cytoplasm and the extracellular vesicle-mediated intercellular exchange of telomeric TTAGGG repeats could exert an anti-inflammatory activity by preventing the activation of the misplaced nucleic acid-sensing pathway. Many pharmacological and genetic strategies have been developed to prevent telomere shortening or to achieve telomere elongation. Recently, it was demonstrated that telomere elongation can be obtained – without genetic manipulation – by culturing mice embryonic stem cells into appropriate media. Based on this observation, we hypothesize that environmental factors could affect the initial length of telomeres by modulating the activity of telomerase during the early stages of pregnancy. Therefore, organisms with longer telomeres could exploit the anti-inflammatory activity of telomeric sequences over an extended time span, eventually delaying the development and progression of age-related diseases.     

Lack of correlation between telomere length and telomerase activity and expression in leukemic cells

The expression of three components of telomerase complex (hTR, hTERT, TP1) along with telomerase activity and telomere length in leukemic cells was investigated. Cells were isolated from peripheral blood and/or bone marrow of children with acute lymphoblastic (ALL) and non-lymphoblastic (ANLL) leukemia. Expression of three components of telomerase as well as telomerase activity was found in all leukemic cells. Chemiluminescent detection of terminal restriction fragments (TRF) from DNA isolated from ALL cells showed variable patterns expressing considerable heterogeneity of telomere length. The ALL cells appeared to have both long and short telomere lengths, in contrast to normal peripheral lymphocytes, which produced limited pattern of TRF. The ANLL cells produced predominantly short telomere pattern despite high telomerase activity and expression. It can be concluded that high telomerase activity and expression in leukemic cells is not always correlated with long telomeres (TRF pattern).     

Alcoholics show reduced telomere length in the oesophagus

Telomeres are repetitive G-rich DNA sequences located at the ends of chromosomes. Chromosomal and genomic instability due to telomere dysfunction plays an important role in carcinogenesis. To study telomere shortening in the oesophageal epithelium of alcoholics, we measured the telomere lengths of basal and parabasal cells in comparison with those of non-alcoholics using Q-FISH and our original software, Tissue Telo, and also assessed histological inflammation. Telomeres in basal cells were significantly shorter in alcoholics than in age-matched normal controls. Prominent histological findings of chronic inflammation were not evident in either alcoholics or non-alcoholics. Our finding that telomeres in the oesophageal epithelium are shorter in alcoholics than in non-alcoholics indicates that telomere shortening may be associated with the frequent occurrence of squamous cell carcinoma in alcoholics. Further studies to clarify the reason for the large annual loss of telomere length with rapid turnover or lower telomerase activity in the oesophageal epithelium of alcoholics will be necessary.     

Telomeres in evolution and evolution of telomeres

This paper examines telomeres from an evolutionary perspective. In the monocot plant order Asparagales two evolutionary switch-points in telomere sequence are known. The first occurred when the Arabidopsis-type telomere was replaced by a telomere based on a repeat motif more typical of vertebrates. The replacement is associated with telomerase activity, but the telomerase has low fidelity and this may have implications for the binding of telomeric proteins. At the second evolutionary switch-point, the telomere and its mode of synthesis are replaced by an unknown mechanism. Elsewhere in plants (Sessia, Vestia, Cestrum) and in arthropods, the telomere “typical” of the group is lost. Probably many other groups with “unusual” telomeres will be found. We question whether telomerase is indeed the original end-maintenance system and point to other candidate processes involving t-loops, t-circles, rolling circle replication and recombination. Possible evolutionary outcomes arising from the loss of telomerase activity in alternative lengthening of telomere (ALT) systems are discussed. We propose that elongation of minisatellite repeats using recombination/replication processes initially substitutes for the loss of telomerase function. Then in more established ALT groups, subtelomeric satellite repeats may replace the telomeric minisatellite repeat whilst maintaining the recombination/replication mechanisms for telomere elongation. Thereafter a retrotransposition-based end-maintenance system may become established. The influence of changing sequence motifs on the properties of the telomere cap is discussed. The DNA and protein components of telomeres should be regarded – as with any other chromosome elements – as evolving and co-evolving over time and responding to changes in the genome and to environmental stresses. We describe how telomere dysfunction, resulting in end-to-end chromosome fusions, can have a profound effect on chromosome evolution and perhaps even speciation.     

Tales of tails: regulation of telomere length and telomerase activity during lymphocyte development, differentiation, activation, and aging

Summary: Telomerase activity and the regulation of telomere length are factors which have been implicated in the control of cellular replication. These variables have been examined during human lymphocyte development, differentiation, activation, and aging. It was found that telomere length of peripheral blood CD4⁺ T cells decreases with age as well as with differentiation from naive to memory cells in vivo , and decreases with cell division in vitro. These results provide evidence that telomere length correlates with lymphocyte replicative history and residual replicative potential. In contrast, telomere length appears to increase during tonsil B-cell differentiation and germinal center (GC) formation in vivo. It was also found that telomerase activity is highly regulated during T-cell development and B-cell differentiation in vivo , with high levels of telomerase activity expressed in thymocytes and GC B cells, and low levels of telomerase activity in resting mature peripheral blood lymphocytes. Finally, resting lymphocytes retain the ability to upregulate telomerase activity upon activation, and this capacity does not appear to decline with age. Although the precise role of telomerase in lymphocyte function remains to be elucidated, telomerase may contribute to protection from telomere shortening in T and B lymphocytes, and may thus play a critical role in lymphocyte development, differentiation and activation. The future study of study telomerase and its regulation of telomere length may enhance our understanding of bow the replicative lifespan is regulated in lymphocytes.     

Effects of telomerase modulation in human hematopoietic progenitor cells

Loss of telomeric repeats has been causally linked to replicative senescence and aging in human cells. In contrast to normal somatic cells, which are telomerase-negative, hematopoietic stem cells have low levels of telomerase, which can be transiently upregulated upon cytokine stimulation. To examine whether ectopic expression of telomerase can overcome telomere erosion in hematopoietic progenitor cells, we overexpressed telomerase in CD34+ and AC133+ cord blood (CB) cells using retroviral vectors containing hTERT, the catalytic component of telomerase. Although the hTERT-transduced CB cells exhibited significantly elevated telomerase activity (approximately 10-fold), the mean telomere length was only increased up to 600 bp, which was in contrast to hTERT-transduced fibroblast cells gaining more than 2-kb telomeric repeats. Moreover, ectopic telomerase activity did not prevent overall telomere shortening, which was in the range of 1.3 kb in serum-free expansion culture. We also blocked endogenous telomerase activity by ectopic expression of dominant-negative hTERT. Whereas CB cells with absent telomerase activity showed reduced absolute numbers of colony-forming cells, we observed increased rates only for burst-forming units erythroid when the enzyme was overexpressed. These results suggest that telomere shortening in human hematopoietic progenitor cells cannot be compensated by increased levels of telomerase alone and is likely to be dependent on other factors, such as telomere binding proteins. Furthermore, telomerase function seems to be directly associated with the proliferative capacity of stem cells and may exert an additional role in lineage differentiation.     

替代性端粒延长机制与端粒

端粒的维持除了可以通过端粒酶的作用外,还可以通过其他途径.这种不依赖端粒酶的延长途径被称为替代性端粒延长机制.替代性端粒延长机制与端粒酶途径之间存在着抑制效应.二者延长端粒的效果不一样,以致其后续效应对肿瘤演变产生不同的影响.对替代性端粒延长机制的理解有助于阐明肿瘤的发生,并对肿瘤的预防和治疗提出新的建议.     

SUMO修饰与端粒维持

端粒(telomere)长度维持在一定的范围内是细胞正常生理功能的一个重要的基础,端粒长度的变化可导致两个截然相反的病理生理过程-癌变和衰老,端粒过长可引起细胞永生化而癌变,端粒进行性缩短则有丝分裂能力下降导致细胞衰老[1].端粒的长度和结构依赖端粒酶的活性及端粒蛋白复合体(shelterin)的调节[2],端粒酶的激活可引起端粒DNA序列增加而延长细胞的寿命.泛素样小分子修饰(small ubiquin-like modifier,SUMO修饰)在不同的端粒维持机制中的作用途径不同,SUMO修饰可激活端粒酶的活性,促进依赖端粒酶合成端粒的途径,SUMO修饰也可促进同源重组途径合成端粒的能力[3],增加端粒的长度,在保持端粒的长度上发挥重要的调节作用.     

The telomere lengthening mechanism in telomerase-negative immortal human cells does not involve the telomerase RNA subunit

According to the telomere hypothesis of senescence, the progressive shortening of telomeres that occurs upon division of normal somatic cells eventually leads to cellular senescence. The immortalisation of human cells is associated with the acquisition of a telomere maintenance mechanism which is usually dependent upon expression of the enzyme telomerase. About one third of in vitro immortalised human cell lines, however, have no detectable telomerase but contain telomeres that are abnormally long. The nature of the alternative telomere maintenance mechanism (referred to as ALT, for Alternative Lengthening of Telomeres) that must exist in these telomerase-negative cells has not been elucidated. It has previously been shown that abnormal lengthening of yeast telomeres may occur due to mutations in the yeast telomerase RNA gene. That this is not the mechanism of the abnormally long telomeres in ALT cell lines was demonstrated by the finding that seven of seven ALT lines have wild-type human telomerase RNA (hTR) sequence, including a novel polymorphism that is present in 30% of normal individuals. We found that two ALT cell lines have no detectable expression of the hTR gene. This shows that the ALT mechanism in these cell lines is not dependent on hTR. Expression of exogenous hTR via infection of these cells with a recombinant hTR-adenovirus vector did not result in telomerase activity, indicating that their lack of telomerase activity is not due to absence of hTR expression. We conclude that the ALT mechanism is not dependent on the expression of hTR, and does not involve mutations in the hTR sequence.      

Molecular basis of telomere syndrome caused by CTC1 mutations

Mutations in CTC1 lead to the telomere syndromes Coats Plus and dyskeratosis congenita (DC), but the molecular mechanisms involved remain unknown. CTC1 forms with STN1 and TEN1 a trimeric complex termed CST, which binds ssDNA, promotes telomere DNA synthesis, and inhibits telomerase-mediated telomere elongation. Here we identify CTC1 disease mutations that disrupt CST complex formation, the physical interaction with DNA polymerase α-primase (polα-primase), telomeric ssDNA binding in vitro, accumulation in the nucleus, and/or telomere association in vivo. While having diverse molecular defects, CTC1 mutations commonly lead to the accumulation of internal single-stranded gaps of telomeric DNA, suggesting telomere DNA replication defects as a primary cause of the disease. Strikingly, mutations in CTC1 may also unleash telomerase repression and telomere length control. Hence, the telomere defect initiated by CTC1 mutations is distinct from the telomerase insufficiencies seen in classical forms of telomere syndromes, which cause short telomeres due to reduced maintenance of distal telomeric ends by telomerase. Our analysis provides molecular evidence that CST collaborates with DNA polα-primase to promote faithful telomere DNA replication.