Telomere length measurement-caveats and a critical assessment of the available technologies and tools

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects.     

Inheritance of telomere length in a bird

Telomere dynamics are intensively studied in human ageing research and epidemiology, with many correlations reported between telomere length and age-related diseases, cancer and death. While telomere length is influenced by environmental factors there is also good evidence for a strong heritable component. In human, the mode of telomere length inheritance appears to be paternal and telomere length differs between sexes, with females having longer telomeres than males. Genetic factors, e.g. sex chromosomal inactivation, and non-genetic factors, e.g. antioxidant properties of oestrogen, have been suggested as possible explanations for these sex-specific telomere inheritance and telomere length differences. To test the influence of sex chromosomes on telomere length, we investigated inheritance and sex-specificity of telomere length in a bird species, the kakapo (Strigops habroptilus), in which females are the heterogametic sex (ZW) and males are the homogametic (ZZ) sex. We found that, contrary to findings in humans, telomere length was maternally inherited and also longer in males. These results argue against an effect of sex hormones on telomere length and suggest that factors associated with heterogamy may play a role in telomere inheritance and sex-specific differences in telomere length.     

Telomere regulation: lessons learnt from mice and men,potential opportunities in horses

Telomeres are genetically conserved nucleoprotein complexes located at the ends of chromosomes that preserve genomic stability. In large mammals, somatic cell telomeres shorten with age, owing to the end replication problem and lack of telomere-lengthening events (e.g. telomerase and ALT activity). Therefore, telomere length reflects cellular replicative reserve and mitotic potential. Environmental insults can accelerate telomere attrition in response to cell division and DNA damage. As such, telomere shortening is considered one of the major hallmarks of ageing. Much effort has been dedicated to understanding the environmental perturbations that accelerate telomere attrition and therapeutic strategies to preserve or extend telomeres. As telomere dynamics seem to reflect cumulative cellular stress, telomere length could serve as a biomarker of animal welfare. The assessment of telomere dynamics (i.e. rate of shortening) in conjunction with telomere-regulating genes and telomerase activity in racehorses could monitor long-term animal health, yet it could also provide some unique opportunities to address particular limitations with the use of other animal models in telomere research. Considering the ongoing efforts to optimise the health and welfare of equine athletes, the purpose of this review is to discuss the potential utility of assessing telomere length in Thoroughbred racehorses. A brief review of telomere biology in large and small mammals will be provided, followed by discussion on the biological implications of telomere length and environmental (e.g. lifestyle) factors that accelerate or attenuate telomere attrition. Finally, the utility of quantifying telomere dynamics in horses will be offered with directions for future research.     

Fission yeast telomeres forecast the end of the crisis

Recent years have placed fission yeast at the forefront of telomere research, as this organism combines a high level of conservation with human telomeres and precise genetic manipulability. Here we highlight some of the latest knowledge of fission yeast telomere maintenance and dysfunction, and illustrate how principles arising from fission yeast research are raising novel questions about telomere plasticity and function in all eukaryotes.     

Brh2 and Rad51 promote telomere maintenance in Ustilago maydis,a new model system of DNA repair proteins at telomeres

Recent studies implicate a number of DNA repair proteins in mammalian telomere maintenance. However, because several key repair proteins in mammals are missing from the well-studied budding and fission yeast, their roles at telomeres cannot be modeled in standard fungi. In this report, we explored the dimorphic fungus Ustilago maydis as an alternative model for telomere research. This fungus, which belongs to the phylum Basidiomycota, has a telomere repeat unit that is identical to the mammalian repeat, as well as a constellation of DNA repair proteins that more closely mimic the mammalian collection. We showed that the two core components of homology-directed repair (HDR) in U. maydis, namely Brh2 and Rad51, both promote telomere maintenance in telomerase positive cells, just like in mammals. In addition, we found that Brh2 is localized to telomeres in vivo, suggesting that it acts directly at chromosome ends. We surveyed a series of mutants with DNA repair defects, and found many of them to have short telomeres. Our results indicate that factors involved in DNA repair are probably also needed for optimal telomere maintenance in U. maydis, and that this fungus is a useful alternative model system for telomere research.     

Telomere length predicts survival independent of genetic influences

Telomeres prevent the loss of coding genetic material during chromosomal replication. Previous research suggests that shorter telomere length may be associated with lower survival. Because genetic factors are important for individual differences in both telomere length and mortality, this association could reflect genetic or environmental pleiotropy rather than a direct biological effect of telomeres. We demonstrate through within-pair analyses of Swedish twins that telomere length at advanced age is a biomarker that predicts survival beyond the impact of early familial environment and genetic factors in common with telomere length and mortality. Twins with the shortest telomeres had a three times greater risk of death during the follow-up period than their co-twins with the longest telomere measurements [hazard ratio (RR) = 2.8, 95% confidence interval 1.1–7.3, P  = 0.03].     

Predicted elements of telomere organization and function in Ustilago maydis

Telomeres are specialized caps of nucleoprotein complexes located at the chromosome termini. They consist of short DNA repeats and of an assortment of associated proteins whose function is currently under intense investigation in model systems. These specialized structures protect the linear ends of eukaryotic chromosomes against DNA repair and degradation activities, and serve as the substrate for telomerase, the ribonucleoprotein complex that synthesises the telomere repeats. The pivotal role of the telomeres in the maintenance of cell viability in several model eukaryotes, including humans, greatly promoted research in telomere biology. Studies on telomere structure and function in fungi other than model systems are limited to providing information on the telomeric repeat sequences. Here, we have summarized the current knowledge on the organization of chromosome ends and on the proteins participating in telomere function in model systems including recent information obtained for filamentous fungi. We also describe Ustilago maydis genes that are potential homologs of proteins known from other systems to participate in telomere biology.     

Proximate determinants of telomere length in sand lizards (Lacerta agilis)

Telomeres are repeat sequences of non-coding DNA that cap the ends of chromosomes and contribute to their stability and the genomic integrity of cells. In evolutionary ecology, the main research target regarding these genomic structures has been their role in ageing and as a potential index of age. However, research on humans shows that a number of traits contribute to among-individual differences in telomere length, in particular traits enhancing cell division and genetic erosion, such as levels of free radicals and stress. In lizards, tail loss owing to predation attempts results in a stress-induced shift to a more cryptic lifestyle. In sand lizard (Lacerta agilis) males, telomere length was compromised by tail regrowth in a body size-related manner, so that small males, which already exhibit more cryptic mating tactics, were less affected than larger males. Tail regrowth just fell short of having a significant relationship with telomere length in females, and so did age in males. In females, there was a significant positive relationship between age and telomere length. We conclude that the proximate effect of compromised antipredation and its associated stress seems to have a more pronounced effect in males than in females and that age-associated telomere dynamics differ between the sexes.     

鸡的端粒生物学研究

端粒是线性染色体末端的核蛋白"帽子"结构,其长度由端粒酶来维持。端粒对于维持基因组的稳定、防止细胞衰老和肿瘤发生具有重要的作用。鸡是遗传和发育研究的经典模式动物,随着鸡基因组学研究的不断深入,鸡的端粒和端粒酶研究取得了很大进展。文章综述了近年来鸡的端粒生物学研究进展,并提出了未来的研究方向。     

How will telomeric complex be further contributed to our longevity? — The potential novel biomarkers of telomere complex counteracting both aging and cancer

With the smooth move towards the coming expected clinical reports of anticancer pharmaceutical molecules targeting telomeres and telomerase, and also with the exciting success in the extension of lifespan by regulating telomerase activity without increased onset of oncogenesis in laboratory mouse models (Garcia-Cao et al., 2006; Jaskelioff et al., 2011), we are convinced that targeting telomeres based on telomerase will be a potential approach to conquer both aging and cancer and the idea of longevity seems to be no more mysterious. More interestingly, emerging evidences from clinical research reveal that other telomeric factors, like specifi c telomeric binding proteins and nonspecific telomere associated proteins also show crucial importance in aging and oncogenesis. This stems from their roles in the stability of telomere structure and in the inhibition of DNA damage response at telomeres. Uncapping these proteins from chromosome ends leads to dramatic telomere loss and telomere dysfunction which is more abrupt than those induced by telomerase inactivation. Abnormal expression of these factors results in developmental failure, aging and even oncogenesis evidenced by several experimental models and clinical cases, indicating telomere specifi c proteins and its associated proteins have complimentary roles to telomerase in telomere protection and controlling cellular fate. Thus, these telomeric factors might be potential clinical biomarkers for early detection or even therapeutic targets of aging and cancer. Future studies to elucidate how these proteins function in telomere protection might benefit patients suffering aging or cancer who are not sensitive to telomerase mediation.     

Age-Related Declines and Disease-Associated Variation in Immune Cell Telomere Length in a Wild Mammal

Immunosenescence, the deterioration of immune system capability with age, may play a key role in mediating age-related declines in whole-organism performance, but the mechanisms that underpin immunosenescence are poorly understood. Biomedical research on humans and laboratory models has documented age and disease related declines in the telomere lengths of leukocytes (‘immune cells’), stimulating interest their having a potentially general role in the emergence of immunosenescent phenotypes. However, it is unknown whether such observations generalise to the immune cell populations of wild vertebrates living under ecologically realistic conditions. Here we examine longitudinal changes in the mean telomere lengths of immune cells in wild European badgers (Meles meles). Our findings provide the first evidence of within-individual age-related declines in immune cell telomere lengths in a wild vertebrate. That the rate of age-related decline in telomere length appears to be steeper within individuals than at the overall population level raises the possibility that individuals with short immune cell telomeres and/or higher rates of immune cell telomere attrition may be selectively lost from this population. We also report evidence suggestive of associations between immune cell telomere length and bovine tuberculosis infection status, with individuals detected at the most advanced stage of infection tending to have shorter immune cell telomeres than disease positive individuals. While male European badgers are larger and show higher rates of annual mortality than females, we found no evidence of a sex difference in either mean telomere length or the average rate of within-individual telomere attrition with age. Our findings lend support to the view that age-related declines in the telomere lengths of immune cells may provide one potentially general mechanism underpinning age-related declines in immunocompetence in natural populations.     

Genomic instability and telomere fusion of canine osteosarcoma cells

Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA.     

Preferential extension of short telomeres induced by low extracellular pH

The majority of tumor cells overcome proliferative limit by expressing telomerase. Whether or not telomerase preferentially extends the shortest telomeres is still under debate. When human cancer cells are cultured at neutral pH, telomerase extends telomeres in telomere length-independent manner. However, the microenvironment of tumor is slightly acidic, and it is not yet known how this influences telomerase action. Here, we examine telomere length homeostasis in tumor cells cultured at pHe 6.8. The results indicate that telomerase preferentially extends short telomeres, such that telomere length distribution narrows and telomeres become nearly uniform in size. After growth at pHe 6.8, the expression of telomerase, TRF1, TRF2 and TIN2 decreases, and the abundance of Cajal bodies decreases. Therefore, telomerase are insufficient for extending every telomere and shorter telomeres bearing less shelterin proteins are more accessible for telomerase recruitment. The findings support the ‘protein-counting mechanism’ in which extended and unextended state of telomere is determined by the number of associated shelterin proteins and the abundance of telomerase. Decreased expression of telomerase and preferential extension of short telomeres have important implications for tumor cell viability, and generate a strong rationale for research on telomerase-targeted anti-cancer therapeutics.     

Telomeric DNA sequence and structure following de novo telomere synthesis in Euplotes crassus.

To learn more about the mechanism of de novo telomere synthesis, we have characterized the sequence and structure of newly synthesized telomeres from Euplotes crassus. E. crassus is a particularly useful organism for studying telomere synthesis because millions of telomeres are made in each cell at a well-defined time during the sexual stage of the life cycle. These newly synthesized telomeres are approximately 50 bp longer than mature macronuclear telomeres. We have investigated the structure of the newly synthesized telomeres and have found that they are much more heterogeneous in length than mature telomeres. Most of the heterogeneity is present on the G-rich strand, indicating that the length of this strand is rather loosely controlled. In contrast, the length of the C-rich strand is much less variable, suggesting that synthesis of this strand is the more precisely regulated step in telomere addition. The G-rich strand exhibits variability both in the total number of G4T4 repeats and in the identity of the terminal nucleotide. In most cases, the G-rich strnd extends beyond the C-rich strand to leave a 3' overhang. While the size of this overhang is variable, the median length is 10 nucleotides. This research provides the first detailed picture of a newly synthesized telomere and has allowed us to formulate a model to describe the various steps involved in de novo telomere synthesis.     

Sex differences in telomeres and lifespan

Males and females often age at different rates resulting in longevity 'gender gaps', where one sex outlives the other. Why the sexes have different lifespans is an age-old question, still fiercely debated today. One cellular process related to lifespan, which is known to differ according to sex, is the rate at which the protective telomere chromosome caps are lost. In humans, men have shorter lifespans and greater telomere shortening. This has led to speculation in the medical literature that sex-specific telomere shortening is one cause of sex-specific mortality. However, telomere shortening may be a cause for and/or a consequence of the processes that govern survival, and to infer general principles from single-taxon studies may be misleading. Here, we review recent work on telomeres in a variety of animal taxa, including those with reverse sexual lifespan dimorphism (i.e., where males live longer), to establish whether sex-specific survival is generally associated with sex differences in telomere dynamics. By doing this, we attempt to tease apart the potential underlying causes for sex differences in telomere lengths in humans and highlight targets for future research across all taxa.     

Early life stress and telomere length: Investigating the connection and possible mechanisms

How can adverse experiences in early life, such as maltreatment, exert such powerful negative effects on health decades later? The answer may lie in changes to DNA. New research suggests that exposure to stress can accelerate the erosion of DNA segments called telomeres. Shorter telomere length correlates with chronological age and also disease morbidity and mortality. Thus, telomere erosion is a potential mechanism linking childhood stress to health problems later in life. However, an array of mechanistic, methodological, and basic biological questions must be addressed in order to translate telomere discoveries into clinical applications for monitoring health and predicting disease risk. This paper covers the current state of the science and lays out new research directions.     

Techniques in plant telomere biology

The role model systems have played in understanding telomere biology has been enormous, and understanding has rapidly transferred to human telomere research. Most work using model organisms to study telomerase and nontelomerase-based telomere-maintenance systems has centered on yeasts, ciliates, and insects. But it is now timely to put considerably more effort into plant models for a number of reasons: (i) the rice and Arabidopsis genome sequencing projects make data mining possible; (ii) extensive collections of insertion mutants of Arabidopsis thaliana enable phenotypic effects of protein gene knockouts to be analyzed, including for those genes involved in telomere structure, function (including, for example, in meiosis), and maintenance; and (iii) the variability of plant telomeres is considerable and ranges from the telomerase-mediated synthesis of the Arabidopsis-type (TTTAGGG) and vertebrate-type (TTAGGG) repeats to sequences synthesized by telomerase-independent mechanism(s) that are still to be discovered. Here we describe how the understanding of telomere biology has been advanced by methods used to isolate telomeric sequences and prove that the putative sequences isolated are indeed telomeric. We show how assays designed to prove the activity of telomerase [e.g., telomeric repeat amplification protocol (TRAP)] lead not only to an understanding of telomere structure and function, but also to the understanding of cell activity in development and in the cell cycle. We review how assays designed to reveal protein/protein and protein/nucleic acid interactions promote understanding of the structure and activities of plant telomeres. Together, the data are making significant contributions to telomere biology in general and could have medical implications.     

Sex differences in telomeres and lifespan in Soay sheep: From the beginning to the end

There is tremendous diversity in ageing rates and lifespan not only among taxa but within species, and particularly between the sexes. Women often live longer than men, and considerable research on this topic has revealed some of the potential biological, psychological and cultural causes of sex differences in human ageing and lifespan. However, sex differences in lifespan are widespread in nonhuman animals suggesting biology plays a prominent role in variation in ageing and lifespan. Recently, evolutionary biologists have borrowed techniques from biomedicine to identify whether similar mechanisms causing or contributing to variation in ageing and lifespan in humans and laboratory animals also operate in wild animals. Telomeres are repetitive noncoding DNA sequences capping the ends of chromosomes that are important for chromosomal stability but that can shorten during normal cell division and exposure to stress. Telomere shortening is hypothesized to directly contribute to the ageing process as once telomeres shorten to some length, the cells stop dividing and die. Men tend to have shorter telomeres and faster rates of telomere attrition with age than women, suggesting one possible biological cause of sex differences in lifespan. In this issue of Molecular Ecology, Watson et al. ( 2017 ) show that telomere lengths in wild Soay sheep are similar between females and males near the beginning of life but quickly diverge with age because males but not females showed reduced telomere lengths at older ages. The authors further show that some of the observed sex difference in telomere lengths in old age may be due to male investment in horn growth earlier in life, suggesting that sexually dimorphic allocation to traits involved in sexual selection might underlie sex differences in telomere attrition. This study provides a rare example of how biological mechanisms potentially contributing to sex differences in lifespan in humans may also operate in free‐living animals. However, future studies using a longitudinal approach are necessary to confirm these observations and identify the ultimate and proximate causes of any sex differences in telomere lengths. Collaborations between evolutionary biologists and gerontologists are especially needed to identify whether telomere lengths have a causal role in ageing, particularly in natural conditions, and whether this directly contributes to sex differences in lifespan.