Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination

Progressive telomere shortening from cell division (replicative aging) provides a barrier for human tumor progression. This program is not conserved in laboratory mice, which have longer telomeres and constitutive telomerase. Wild species that do/do not use replicative aging have been reported, but the evolution of different phenotypes and a conceptual framework for understanding their uses of telomeres is lacking. We examined telomeres/telomerase in cultured cells from > 60 mammalian species to place different uses of telomeres in a broad mammalian context. Phylogeny‐based statistical analysis reconstructed ancestral states. Our analysis suggested that the ancestral mammalian phenotype included short telomeres (< 20 kb, as we now see in humans) and repressed telomerase. We argue that the repressed telomerase was a response to a higher mutation load brought on by the evolution of homeothermy. With telomerase repressed, we then see the evolution of replicative aging. Telomere length inversely correlated with lifespan, while telomerase expression co‐evolved with body size. Multiple independent times smaller, shorter‐lived species changed to having longer telomeres and expressing telomerase. Trade‐offs involving reducing the energetic/cellular costs of specific oxidative protection mechanisms (needed to protect < 20 kb telomeres in the absence of telomerase) could explain this abandonment of replicative aging. These observations provide a conceptual framework for understanding different uses of telomeres in mammals, support a role for human‐like telomeres in allowing longer lifespans to evolve, demonstrate the need to include telomere length in the analysis of comparative studies of oxidative protection in the biology of aging, and identify which mammals can be used as appropriate model organisms for the study of the role of telomeres in human cancer and aging.     

Telomerase therapeutics for degenerative diseases

Telomerase is active in early embryonic and fetal development but is down-regulated in all human somatic tissues before birth. Since telomerase is virtually absent or only transiently active in normal somatic cells throughout postnatal life, telomere length gradually decreases as a function of age in most human tissues. Although telomerase repression likely evolved as a tumor suppressor mechanism, a growing body of evidence from epidemiology and genetic studies point to a role of telomerase repression and short telomeres in a broad spectrum of diseases: (a) Humans with shorter than average telomere length are at increased risk of dying from heart disease, stroke, or infection; (b) Patients with Dyskeratosis congenita are born with shortened telomeres due to mutations in telomerase components, suffer from a variety of proliferative tissue disorders, and typically die early of bone marrow failure; and (c) Individuals with long-term chronic stress or infections have accelerated telomere shortening compared to age-matched counterparts. Telomerase activation may prove useful in the treatment of diseases associated with telomere loss. While human cells dividing in culture lose telomeric DNA and undergo changes that mirror certain age- or disease-associated changes in vivo, telomerase transduced cells have extended replicative capacities, increased resistance to stress, improved functional activities in vitro and in vivo, and no loss of differentiation capacity or growth control. In addition, telomerase transduction in vivo can prevent telomere dysfunction and cirrhotic changes in liver of telomerase knockout mice. Thus, pharmacological activation of telomerase has significant potential for the treatment of a broad spectrum of chronic or degenerative diseases.     

Upregulation of telomerase function during tissue regeneration

Telomerase plays a primary role in the maintenance of telomeres in immortal, germ, and tumor cells in humans but is lacking in most somatic cells and tissues. However, many species, including fish and inbred mice, express telomerase in most cells and tissues. Little is known about the expression of telomerase in aquatic species, although the importance of telomerase for longevity has been suggested. We compared telomerase activity and telomere lengths among a broad range of tissues from aquatic species and found telomerase at significant levels in both long- and short-lived aquatic species, suggesting constitutive telomerase expression has an alternative function. Telomere lengths in these aquatic species were comparable to those observed in normal human tissues and cell strains. Given that a host of aquatic species with short life spans have telomerase and a tremendous capacity to regenerate, we tested the hypothesis that telomerase upregulation is important for tissue regeneration. During regeneration, telomerase activity was upregulated and telomere lengths are maintained with the shortest telomeres being elongated, indicating the importance for maintaining telomere length and integrity during tissue regeneration. Thus, the expression of telomerase in aquatic animals is likely not related to longevity but to their ability to regenerate injured tissue.     

Telomere biology: Cancer firewall or aging clock?

It has been a decade since the first surprising discovery that longer telomeres in humans are statistically associated with longer life expectancies. Since then, it has been firmly established that telomere shortening imposes an individual fitness cost in a number of mammalian species, including humans. But telomere shortening is easily avoided by application of telomerase, an enzyme which is coded into nearly every eukaryotic genome, but whose expression is suppressed most of the time. This raises the question how the sequestration of telomerase might have evolved. The predominant assumption is that in higher organisms, shortening telomeres provide a firewall against tumor growth. A more straightforward interpretation is that telomere attrition provides an aging clock, reliably programming lifespans. The latter hypothesis is routinely rejected by most biologists because the benefit of programmed lifespan applies only to the community, and in fact the individual pays a substantial fitness cost. There is a long-standing skepticism that the concept of fitness can be applied on a communal level, and of group selection in general. But the cancer hypothesis is problematic as well. Animal studies indicate that there is a net fitness cost in sequestration of telomerase, even when cancer risk is lowered. The hypothesis of protection against cancer has never been tested in animals that actually limit telomerase expression, but only in mice, whose lifespans are not telomerase-limited. And human medical evidence suggests a net aggravation of cancer risk from the sequestration of telomerase, because cells with short telomeres are at high risk of neoplastic transformation, and they also secrete cytokines that exacerbate inflammation globally. The aging clock hypothesis fits well with what is known about ancestral origins of telomerase sequestration, and the prejudices concerning group selection are without merit. If telomeres are an aging clock, then telomerase makes an attractive target for medical technologies that seek to expand the human life- and health-spans.     

Telomeres and human disease: ageing, cancer and beyond

Telomere length and telomerase activity are important factors in the pathobiology of human disease. Age-related diseases and premature ageing syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. Altered functioning of both telomerase and telomere-interacting proteins is present in some human premature ageing syndromes and in cancer, and recent findings indicate that alterations that affect telomeres at the level of chromatin structure might also have a role in human disease. These findings have inspired a number of potential therapeutic strategies that are based on telomerase and telomeres.     

Telomerase abrogates aneuploidy‐induced telomere replication stress,senescence and cell depletion

The causal role of aneuploidy in cancer initiation remains under debate since mutations of euploidy‐controlling genes reduce cell fitness but aneuploidy strongly associates with human cancers. Telomerase activation allows immortal growth by stabilizing telomere length, but its role in aneuploidy survival has not been characterized. Here, we analyze the response of primary human cells and murine hematopoietic stem cells (HSCs) to aneuploidy induction and the role of telomeres and the telomerase in this process. The study shows that aneuploidy induces replication stress at telomeres leading to telomeric DNA damage and p53 activation. This results in p53/Rb‐dependent, premature senescence of human fibroblast, and in the depletion of hematopoietic cells in telomerase‐deficient mice. Endogenous telomerase expression in HSCs and enforced expression of telomerase in human fibroblasts are sufficient to abrogate aneuploidy‐induced replication stress at telomeres and the consequent induction of premature senescence and hematopoietic cell depletion. Together, these results identify telomerase as an aneuploidy survival factor in mammalian cells based on its capacity to alleviate telomere replication stress in response to aneuploidy induction.     

Role of the TRF2 Telomeric Protein in Cancer and Aging

Telomeres are the special heterochromatin that forms the ends of chromosomes, consisting of TTAGGG repeats and associated proteins. Telomeres protect the ends from degradation and recombination, and are essential for chromosomal stability. Both a minimal length of telomere repeats and the telomere-binding proteins are required for telomere protection. Telomerase is a DNA polymerase that specifically elongates telomeres, in this way regulating telomere length and function. A minimal telomere length is required to maintain tissue homeostasis. On one hand, critically short telomeres trigger loss of cell viability and premature death in mice deficient for telomerase activity. Furthermore, altered functioning of telomerase and telomere-interacting proteins is present in some human premature ageing syndromes and cancer. A new mouse model with critically short telomeres has been generated by over-expressing the TRF2 telomere-binding protein, K5-TRF2 mice. These mice show short telomeres in the presence of telomerase activity, leading to premature aging and increased cancer. Short telomeres in TRF2 mice can be rescued in the absence of the XPF nuclease, indicating that this enzyme rapidly degrades telomeres in the presence of increased TRF2 expression. K5-TRF2 mice represent a new tool to understand the consequences of critical telomere shortening a telomerase-proficient genetic background, more closely resembling human cancer and aging pathologies.     

Species-specific and sequence-specific recognition of the dG-rich strand of telomeres by yeast telomerase.

A gel mobility shift assay was developed to examine recognition of yeast telomeres by telomerase. An RNase-sensitive G-rich strand-specific binding activity can be detected in partially purified yeast telomerase fractions. The binding activity was attributed to telomerase, because it co-purifies with TLC1 RNA and telomerase activity over three different chromatographic steps and because the complex co-migrates with TLC1 RNA when subjected to electrophoresis through native gels. Analysis of the binding specificity of yeast telomerase indicates that it recognizes the G-rich strand of yeast telomeres with high affinity and specificity. The K d for the interaction is approximately 3 nM. Single-stranded G-rich telomeres from other species, such as human and Tetrahymena, though capable of being extended by yeast telomerase in polymerization assays at high concentrations, bind the enzyme with at least 100-fold lower affinities. The ability of a sequence to be bound tightly by yeast telomerase in vitro correlates with its ability to seed telomere formation in vivo. The implications of these findings for regulation of telomerase activity are discussed.     

A Cajal body-independent pathway for telomerase trafficking in mice

The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.     

Telomerase recruitment requires both TCAB1 and Cajal bodies independently

The ability of most cancer cells to grow indefinitely relies on the enzyme telomerase and its recruitment to telomeres. In human cells, recruitment depends on the Cajal body RNA chaperone TCAB1 binding to the RNA subunit of telomerase (hTR) and is also thought to rely on an N-terminal domain of the catalytic subunit, hTERT. We demonstrate that coilin, an essential structural component of Cajal bodies, is required for endogenous telomerase recruitment to telomeres but that overexpression of telomerase can compensate for Cajal body absence. In contrast, recruitment of telomerase was sensitive to levels of TCAB1, and this was not rescued by overexpression of telomerase. Thus, although Cajal bodies are important for recruitment, TCAB1 has an additional role in this process that is independent of these structures. TCAB1 itself localizes to telomeres in a telomerase-dependent but Cajal body-independent manner. We identify a point mutation in hTERT that largely abolishes recruitment yet does not affect association of telomerase with TCAB1, suggesting that this region mediates recruitment by an independent mechanism. Our results demonstrate that telomerase has multiple independent requirements for recruitment to telomeres and that the function of TCAB1 is to directly transport telomerase to telomeres.     

Molecular steps of G‐overhang generation at human telomeres and its function in chromosome end protection

Telomeric G‐overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G‐overhang generation at human telomeres is poorly understood. Here, we show that G‐overhangs can undergo cell cycle‐regulated changes independent of telomerase activity. G‐overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C‐strand fill‐in, whereas the sizes of G‐overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C‐strands are precisely defined throughout the cell cycle, indicating that C‐strand resection is strictly regulated. We demonstrate that C‐strand fill‐in is mediated by DNA polymerase α (polα) and controlled by cyclin‐dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G‐overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G‐overhangs and an increase in telomeric DNA damage. Our results suggest that G‐overhang generation at human telomeres is regulated by multiple tightly controlled processes and C‐strand fill‐in is under the control of polα and CDK1.     

Telomeric recombination induced by dysfunctional telomeres

Telomere maintenance is essential for cellular immortality, and most cancer cells maintain their telomeres through the enzyme telomerase. Telomeres and telomerase represent promising anticancer targets. However, 15% of cancer cells maintain their telomeres through alternative recombination-based mechanisms, and previous analyses showed that recombination-based telomere maintenance can be activated after telomerase inhibition. We determined whether telomeric recombination can also be promoted by telomere dysfunction. We report for the first time that telomeric recombination can be induced in human telomerase-positive cancer cells with dysfunctional telomeres.     

Human telomeres have different overhang sizes at leading versus lagging strands

G-rich 3' telomeric overhangs are required both for forming the distinct telomere structures to protect chromosome ends and for extending telomeres by telomerase. However, little is known about the molecular mechanisms generating telomere overhangs in human cells. We show here that cultured normal human diploid cells have longer G overhangs at telomeres generated by lagging-strand synthesis than by leading-strand synthesis. We also demonstrate that telomerase expression results in elongated overhangs at the leading daughter telomeres. Thus, the overhangs at the leading and lagging daughter telomeres are generated differently in human cells, and telomerase may preferentially affect overhangs generated at the telomeres produced by leading-strand synthesis.