Chromatin regulation and non-coding RNAs at mammalian telomeres

In eukaryotes, terminal chromosome repeats are bound by a specialized nucleoprotein complex that controls telomere length and protects chromosome ends from DNA repair and degradation. In mammals the “shelterin” complex mediates these central functions at telomeres. In the recent years it has become evident that also the heterochromatic structure of mammalian telomeres is implicated in telomere length regulation. Impaired telomeric chromatin compaction results in a loss of telomere length control. Progressive telomere shortening affects chromatin compaction at telomeric and subtelomeric repeats and activates alternative telomere maintenance mechanisms. Dynamics of chromatin structure of telomeres during early mammalian development and nuclear reprogramming further indicates a central role of telomeric heterochromatin in organismal development. In addition, the recent discovery that telomeres are transcribed, giving rise to UUAGGG-repeat containing TelRNAs/TERRA, opens a new level of chromatin regulation at telomeres. Understanding the links between the epigenetic status of telomeres, TERRA/TelRNA and telomere homeostasis will open new avenues for our understanding of organismal development, cancer and ageing.     

Identification of the determinants for the specific recognition of single-strand telomeric DNA by Cdc13

The single-strand overhang present at telomeres plays a critical role in mediating both the capping and telomerase regulation functions of telomeres. The telomere end-binding proteins, Cdc13 in Saccharomyces cerevisiae, Pot1 in higher eukaryotes, and TEBP in the ciliated protozoan Oxytricha nova, exhibit sequence-specific binding to their respective single-strand overhangs. S. cerevisiae telomeres are composed of a heterogeneous mixture of GT-rich telomeric sequence, unlike in higher eukaryotes which have a simple repeat that is maintained with high fidelity. In yeast, the telomeric overhang is recognized by the essential protein Cdc13, which coordinates end-capping and telomerase activities at the telomere. The Cdc13 DNA-binding domain (Cdc13-DBD) binds these telomere sequences with high affinity (3 pM) and sequence specificity. To better understand the basis for this remarkable recognition, we have investigated the binding of the Cdc13-DBD to a series of altered DNA substrates. Although an 11-mer of GT-rich sequence is required for full binding affinity, only three of these 11 bases are recognized with high specificity. This specificity differs from that observed in the other known telomere end-binding proteins, but is well suited to the specific role of Cdc13 at yeast telomeres. These studies expand our understanding of telomere recognition by the Cdc13-DBD and of the unique molecular recognition properties of ssDNA binding.     

Dyskeratosis congenita as a disorder of telomere maintenance

Since 1998, there have been great advances in our understanding of the pathogenesis of dyskeratosis congenita (DC), a rare inherited bone marrow failure and cancer predisposition syndrome with prominent mucocutaneous abnormalities and features of premature aging. DC is now characterized molecularly by the presence of short age-adjusted telomeres. Mutations in seven genes have been unequivocally associated with DC, each with a role in telomere length maintenance. These observations, combined with knowledge that progressive telomere shortening can impose a proliferative barrier on dividing cells and contribute to chromosome instability, have led to the understanding that extreme telomere shortening drives the clinical features of DC. However, some of the genes implicated in DC encode proteins that are also components of H/ACA-ribonucleoprotein enzymes, which are responsible for the post-translational modification of ribosomal and spliceosomal RNAs, raising the question whether alterations in these activities play a role in the pathogenesis of DC. In addition, recent reports suggest that some cases of DC may not be characterized by short age-adjusted telomeres. This review will highlight our current knowledge of the telomere length defects in DC and the factors involved in its development.     

Telomere instability in a human cancer cell line.

Telomere maintenance is essential in immortal cancer cells to compensate for DNA lost from the ends of chromosomes, to prevent chromosome fusion, and to facilitate chromosome segregation. However, the high rate of fusion of chromosomes near telomeres, termed telomere association, in many cancer cell lines has led to the proposal that some cancer cells may not efficiently perform telomere maintenance. Deficient telomere maintenance could play an important role in cancer because telomere associations and nondisjunction have been demonstrated to be mechanisms for genomic instability. To investigate this possibility, we have analyzed the telomeres of the human squamous cell carcinoma cell line SQ-9G, which has telomere associations in approximately 75% of the cells in the population. The absence of detectable telomeric repeat sequences at the sites of these telomere associations suggests that they result from telomere loss. The analysis of telomere length by quantitative in situ hybridization demonstrated that, compared to the human squamous cell carcinoma cell line SCC-61 which has few telomere associations, SQ-9G has more extensive heterogeneity in telomere length and more telomeres without detectable telomeric repeat sequences. The dynamics of the changes in telomere length also demonstrated a higher rate of fluctuation in telomere length, both on individual telomeres and coordinately on all telomeres. These results demonstrate that telomere maintenance can play a role in the genomic instability seen in cancer cells.     

Telomerase enzymatic component hTERT shortens long telomeres in human cells

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

Those dam-aged telomeres!

Telomere integrity plays a crucial role in the capacity for continuous cell proliferation. In some circumstances, shortened telomeres contribute to cell arrest or death, but in others, shortened telomeres may actually enhance the incidence and spectrum of tumors. Resolution of this apparent paradox requires a more detailed understanding of a non-functional telomere. Recent evidence reveals that critically shortened or uncapped telomeres share molecular hallmarks of damaged DNA. It is likely that the cellular response to this DNA damage, influenced by the nature of the damage itself, affects the outcome of loss of telomere function.     

Rap1 affects the length and heterogeneity of human telomeres

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

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.     

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.     

Drosophila telomeres: A variation on the telomerase theme

In Drosophila, the role of telomerase is carried out by three specialized retrotransposable elements, HeT-A, TART and TAHRE. Telomeres contain long tandem head-to-tail arrays of these elements. Within each array, the three elements occur in random, but polarized, order. Some are truncated at the 5' end, giving the telomere an enriched content of the large 3' untranslated regions, which distinguish these telomeric elements from other retrotransposons. Thus, Drosophila telomeres resemble other telomeres because they are long arrays of repeated sequences, albeit more irregular arrays than those produced by telomerase. The telomeric retrotransposons are reverse-transcribed directly onto the end of the chromosome, extending the end by successive transpositions. Their transposition uses exactly the same method by which telomerase extends chromosome ends--copying an RNA template. In addition to these similarities in structure and maintenance, Drosophila telomeres have strong functional similarities to other telomeres and, as variants, provide an important model for understanding general principles of telomere function and evolution.     

Telomere erosion in human pluripotent stem cells leads to ATR-mediated mitotic catastrophe

It is well established that short telomeres activate an ATM-driven DNA damage response that leads to senescence in terminally differentiated cells. However, technical limitations have hampered our understanding of how telomere shortening is signaled in human stem cells. Here, we show that telomere attrition induces ssDNA accumulation (G-strand) at telomeres in human pluripotent stem cells (hPSCs), but not in their differentiated progeny. This led to a unique role for ATR in the response of hPSCs to telomere shortening that culminated in an extended S/G2 cell cycle phase and a longer period of mitosis, which was associated with aneuploidy and mitotic catastrophe. Loss of p53 increased resistance to death, at the expense of increased mitotic abnormalities in hPSCs. Taken together, our data reveal an unexpected dominant role of ATR in hPSCs, combined with unique cell cycle abnormalities and, ultimately, consequences distinct from those observed in their isogenic differentiated counterparts.     

Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway

Functional telomeres are required to maintain the replicative ability of cancer cells and represent putative targets for G-quadruplex (G4) ligands. Here, we show that the pentacyclic acridinium salt RHPS4, one of the most effective and selective G4 ligands, triggers damages in cells traversing S phase by interfering with telomere replication. Indeed, we found that RHPS4 markedly reduced BrdU incorporation at telomeres and altered the dynamic association of the telomeric proteins TRF1, TRF2 and POT1, leading to chromosome aberrations such as telomere fusions and telomere doublets. Analysis of the molecular damage pathway revealed that RHPS4 induced an ATR-dependent ATM signaling that plays a functional role in the cellular response to RHPS4 treatment. We propose that RHPS4, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing resulting in telomere dysfunction and activation of a replication stress response pathway. The detailed understanding of the molecular mode of action of this class of compounds makes them attractive tools to understand telomere biology and provides the basis for a rational use of G4 ligands for the therapy of cancer.     

Short Telomeres are Sufficient to Cause the Degenerative Defects Associated with Aging

Telomerase function is critical for telomere maintenance. Mutations in telomerase components lead to telomere shortening and progressive bone marrow failure in the premature aging syndrome dyskeratosis congenita. Short telomeres are also acquired with aging, yet the role that they play in mediating age-related disease is not fully known. We generated wild-type mice that have short telomeres. In these mice, we identified hematopoietic and immune defects that resembled those present in dyskeratosis congenita patients. When mice with short telomeres were interbred, telomere length was only incrementally restored, and even several generations later, wild-type mice with short telomeres still displayed degenerative defects. Our findings implicate telomere length as a unique heritable trait that, when short, is sufficient to mediate the degenerative defects of aging, even when telomerase is wild-type.     

Telomere dysfunction in aging and cancer

Telomeres are unique DNA-protein structures that contain noncoding TTAGGG repeats and telomere-associated proteins. These specialized structures are essential for maintaining genomic integrity. Alterations that lead to the disruption of telomere maintenance result in chromosome end-to-end fusions and/or ends being recognized as double-strand breaks. A large body of evidence suggests that the cell responds to dysfunctional telomeres by undergoing senescence, apoptosis, or genomic instability. In conjunction with other predisposing mechanisms, the genomic instability encountered in preimmortal cells due to dysfunctional or uncapped telomeres might lead to cancer. Furthermore, telomere dysfunction has been proposed to play critical roles in aging as well as cancer progression. Conversely, recent evidence has shown that targeting telomere maintenance mechanisms and inducing telomere dysfunction in cancer cells by inhibiting telomerase can lead to catastrophic events including rapid cell death and increased sensitivity to other cancer therapeutics. Thus, given the major role telomeres play during development, it is important to continue our understanding telomere structure, function and maintenance. Herein, we provide an overview of the emerging knowledge of telomere dysfunction and how it relates to possible links between aging and cancer.     

Evolving views of telomerase and cancer

The maintenance of telomeres, nucleoprotein structures that constitute the ends of eukaryotic chromosomes, regulates many crucial cellular functions and might, in multicellular organisms, participate in the control of complex phenotypes such as aging and cancer. Stabilization of telomere length is strongly associated with cellular immortalization, and constitutive telomerase activation occurs in most human cancers. Such observations form the basis for the prevailing model that postulates that alterations in telomere biology both suppress and facilitate malignant transformation by regulating genomic stability and cell life span. However, recent findings suggest that telomere maintenance might not be an obligate requirement for initial tumor formation in some settings and that telomerase activation contributes to tumorigenesis independently of its role in maintaining telomere length. These recent developments indicate that our understanding of telomere biology remains incomplete and implicate additional complexity in the relationships among telomeres, telomerase and cancer.     

Human telomerase can immortalize Indian muntjac cells

The mechanisms of replicative senescence by telomere shortening are not fully understood. The Indian muntjac has the fewest chromosomes of all mammals, greatly simplifying the analysis of each telomere over time. In this study, telomere shortening was observed throughout the life span of cultured normal muntjac cells by quantitative fluorescence in situ hybridization and terminal restriction fragment analysis. Ectopic expression of the human telomerase catalytic subunit in these cells reconstituted telomerase activity, extended telomere lengths, and immortalized the cells, demonstrating that the Indian muntjac cells can serve as a telomere-based replicative senescence model for human cells. In one strain, two chromosome ends had significantly shorter telomeres than the other ends, which led to a variety of chromosome abnormalities. Near senescence, additional ends became telomere signal free, and chromosome aberrancies increased dramatically. Interstitial telomere sequences coincided with fragile sites, suggesting that these remnants of chromosome fusion events might contribute to genome instability. One SV40-immortalized cell line lacked telomerase, and its genetic instability was corrected by the ectopic expression of telomerase, confirming that too-short telomeres were the source of abnormalities. Indian muntjac cells provide an excellent system for understanding the mechanism of replicative senescence and the role of telomerase in the elongation of individual telomeres.     

Mice with bad ends: mouse models for the study of telomeres and telomerase in cancer and aging

Telomeres are capping structures at the ends of eukaryotic chromosomes, which consist of repetitive DNA bound to an array of specialized proteins. Telomeres are part of the constitutive heterochromatin and are subjected to epigenetic modifications. The function of telomeres is to prevent chromosome ends from being detected as damaged DNA. Both the length of telomere repeats and the integrity of the telomere-binding proteins are important for telomere protection. Telomere length is regulated by telomerase, by the telomere-binding proteins, as well as by activities that modify the state of the chromatin. Various mouse models with altered levels of telomerase activity, or mutant for different telomere-binding proteins, have been recently generated. Here, I will discuss how these different mouse models have contributed to our understanding on the role of telomeres and telomerase in cancer and aging.