Developmental pattern of telomerase expression in the sand scallop, Euvola ziczac

Abstract. Telomerase is a ribonucleoprotein that can maintain telomeres, the repetitive sequences of DNA found at the end of eukaryotic chromosomes, and confer long-term proliferative capacity on cells. Telomerase expression is essential during periods of intense cell division such as the early developmental process. In later development, some species retain telomerase activity while others repress telomerase activity in what is thought to be a tumor-protective mechanism. Despite the importance of telomerase expression in both development and neoplastic disease, no studies to date have characterized its expression in bivalves. We present the first report of telomerase expression in a bivalve species, the sand scallop, Euvola ziczac. Telomerase activity was detected throughout the early stages of development and in all adult tissues examined. Analysis of DNA isolated from adult tissues indicated long telomeres, with terminal restriction fragment lengths >20 kb in both somatic and germ tissues. Ubiquitous telomerase expression throughout development and into adulthood would suggest a lack of telomere-related senescence and suggests that these scallops do not use telomerase repression as a mechanism to suppress the formation of neoplasm.     

Telomeric repeat sequences

Chromosomes not only carry transcribed genes and their regulatory DNA sequences, but also contain regions that are required for the stability and maintenace of the chromosome as a unit. These include centromeres, telomeres and origins of replication. It is clear for replication origins and centromeres that the positions of these chromosomal organelles are determined by sites of the appropriate DNA sequences, but also that functional performance requires one or more contributing proteins. Telomeres are also structurally complex, with one or more DNA components, including simple telomeric repeats and more complex telomere-associated sequences, as well as one or more specific proteins that recognize these sequences. Accumulating evidence suggests that the simple telomeric repeats are required in most, but not all species, although they are not sufficient to determine the chromosomal position of a telomere.     

Chromosome ends: different sequences may provide conserved functions

The structures of specific chromosome regions, centromeres and telomeres, present a number of puzzles. As functions performed by these regions are ubiquitous and essential, their DNA, proteins and chromatin structure are expected to be conserved. Recent studies of centromeric DNA from human, Drosophila and plant species have demonstrated that a hidden universal centromere-specific sequence is highly unlikely. The DNA of telomeres is more conserved consisting of a tandemly repeated 6-8 bp Arabidopsis-like sequence in a majority of organisms as diverse as protozoan, fungi, mammals and plants. However, there are alternatives to short DNA repeats at the ends of chromosomes and for telomere elongation by telomerase. Here we focus on the similarities and diversity that exist among the structural elements, DNA sequences and proteins, that make up terminal domains (telomeres and subtelomeres), and how organisms use these in different ways to fulfil the functions of end-replication and end-protection.     

Telomere lengthening early in development

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.     

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.     

A telomeric satellite in Drosophila virilis and its sibling species

Telomere elongation by telomerase is the most widespread mechanism among eukaryotes. However, alternative mechanisms such as homologous recombination between terminal satellite DNAs are probably used in lower dipteran insects and in some plants. Drosophila melanogaster uses the very unusual telomere elongation pathway of transposition of telomere-specific retrotransposable elements. The uniqueness of this telomere elongation mechanism raises the question of its origin. We, therefore, analyzed sequences located at telomeres of fairly distantly related Drosophila species, and in this paper we describe the characterization of complex satellite DNA sequences located at the telomeres of D. virilis and other species in the virilis group. We suggest an involvement of these DNA satellites in telomere elongation by homologous recombination similar to that found in lower dipterans. Our findings raise the possibility that telomere elongation by specific retrotransposons as found in D. melanogaster and its sibling species is a recent event in the evolution of dipteran insects.     

Telomere maintenance in Drosophila: Rapid transposon evolution at chromosome ends

The maintenance of terminal sequences is an important role of the telomere, since it prevents the loss of internal regions that encode essential genes. In most eukaryotes, this is accomplished by the telomerase. However, telomere length can also be maintained by other mechanisms, such as homologous recombination and transposition of telomeric retrotransposons to the chromosome ends. A remarkable situation is the case of Drosophila, where telomerase was lost, and thus telomeres managed to be maintained by occasional retrotransposition of telomeric elements to the receding ends. In the recent analysis of 12 Drosophila genomes, ¬¬the multiplicity of autonomous and non-autonomous telomere-specific retrotransposons has revealed extensive and rapid evolution of telomeric DNA. The phylogenetic relationship among these telomeric retrotransposons is congruent with the species phylogeny, suggesting that they have been vertically transmitted from a common ancestor. In this review, we also suggest that the formation of a non-canonical DNA structure at Drosophila telomeres could be the way to protect the ends.     

Telomere elongation in immortal human cells without detectable telomerase activity.

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.     

Telomere length mediates the effects of telomerase on the cellular response to genotoxic stress

Telomerase inhibition may be a novel anti-cancer strategy that can be used in combination with conventional therapies, such as DNA damaging agents. There are conflicting reports as to whether and to what extent telomerase and telomere length influence the sensitivity of cells to genotoxins. To understand the relationship between telomere length, telomerase expression, and sensitivity to genotoxic stress, we expressed the catalytic subunit of telomerase, hTERT, in human fibroblasts having different telomere lengths. We show that telomerase confers resistance to ionizing radiation, bleomycin, hydrogen peroxide, and etoposide only in cells with short, presumably near-dysfunctional, telomeres. This resistance depended on the ability of telomerase to elongate the short telomeres, and telomerase did not protect cells with long telomeres. Interestingly, although long telomeres had no effect on sensitivity to etoposide and bleomycin, they exacerbated sensitivity to hydrogen peroxide, supporting the idea that, compared to other types of DNA damage, telomeres are particularly vulnerable to oxidative damage. Our findings identify a mechanism and conditions under which telomerase and telomeres affect the response of human cells to genotoxic agents and may have important implications for anti-cancer interventions.     

Targeting the telosome: Therapeutic implications

Since telomere integrity is required to guarantee the unlimited replicative potential of cancer cells, telomerase, the enzyme responsible for telomere length maintenance in most human tumors, and lately also telomeres themselves have become extremely attractive targets for new anticancer interventions. At the current status of knowledge, it is still not possible to define the best therapeutic target between telomerase and telomeres. It is noteworthy that interfering with telomeres, through direct targeting of telomeric DNA or proteins involved in the telosome complex, could negatively affect the proliferative potential not only of tumors expressing telomerase activity but also of those that maintain their telomeres through alternative lengthening or still unknown mechanisms. This review presents the different therapeutic approaches proposed thus far and developed in preclinical tumor models and discusses the perspectives for their use in the clinical setting.     

Telomeres and telomerase dance to the rhythm of the cell cycle

The stability of the ends of linear eukaryotic chromosomes is ensured by functional telomeres, which are composed of short, species-specific direct repeat sequences. The maintenance of telomeres depends on a specialized ribonucleoprotein (RNP) called telomerase. Both telomeres and telomerase are dynamic entities with different physical behaviors and, given their substrate-enzyme relation, they must establish a productive interaction. Regulatory mechanisms controlling this interaction are key missing elements in our understanding of telomere functions. Here, we review the dynamic properties of telomeres and the maturing telomerase RNPs, and summarize how tracking the timing of their dance during the cell cycle will yield insights into chromosome stability mechanisms. Cancer cells often display loss of genome integrity; therefore, these issues are of particular interest for our understanding of cancer initiation or progression.     

De novo telomere addition by Tetrahymena telomerase in vitro.

Previous molecular genetic studies have shown that during programmed chromosomal healing, telomerase adds telomeric repeats directly to non-telomeric sequences in Tetrahymena, forming de novo telomeres. However, the biochemical mechanism underlying this process is not well understood. Here, we show for the first time that telomerase activity is capable in vitro of efficiently elongating completely non-telomeric DNA oligonucleotide primers, consisting of natural telomere-adjacent or random sequences, at low primer concentrations. Telomerase activity isolated from mated or vegetative cells had indistinguishable specificities for nontelomeric and telomeric primers. Consistent with in vivo results, the sequence GGGGT... was the predominant initial DNA sequence added by telomerase in vitro onto the 3' end of the non-telomeric primers. The 3' and 5' sequences of the primer both influenced the efficiency and pattern of de novo telomeric DNA addition. Priming of telomerase by double-stranded primers with overhangs of various lengths showed a requirement for a minimal 3' overhang of 20 nucleotides. With fully single-stranded non-telomeric primers, primer length up to approximately 30 nucleotides strongly affected the efficiency of telomeric DNA addition. We propose a model for the primer binding site of telomerase for non-telomeric primers to account for these length and structural requirements. We also propose that programmed de novo telomere addition in vivo is achieved through a hitherto undetected intrinsic ability of telomerase to elongate completely non-telomeric sequences.     

The highly variable pentameric repeats of the AT-rich germline limited DNA in Parascaris univalens are the telomeric repeats of somatic chromosomes.

We have characterized the organization of the germline limited DNA of P. univalens by means of sequence analysis. The repeat unit of this satellite DNA is the pentanucleotide 5'TTGCA, although there is a high degree of sequence variation. Repeat variants are not arranged in tandem but in a disperse, nonrandom manner. In the somatic genome which arises from the germline genome through extensive genomic rearrangement early in development, copies of these pentamers represent the telomeric repeats, indicated by their sensitivity to Bal 31 and their presence in a somatic endlibrary. Unlike telomeric sequences from other species the P. univalens telomeres do not display consecutive guanines and no strand bias for that base, recently suggested as universal features of eukaryotic telomeres. Investigation of fragments that carry pentameric repeats along with sequences of different type identifies a 5 bp consensus sequence at the junction point. We suggest a model in which pentameric repeats originate via amplification by a terminal transferase (telomerase) in both the germline and the somatic genome.     

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 biology: integrating chromosomal end protection with DNA damage response

Telomeres play the key protective role at chromosomes. Many studies indicate that loss of telomere function causes activation of DNA damage response. Here, we review evidence supporting interdependence between telomere maintenance and DNA damage response and present a model in which these two pathways are combined into a single mechanism for protecting chromosomal integrity. Proteins directly involved in telomere maintenance and DNA damage response include Ku, DNA-PKcs, RAD51D, PARP-2, WRN and RAD50/MRE11/NBS1 complex. Since most of these proteins participate in the repair of DNA double-strand breaks (DSBs), this was perceived by many authors as a paradox, given that telomeres function to conceal natural DNA ends from mechanisms that detect and repair DSBs. However, we argue here that the key function of one particular DSB protein, Ku, is to prevent or control access of telomerase, the enzyme that synthesises telomeric sequences, to both internal DSBs and natural chromosomal ends. This view is supported by observations that Ku has a high affinity for DNA ends; it acts as a negative regulator of telomerase and that telomerase itself can target internal DSBs. Ku then directs other DSB repair/telomere maintenance proteins to either repair DSBs at internal chromosomal sites or prevent uncontrolled elongation of telomeres by telomerase. This model eliminates the above paradox and provides a testable scenario in which the role of DSB repair proteins is to protect chromosomal integrity by balancing repair activities and telomere maintenance. In our model, a close association between telomeres and different DNA damage response factors is not an unexpected event, but rather a logical result of chromosomal integrity maintenance activities. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK     

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.