Posttranslational control of telomere maintenance and the telomere damage response

Telomeres help maintain genome integrity by protecting natural chromosome ends from being recognized as damaged DNA. When telomeres become dysfunctional, they limit replicative lifespan and prevent outgrowth of potentially cancerous cells by activating a DNA damage response that forces cells into senescence or apoptosis. On the other hand, chromosome ends devoid of proper telomere protection are subject to DNA repair activities that cause end-to-end fusions and, when cells divide, extensive genomic instability that can promote cancer. While telomeres represent unique chromatin structures with important roles in cancer and aging, we have limited understanding of the way telomeres and the response to their malfunction are controlled at the level of chromatin. Accumulating evidence indicates that different types of posttranslational modifications act in both telomere maintenance and the response to telomere uncapping. Here, we discuss the latest insights on posttranslational control of telomeric chromatin, with emphasis on ubiquitylation and SUMOylation events.     

An interstitial telomere array proximal to the distal telomere of mouse chromosome 13

Lambda clones of mouse DNA from BALB/c and C57BL/10, each containing an array of telomere hexamers, were localized by FISH to a region close to the telomere of Chr 13. Amplification of mouse genomic DNA with primers flanking SSRs within the cloned DNA showed several alleles, which were used to type eight sets of RI strains. The two lambda clones contained allelic versions of the interstitial telomere array, Tel-rs4, which is 495 bp in C57BL/10 and which includes a variety of sequence changes from the consensus telomere hexamer. Comparison of the segregation of the amplification products of the SSRs with the segregation of other loci in an interspecies backcross (C57BL/6JEi × SPRET/Ei) F₁× SPRET/Ei shows recombination suppression, possibly associated with ribosomal DNA sequences present on distal Chr 13 in Mus spretus, when compared with recombination in an interstrain backcross, (C57BL/6J × DBA/J) F₁× C57BL/6J, and with the MIT F₂ intercross. Analysis of recombination in females using a second interstrain backcross, (ICR/Ha × C57BL/6Ha) F₁× C57BL/6Ha, also indicates recombination suppression when compared with recombination in males of the same strains, using backcross C57BL/6Ha × (ICR/Ha × C57BL/6Ha) F₁. Thus, more than one cause may contribute to recombination suppression in this region. The combined order of the loci typed was D13Mit37–D13Mit30–D13Mit148–(D13Rp1, 2, 3, 4, Tel-rs4)–D13Mit53–D13Mit196–D13Mit77–(D13Mit78, 35). Data from crosses where apparently normal frequencies of recombination occur suggest that the telomere array is about 6 map units proximal to the most distal loci on Chr 13. This distance is consistent with evidence from markers identified in two YAC clones obtained from the region. Received: 24 September 1996/Accepted: 20 January 1997     

The role of telomere trimming in normal telomere length dynamics

Telomeres consist of repetitive DNA and associated proteins that protect chromosome ends from illicit DNA repair. It is well known that telomeric DNA is progressively eroded during cell division, until telomeres become too short and the cell stops dividing. There is a second mode of telomere shortening, however, which is a regulated form of telomere rapid deletion (TRD) termed telomere trimming that is reviewed here. Telomere trimming appears to involve resolution of recombination intermediate structures, which shortens the telomere by release of extrachromosomal telomeric DNA. This has been detected in human and in mouse cells and occurs both in somatic and germline cells, where it sets an upper limit on telomere length and contributes to a length equilibrium set-point in cells that have a telomere elongation mechanism. Telomere trimming thus represents an additional mechanism of telomere length control that contributes to normal telomere dynamics and cell proliferative potential.     

Tiny telomere DNA

We describe the design, synthesis and biophysical characterization of a novel DNA construct in which a folded quadruplex structure is joined to a standard double helix. Circular dichroism, gel electrophoresis, three-dimensional UV melting and differential scanning calorimetry were all used to characterize the structure. Rigorous molecular dynamics simulations were used to build a plausible atomic-level structural model of the DNA construct. This novel DNA construct provides a model for the duplex–quadruplex junction region at the end of chromosomal DNA and offers a system for the study of structure-selective ligand binding.     

Mammalian telomere biology

The review considers the function of the important chromosome regions telomeres in normal and immortal cells. Telomeres are dynamic nucleoprotein structures that cap the ends of eukaryotic chromosomes, protecting them from degradation and end-to-end fusion. The functional state of telomeres depends on many interrelated parameters such as telomerase activity, the status of the telomere safety complex shelterin, and telomere-associated proteins (replication, recombination, DNA break repair factors, etc.). Special attention is paid to the mechanisms that control the telomere length in normal and immortal cells as well as in cells containing or lacking active telomerase. The features attributed to an alternative telomere length control are analyzed, in particular, in view of a recently discovered additional mechanism of telomere shortening by t-cycle trimming. The possibility of expressing both telomerase-dependent and recombinational pathways of telomere length control in normal mammalian cells is considered, as well as the role of shelterin proteins in choosing one of them to be dominant. The review additionally discusses the role of telomeres in the spatial organization of the nucleus during mitosis and meiosis and specific telomere organizations in mammals, including Iberian shrews with their unusual or rare chromosome structures.     

Switching and signaling at the telomere

This review describes the structure of telomeres, the protective DNA-protein complexes at eukaryotic chromosomal ends, and several molecular mechanisms involved in telomere functions. Also discussed are cellular responses to compromising the functions of telomeres and of telomerase, which synthesizes telomeric DNA.     

Spontaneous telomere to telomere fusions occur in unperturbed fission yeast cells

Telomeres protect eukaryotic chromosomes from illegitimate end-to-end fusions. When this function fails, dicentric chromosomes are formed, triggering breakage-fusion-bridge cycles and genome instability. How efficient is this protection mechanism in normal cells is not fully understood. We created a positive selection assay aimed at capturing chromosome-end fusions in Schizosaccharomyces pombe. We placed telomere sequences with a head to head arrangement in an intron of a selectable marker contained on a plasmid. By linearizing the plasmid between the telomere sequences, we generated a stable mini-chromosome that fails to express the reporter gene. Whenever the ends of the mini-chromosome join, the marker gene is reconstituted and fusions are captured by direct selection. Using telomerase mutants, we recovered several fusion events that lacked telomere sequences. The end-joining reaction involved specific homologous subtelomeric sequences capable of forming hairpins, suggestive of ssDNA stabilization prior to fusing. These events occurred via microhomology-mediated end-joining (MMEJ)/single-strand annealing (SSA) repair and also required MRN/Ctp1. Strikingly, we were able to capture spontaneous telomere-to-telomere fusions in unperturbed cells. Similar to disruption of the telomere regulator Taz1/TRF2, end-joining reactions occurred via non-homologous end-joining (NHEJ) repair. Thus, telomeres undergo fusions prior to becoming critically short, possibly through transient deprotection. These dysfunction events induce chromosome instability and may underlie early tumourigenesis.     

A role for sister telomere cohesion in telomere elongation by telomerase

Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC), a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure. Recently a new class of particularly severe DC patients was found to harbor mutations in the shelterin subunit TIN2. The DC-TIN2 mutations were clustered in small domain of unknown function. In a recently published study we showed that the DC mutation cluster in TIN2 harbored a binding site for heterochromatin protein 1 (HP1) and, further, that HP1 binding to TIN2 was required for sister telomere cohesion in S phase and for telomere length maintenance by telomerase. We briefly review and discuss the implications of our findings in this Extra View and present some new data that may shed light on how sister telomere cohesion could influence telomere elongation by telomerase.Key words: telomeres, cohesion, telomerase, TIN2, dyskeratosis congenita     

A role for sister telomere cohesion in telomere elongation by telomerase

Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC) a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure. Recently a new class of particularly severe DC patients was found to harbor mutations in the shelterin subunit TIN2. The DC-TIN2 mutations were clustered in small domain of unknown function. In a recently published study we showed that the DC mutation cluster in TIN2 harbored a binding site for heterochromatin protein 1 (HP1) and further, that HP1 binding to TIN2 was required for sister telomere cohesion in S phase and for telomere length maintenance by telomerase. We briefly review and discuss the implications of our findings in this Extra View, and present some new data that may shed light on how sister telomere cohesion could influence telomere elongation by telomerase.     

DNA methyltransferases control telomere length and telomere recombination in mammalian cells

Here, we describe a role for mammalian DNA methyltransferases (DNMTs) in telomere length control. Mouse embryonic stem (ES) cells genetically deficient for DNMT1, or both DNMT3a and DNMT3b have dramatically elongated telomeres compared with wild-type controls. Mammalian telomere repeats (TTAGGG) lack the canonical CpG methylation site. However, we demonstrate that mouse subtelomeric regions are heavily methylated, and that this modification is decreased in DNMT-deficient cells. We show that other heterochromatic marks, such as histone 3 Lys 9 (H3K9) and histone 4 Lys 20 (H4K20) trimethylation, remain at both subtelomeric and telomeric regions in these cells. Lack of DNMTs also resulted in increased telomeric recombination as indicated by sister-chromatid exchanges involving telomeric sequences, and by the presence of 'alternative lengthening of telomeres' (ALT)-associated promyelocytic leukaemia (PML) bodies (APBs). This increased telomeric recombination may lead to telomere-length changes, although our results do not exclude a potential involvement of telomerase and telomere-binding proteins in the aberrant telomere elongation observed in DNMT-deficient cells. Together, these results demonstrate a previously unappreciated role for DNA methylation in maintaining telomere integrity.     

Identification of the telomere in Trypanosoma cruzi reveals highly heterogeneous telomere lengths in different parasite strains.

Here we describe the cloning and characterisation of the Trypanosoma cruzi telomere. In the Y strain, it is formed by typical GGGTTA repeats with a mean size of approximately 500 bp. Adjacent to the telomere repeats we found a DNA sequence with significant homology to the T.cruzi 85 kDa surface antigen (gp85). Examination of the telomere in nine T.cruzi strains reveals differences in the organisation of chromosome ends. In one group of strains the size of the telomere repeat is relatively homogeneous and short (0.5-1.5 kb) as in the Y strain, while in the other, the length of the repeat is very heterogeneous and significantly longer, ranging in size from 1 to >10 kb. These different strains can be grouped similarly to previously existing classifications based on isoenzyme loci, rRNA genes, mini-exon gene sequences, randomly amplified polymorphic DNA and rRNA promoter sequences, suggesting that differential control of telomere length and organisation appeared as an early event in T. cruzi evolution. Two-dimensional pulsed field gel electrophoresis analysis shows that some chromosomes carry telomeres which are significantly larger than the mean telomere length. Importantly, the T.cruzi telomeres are organised in nucleosomal and non-nucleosomal chromatin.     

An optimized set of human telomere clones for studying telomere integrity and architecture

Telomere-specific clones are a valuable resource for the characterization of chromosomal rearrangements. We previously reported a first-generation set of human telomere probes consisting of 34 genomic clones, which were a known distance from the end of the chromosome ( approximately 300 kb), and 7 clones corresponding to the most distal markers on the integrated genetic/physical map (1p, 5p, 6p, 9p, 12p, 15q, and 20q). Subsequently, this resource has been optimized and completed: the size of the genomic clones has been expanded to a target size of 100-200 kb, which is optimal for use in genome-scanning methodologies, and additional probes for the remaining seven telomeres have been identified. For each clone we give an associated mapped sequence-tagged site and provide distances from the telomere estimated using a combination of fiberFISH, interphase FISH, sequence analysis, and radiation-hybrid mapping. This updated set of telomeric clones is an invaluable resource for clinical diagnosis and represents an important contribution to genetic and physical mapping efforts aimed at telomeric regions.     

A reassessment of the telomere hypothesis of senescence

According to the telomere hypothesis of senescence, the telomeric shortening that accompanies the replication of normal somatic cells acts as the mitotic clock that eventually results in their permanent exit from the cell cycle. Although evidence consistent with the telomere hypothesis continues to accumulate, on the basis of recent findings it is suggested that instead of a single clock mechanism there are multiple inducers of senescence.     

Unique hole-trapping property of the human telomere sequence

The hypothetical protection of genes from oxidative damage provided by the G-rich telomeric overhangs located at the end of chromosomes, which consist, in humans, of single strands of TTAGGG sequence repeats, is investigated here. First principle Moller-Plesset perturbation theory calculations reveal that the TTAGGG human telomere sequence is particularly prone to oxidation and can act as a profound hole trap as deep as a sequence of five consecutive guanines. In addition, we show that the sequence dependence is very important and that modifications in the human telomeric sequence can induce crucial changes in the electronic structure of the sequence, with concomitant increase of the ionization energy. These theoretical results provide, for the first time, quantitative data indicating a high and unique efficiency of the human telomeric sequence as a trap in long-range hole migration which will aid in the design of subsequent experiments.