Mechanisms underlying telomere repeat turnover, revealed by hypervariable variant repeat distribution patterns in the human Xp/Yp telomere.

Sequences immediately adjacent to the human Xp/Yp telomere exhibit a high frequency of base substitutional polymorphisms, together with almost complete linkage disequilibrium, to create only a few diverged haplotypes. This sequence divergence has been used to develop a PCR-based system for mapping the distribution of the telomere (TTAGGG) and variant repeats (TGAGGG and TCAGGG) at the proximal end of the telomere repeat array. The distribution of these repeats is extremely variable. Almost all Xp/Yp telomeres are different, indicating a high mutation rate. Some telomere maps associated with the same flanking haplotype show similarities, identifying subsets of telomeres that share a recent common ancestry. Mechanisms underlying the rapid turnover of repeats at the proximal end of the Xp/Yp telomere include intra-allelic processes, such as slippage during replication. Inter-allelic exchanges may occur occasionally, but telomerase activity probably plays only a minor role in the germline turnover of proximally located telomere and variant repeats.     

Studying the biological and technical sources of variation in telomere length of individual chromosomes.

BACKGROUND: Consistent average length differences between species and chromosome arm differences within species indicate that telomere length is genetically determined. This seems to contradict an observed large variation in lengths of the same human telomere between metaphases of the same individual. We examined the extent to which the variation in the telomeres of the human X and Y chromosomes is heritable, induced, or technical in origin. METHODS: Metaphase chromosomes were stained by fluorescence in situ hybridization with a telomere repeat-specific probe, and fluorescence intensities of the X and Y chromosomes were measured. If telomere length variation is predominantly genetically determined and a 50% probability of meiotic recombination between the pseudo-autosomal regions of Yp and Xp in the father is taken into account, one expects an equal chance that the Yp telomere of a son is derived from his father's Xp or Yp telomere. This implies that the Yp/Yq telomere ratios in fathers and sons will be identical in the absence of paternal meiotic recombination and different when recombination occurs. RESULTS: Among five father-son pairs, four showed similar Yp/Yq ratios (P > 0.05), whereas one pair exhibited a large difference in the Yp/Yq ratio that was attributable to a significantly longer Xp than Yp telomere in the father and a presumptive meiotic exchange between X and Y during paternal meiosis. Further, the Xq telomere exhibited a generally shorter telomere length than the others. CONCLUSIONS: The high variation in telomere length appeared to be intracellular (between sister chromatids) and, hence, technical in nature. We found no measurable induced variation in the cells studied, implying that, if induced variation exists, it is small compared with the technical variation.     

Mutation mechanisms that underlie turnover of a human telomere-adjacent segmental duplication containing an unstable minisatellite

Subterminal regions, juxtaposed to telomeres on human chromosomes, contain a high density of segmental duplications, but relatively little is known about the evolutionary processes that underlie sequence turnover in these regions. We have characterized a segmental duplication adjacent to the Xp/Yp telomere, each copy containing a hypervariable array of the DXYS14 minisatellite. Both DXYS14 repeat arrays mutate at a high rate (0.3 and 0.2% per gamete) but linkage disequilibrium analysis across 27 SNPs and a direct crossover assay show that recombination during meiosis is suppressed. Therefore instability at DXYS14a and b is dominated by intra-allelic processes or possibly conversion limited to the repeat arrays. Furthermore some chromosomes (14%) carry only one copy of the duplicon, including one DXYS14 repeat array that is also highly mutable (1.2% per gamete). To explain these and other observations, we propose there is another low-rate mutation process that causes copy number change in part or all of the duplicon.     

Mammalian 5' C-rich telomeric overhangs are a mark of recombination-dependent telomere maintenance

Recent evidence for 5'-cytosine (C)-rich overhangs at the telomeres of the nematode Caenorhabditis elegans provided the impetus to re-examine the end structure of mammalian telomeres. Two-dimensional (2D) gel electrophoresis, single telomere-length analysis (STELA), and strand-specific exonuclease assays revealed the presence of a 5'-C-rich overhang at the telomeres of human and mouse chromosomes. C-overhangs were prominent in G1/S arrested as well as terminally differentiated cells, indicating that they did not represent replication intermediates. C-rich overhangs were far more prevalent in tumor cells engaged in the alternative lengthening of telomeres (ALT) pathway of telomere maintenance, which relies on the homologous recombination (HR) machinery. Transient siRNA-based depletion of the HR-specific proteins RAD51, RAD52, and XRCC3 resulted in changes in C-overhang levels, implicating the involvement of 5'-C-overhangs in the HR-dependent pathway of telomere maintenance.     

ALT- 端粒延长替代机制

端粒长度和结构的稳定与肿瘤及衰老的发生密切相关, 端粒维持机制是细胞增殖的必要条件, 端粒维持机制的激活是肿瘤细胞演化过程中的一个重要环节。这种端粒维持机制可能是通过重新激活端粒酶, 使细胞快速增殖。在端粒酶失活或不足的情况下, 也存在着一种或多种维持和增加端粒长度的机制, 统称为端粒延长替代机制(Alterative lengthening of telomere, ALT)。其特点包括: 具有不均一的端粒长度, 存在与ALT相关的PML小体(APBs)以及同源重组增加。ALT细胞内存在的ALT相关蛋白及异常活跃的同源重组为ALT机制的激活和维持提供了可能。文章综述了ALT的特征性表型、与端粒酶的相关性及其可能的发生机制。对ALT机制的深入研究将有利于阐明衰老与肿瘤之间的辩证关系。     

Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres

In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3' of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3' terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.     

Mapping Human Telomere Regions with YAC and P1 Clones: Chromosome-Specific Markers for 27 Telomeres Including 149 STSs and 24 Polymorphisms for 14 Proterminal Regions

A YAC library enriched for telomere clones was constructed and screened for the human telomere-specific repeat sequence (TTAGGG). Altogether 196 TYAC library clones were studied: 189 new TYAC clones were isolated, 149 STSs were developed for 132 different TYACs, and 39 P1 clones were identified using 19 STSs from 16 of the TYACs. A combination of mapping methods including fluorescencein situhybridization, somatic cell hybrid panels, clamped homogeneous electric fields, meiotic linkage, and BLASTN sequence analysis was utilized to characterize the resource. Forty-five of the TYACs map to 31 specific telomere regions. Twenty-four linkage markers were developed and mapped within 14 proterminal regions (12 telomeres and 2 terminal bands). The polymorphic markers include 12 microsatellites for 10 telomeres (1q, 2p, 6q, 7q, 10p, 10q, 13q, 14q, 18p, 22q) and the terminal bands of 11q and 12p. Twelve RFLP markers were identified and meiotically mapped to the telomeres of 2q, 7q, 8p, and 14q. Chromosome-specific STSs for 27 telomeres were identified from the 196 TYACs. More than 30,000 nucleotides derived from the TYAC vector-insert junction regions or from regions flanking TYAC microsatellites were compared to reported sequences using BLASTN. In addition to identifying homology with previously reported telomere sequences and human repeat elements, gene sequences and a number of ESTs were found to be highly homologous to the TYAC sequences. These genes include human coagulation factor V (F5), Wee1 protein tyrosine kinase (WEE1), neurotropic protein tyrosine kinase type 2 (NTRK2), glutathioneS-transferase (GST1), and β tubulin (TUBB). The TYAC/P1 resource, derivative STSs, and polymorphisms constitute an enabling resource to further studies of telomere structure and function and a means for physical and genetic map integration and closure.     

Unwinding protein complexes in ALTernative telomere maintenance

Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA‐binding proteins and a number of three dimensional nucleic acid structures including G‐quartets and D‐loops. A number of studies suggest that the BLM and WRN recQ‐like helicases play important roles in recombination‐mediated mechanisms of telomere elongation or A lternative L engthening of T elomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT‐associated nuclear bodies in telomerase‐negative immortalized cell lines and interact with the telomere‐specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double‐strand break repair processes such as non‐homologous end joining, homologous recombination‐mediated repair, resolution of stalled replication forks and synthesis‐dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ‐like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec‐Q‐like helicases and their role in telomere elongation during ALT processes. J. Cell. Biochem. 109: 7–15, 2010. © 2009 Wiley‐Liss, Inc.     

A ‘higher order’ of telomere regulation: telomere heterochromatin and telomeric RNAs

Protection of chromosome ends from DNA repair and degradation activities is mediated by specialized protein complexes bound to telomere repeats. Recently, it has become apparent that epigenetic regulation of the telomric chromatin template critically impacts on telomere function and telomere‐length homeostasis from yeast to man. Across all species, telomeric repeats as well as the adjacent subtelomeric regions carry features of repressive chromatin. Disruption of this silent chromatin environment results in loss of telomere‐length control and increased telomere recombination. In turn, progressive telomere loss reduces chromatin compaction at telomeric and subtelomeric domains. The recent discoveries of telomere chromatin regulation during early mammalian development, as well as during nuclear reprogramming, further highlights a central role of telomere chromatin changes in ontogenesis. In addition, telomeres were recently shown to generate long, non‐coding RNAs that remain associated to telomeric chromatin and will provide new insights into the regulation of telomere length and telomere chromatin. In this review, we will discuss the epigenetic regulation of telomeres across species, with special emphasis on mammalian telomeres. We will also discuss the links between epigenetic alterations at mammalian telomeres and telomere‐associated diseases.     

The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening

Pif1 family helicases are evolutionary conserved 5′–3′ DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.     

Survival mechanisms for Streptomyces linear replicons after telomere damage

The ability of linear replicons to propagate their DNA after telomere damage is essential for perpetuation of the genetic information they carry. We introduced deletions at specific locations within telomeres of streptomycete linear plasmids and investigated mechanisms that enable survival. Here, we report that rescue of such plasmids in Streptomyces lividans occurs by three distinct types of events: (i) repair of the damaged telomere by homologous recombination; (ii) circularization of the plasmid by non-homologous end-to-end joining; and (iii) formation of long palindromic linear plasmids that duplicate the intact telomere by a non-recombinational process. The relative frequency of use of these survival mechanisms depended on the location and length of the telomeric DNA deletion. Repair by intermolecular recombination between the telomeres of chromosomes and plasmids, deletion of additional DNA during plasmid circularization, and insertion of chromosomal DNA fragments into plasmids during end-to-end joining were observed. Our results show that damage to telomeres of Streptomyces linear replicons can promote major structural transformations in these replicons as well as genetic exchange between chromosomes and extrachromosomal DNA. Our findings also suggest that spontaneous circularization of linear Streptomyces chromosomes may be a biological response to instances of telomere damage that cannot be repaired by homologous recombination.     

RTEL1 contributes to DNA replication and repair and telomere maintenance

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.     

The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins

Most cancer cells activate telomerase to elongate telomeres and achieve unlimited replicative potential. Some cancer cells cannot activate telomerase and use telomere homologous recombination (HR) to elongate telomeres, a mechanism termed alternative lengthening of telomeres (ALT). A hallmark of ALT cells is the recruitment of telomeres to PML bodies (termed APBs). Here, we show that the SMC5/6 complex localizes to APBs in ALT cells and is required for targeting telomeres to APBs. The MMS21 SUMO ligase of the SMC5/6 complex SUMOylates multiple telomere-binding proteins, including TRF1 and TRF2. Inhibition of TRF1 or TRF2 SUMOylation prevents APB formation. Depletion of SMC5/6 subunits by RNA interference inhibits telomere HR, causing telomere shortening and senescence in ALT cells. Thus, the SMC5/6 complex facilitates telomere HR and elongation in ALT cells by promoting APB formation through SUMOylation of telomere-binding proteins.     

SUN1 is required for telomere attachment to nuclear envelope and gametogenesis in mice

Prior to the pairing and recombination between homologous chromosomes during meiosis, telomeres attach to the nuclear envelope and form a transient cluster. However, the protein factors mediating meiotic telomere attachment to the nuclear envelope and the requirement of this attachment for homolog pairing and synapsis have not been determined in animals. Here we show that the inner nuclear membrane protein SUN1 specifically associates with telomeres between the leptotene and diplotene stages during meiotic prophase I. Disruption of Sun1 in mice prevents telomere attachment to the nuclear envelope, efficient homolog pairing, and synapsis formation in meiosis. Massive apoptotic events are induced in the mutant gonads, leading to the abolishment of both spermatogenesis and oogenesis. This study provides genetic evidence that SUN1-telomere interaction is essential for telomere dynamic movement and is required for efficient homologous chromosome pairing/synapsis during mammalian gametogenesis.     

Role of mammalian Rad54 in telomere length maintenance

The homologous recombination (HR) DNA repair pathway participates in telomere length maintenance in yeast but its putative role at mammalian telomeres is unknown. Mammalian Rad54 is part of the HR machinery, and Rad54-deficient mice show a reduced HR capability. Here, we show that Rad54-deficient mice also show significantly shorter telomeres than wild-type controls, indicating that Rad54 activity plays an essential role in telomere length maintenance in mammals. Rad54 deficiency also resulted in an increased frequency of end-to-end chromosome fusions involving telomeres compared to the controls, suggesting a putative role of Rad54 in telomere capping. Finally, the study of mice doubly deficient for Rad54 and DNA-PKcs showed that telomere fusions due to DNA-PKcs deficiency were not rescued in the absence of Rad54, suggesting that they are not mediated by Rad54 activity.     

Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination

Mammalian telomeres have heterochromatic features, including trimethylated histone H3 at lysine 9 (H3K9me3) and trimethylated histone H4 at lysine 20 (H4K20me3). In addition, subtelomeric DNA is hypermethylated. The enzymatic activities responsible for these modifications at telomeres are beginning to be characterized. In particular, H4K20me3 at telomeres could be catalyzed by the novel Suv4-20h1 and Suv4-20h2 histone methyltransferases (HMTases). In this study, we demonstrate that the Suv4-20h enzymes are responsible for this histone modification at telomeres. Cells deficient for Suv4-20h2 or for both Suv4-20h1 and Suv4-20h2 show decreased levels of H4K20me3 at telomeres and subtelomeres in the absence of changes in H3K9me3. These epigenetic alterations are accompanied by telomere elongation, indicating a role for Suv4-20h HMTases in telomere length control. Finally, cells lacking either the Suv4-20h or Suv39h HMTases show increased frequencies of telomere recombination in the absence of changes in subtelomeric DNA methylation. These results demonstrate the importance of chromatin architecture in the maintenance of telomere length homeostasis and reveal a novel role for histone lysine methylation in controlling telomere recombination.     

Role for telomere cap structure in meiosis

Telomeres, the natural ends of eukaryotic chromosomes, are essential for the protection of chromosomes from end-to-end fusions, recombination, and shortening. Here we explore their role in the process of meiotic division in the budding yeast, Kluyveromyces lactis. Telomerase RNA mutants that cause unusually long telomeres with deregulated structure led to severely defective meiosis. The severity of the meiotic phenotype of two mutants correlated with the degree of loss of binding of the telomere binding protein Rap1p. We show that telomere size and the extent of potential Rap1p binding to the entire telomere are irrelevant to the process of meiosis. Moreover, we demonstrate that extreme difference in telomere size between two homologous chromosomes is compatible with the normal function of telomeres during meiosis. In contrast, the structure of the most terminal telomeric repeats is critical for normal meiosis. Our results demonstrate that telomeres play a critical role during meiotic division and that their terminal cap structure is essential for this role.     

Phylogeography of Haplotypes of Five Microsatellites Located in a Low-recombination Region of the X Chromosome: Studies Worldwide and in Brazilian Populations

We studied five microsatellites (DXS995, DXS8076, DXS8114, DXS1002 and DXS1050) located in a region of very low recombination rate in the long arm of the human X chromosome (Xq13.3–Xq21.3). No recombination was seen in 291 meioses in CEPH families. To test whether haplotypes composed of the five microsatellites could differentiate among distinct human continental populations, we studied an international panel containing 72 males from Africa, Europe, Asia and the America. Haplotypic diversity was very high within these groups and no haplotypes were shared among them. This led to the hope that we might be able to identify continent-specific lineages. However, in a median joining network there was no clear discrimination of the different continental groups. We then tested whether we could identify X chromosomal lineages from different continental origins in Brazilians. We typed 180 white Brazilians from four different geographical regions and examined their proportions of haplotype sharing with Africans, Asians, Europeans and Amerindians. No phylogeographical patterns emerged from the data. Moreover, there were several instances of the same haplotype being shared by many (and in one instance all) groups, suggesting that recombination might be occurring. We thus studied pairwise the level of linkage disequilibrium (LD) between the microsatellites. No detectable linkage disequilibrium between the most external loci DXS995 and DXS1050 was observed. Thus, even though recombination may be absent on short time spans, as seen in the CEPH pedigrees, on a long term basis it occurs often enough to dissipate all linkage disequilibrium. On the other hand, we observed very strong linkage disequilibrium between the pairs DXS995/DXS8076 and DXS1002/DXS8114, raising the possibility of resequencing the segment between them to identify single nucleotide polymorphisms (SNPs) in their intervals. The combination of X-linked microsatellites and SNPs in strong linkage disequilibrium might provide a powerful new tool to investigate human demographic history.     

Recombination can cause telomere elongations as well as truncations deep within telomeres in wild-type Kluyveromyces lactis cells

In this study, we examined the role of recombination at the telomeres of the yeast Kluyveromyces lactis. We demonstrated that an abnormally long and mutationally tagged telomere was subject to high rates of telomere rapid deletion (TRD) that preferentially truncated the telomere to near-wild-type size. Unlike the case in Saccharomyces cerevisiae, however, there was not a great increase in TRD in meiosis. About half of mitotic TRD events were associated with deep turnover of telomeric repeats, suggesting that telomeres were often cleaved to well below normal length prior to being reextended by telomerase. Despite its high rate of TRD, the long telomere showed no increase in the rate of subtelomeric gene conversion, a highly sensitive test of telomere dysfunction. We also showed that the long telomere was subject to appreciable rates of becoming elongated substantially further through a recombinational mechanism that added additional tagged repeats. Finally, we showed that the deep turnover that occurs within normal-length telomeres was diminished in the absence of RAD52. Taken together, our results suggest that homologous recombination is a significant process acting on both abnormally long and normally sized telomeres in K. lactis.