The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.     

Conservation and characterization of unique porcine interstitial telomeric sequences

Telomeres are composed of TTAGGG repeats and located at the ends of chromosomes. Telomeres protect chromosomes from instability in mammals, including mice and humans. Repetitive TTAGGG sequences are also found at intrachromosomal sites, where they are named as interstitial telomeric sequences (ITSs). Aberrant ITSs are implicated in chromosomal instability and found in cancer cells. Interestingly, in pigs, vertebrate telomere sequences TTAGGG (vITSs) are also localized at the centromeric region of chromosome 6, in addition to the end of all chromosomes. Surprisingly, we found that botanic telomere sequences, TTTAGGG (bITSs), also localize with vITSs at the centromeric regions of pig chromosome 6 using telomere fluorescence in situ hybridization (FISH) and by comparisons between several species. Furthermore, the average lengths of vITSs are highly correlated with those of the terminal telomeres (TTS). Also, pig ITSs show a high incidence of telomere doublets, suggesting that pig ITSs might be unstable and dynamic. Together, our results show that pig cells maintain the conserved telomere sequences that are found at the ITSs from of plants and other vertebrates. Further understanding of the function and regulation of pig ITSs may provide new clues for evolution and chromosomal instability.     

Lack of spontaneous and radiation-induced chromosome breakage at interstitial telomeric sites in murine scid cells

Interstitial telomeric sites (ITSs) in chromosomes from DNA repair-proficient mammalian cells are sensitive to both spontaneous and radiation-induced chromosome breakage. Exact mechanisms of this chromosome breakage sensitivity are not known. To investigate factors that predispose ITSs to chromosome breakage we used murine scid cells. These cells lack functional DNA-PKcs, an enzyme involved in the repair of DNA double-strand breaks. Interestingly, our results revealed lack of both spontaneous and radiation-induced chromosome breakage at ITSs found in scid chromosomes. Therefore, it is possible that increased sensitivity of ITSs to chromosome breakage is associated with the functional DNA double-strand break repair machinery. To investigate if this is the case we used scid cells in which DNA-PKcs deficiency was corrected. Our results revealed complete disappearance of ITSs in scid cells with functional DNA-PKcs, presumably through chromosome breakage at ITSs, but their unchanged frequency in positive and negative control cells. Therefore, our results indicate that the functional DNA double-strand break machinery is required for elevated sensitivity of ITSs to chromosome breakage. Interestingly, we observed significant differences in mitotic chromosome condensation between scid cells and their counterparts with restored DNA-PKcs activity suggesting that lack of functional DNA-PKcs may cause a defect in chromatin organization. Increased condensation of mitotic chromosomes in the scid background was also confirmed in vivo. Therefore, our results indicate a previously unanticipated role of DNA-PKcs in chromatin organisation, which could contribute to the lack of ITS sensitivity to chromosome breakage in murine scid cells.     

Absence of yKu/Hdf1 but not myosin-like proteins alters chromosome dynamics during prophase I in yeast

Abstract Meiosis is central to the formation of haploid gametes or spores in that it segregates homologous chromosomes and halves the chromosome number. A prerequisite of this genome bisection is the pairing of homologous chromosomes during the first meiotic prophase. When budding yeast cells are induced to undergo meiosis, this has profound consequences for nuclear structure: after premeiotic DNA replication centromeres disperse, while telomeres move about the nuclear periphery and temporarily cluster during the leptotene/zygotene transition (bouquet stage) of the prophase to first meiotic division. In vegetative cells, Hdf1p (yKu) and the myosin-like proteins Mlp1p and Mlp2p have been suggested to contribute to the organization of silent chromatin, tethering of telomeres to the nuclear periphery, DNA repair, and telomere maintenance. Here, we investigated by molecular cytology whether yKu and Mlp proteins contribute to telomere and chromosome dynamics in meiosis. It was found that mlp1 Δ mlp2 Δ double-mutant cells undergo centromere dispersion, telomere clustering, homologue pairing, and sporulation like wild type. On the other hand, cells deficient for yKu underwent meiosis-specific chromosomal events with a delay, while they eventually sporulated like wild type. These results suggest that the absence of yKu not only affects vegetative nuclear architecture ( Laroche et al., 1998 ) but also interferes with the ordered occurrence of chromosome dynamics during first meiotic prophase.     

On the chromatin structure of eukaryotic telomeres

Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed.Key words: telomeres, subtelomeres, euchromatin, heterochromatin, ChIP, immunolocalizationTelomeric DNA usually contains tandem repeats of a short GC rich motif. The number of repeats and, therefore, the length of telomeres is subject to regulation and influences relevant biological processes like aging and cancer.^1–3 In situ hybridization studies have revealed that telomeric repeats are also present at interstitial chromosomal loci.^4,5 An analysis of the genome sequence from different eukaryotes indicates that ITSs have a widespread distribution in different model systems including zebrafish, chicken, opossum, mouse, dog, cattle, horse, human, rice, poplar or Arabidopsis (see Fig. 1 for an example; www.ncbi.nlm.nih.gov/mapview). These ITSs have been related to chromosomal aberrations, fragile sites, hot spots for recombination and diseases caused by genomic instability, although their functions remain unknown.⁶Open in a separate windowFigure 1Distribution of the main telomeric repeat arrays in the genome of several model organisms. These representations have been performed by using the megaBLAST program and the all assemblies genomic databases at NCBI (www.ncbi.nlm.nih.gov/mapview). Searches for homology with 100 tandem telomeric repeats were done using the default parameters except that the expected threshold was set to 10 and the filters were turned off. Chromosomes are represented as vertical bars and numbered at the bottom. The horizontal bars represent the telomeric repeat arrays. Colors indicate the BLAST scores (red ≥200; pink 80–200; green 50–80).Telomeres and ITSs have probably cross talk through evolution. In some instances, ITSs could have been generated by telomeric fusions. Pioneering studies performed by Hermann J. Muller in Drosophila and Barbara McClintock in maize showed that newly formed chromosome ends tend to fuse giving rise to the so-called breakage-fusion-bridge cycle.^7,8 This cycle can lead to stable chromosomal reorganizations after healing of the broken ends. In addition, Muller and McClintock found that, unlike these newly formed broken chromosome ends, natural chromosomal ends are quite stable and do not tend to fuse.⁹ It is currently known that telomere dysfunction due to mutations that cause telomeric shortening or abolish the expression of certain telomeric proteins can lead to telomeric fusions, anaphase bridges and genome reorganizations.^1–3,10,11 Therefore, telomeric shortening or alterations of telomeric chromatin structure might be expected to generate ITSs through evolution by promoting telomeric fusions.¹² ITSs might also originate through the activity of telomerase during the repair process of double strand breaks or by recombination.^13–16 In addition, telomerase activity might lead to the formation of new telomeres by healing of chromosome breaks within internal telomeric repeats and even within other sequences.^17–19 This process of healing involves the acquisition of telomeric chromatin structure.DNA folds into two major chromatin organizations inside the cell nucleus: heterochromatin and euchromatin. Heterochromatin is highly condensed in interphase nuclei and is usually associated with repetitive and silent DNA. By contrast, euchromatin has an open conformation and is often related to the capacity to be transcribed. Both kinds of chromatin exhibit defined epigenetic modifications that influence their biochemical behavior. Thus, the study of these epigenetic marks is an issue of major interest.The chromatin structures of telomeres and ITSs might be different. Therefore, they should be studied independently. Chromatin structure analyses are usually performed by immunocytolocalization or by chromatin immunoprecipitation (ChIP).^20–23 Special care should be taken when the epigenetic status of telomeres is analyzed by immunocytolocalization. This technique does not allow differentiating between telomeres and subtelomeric regions. Since subtelomeric regions are known to be heterochromatic in many eukaryotic organisms, heterochromatic marks should be immunolocalized at the chromosome ends of these organisms. However, these marks could correspond to subtelomeric regions and not to telomeres.The ChIP technique implies the immunoprecipitation of chromatin with specific antibodies and the further analysis of the immunoprecipitated DNA. DNA sequences immunoprecipitated by a specific antibody are thought to associate in vivo with the feature recognized by this antibody. Whereas the enrichment of single copy sequences in the immunoprecipitated DNA has been usually analyzed by quantitative PCR, the analyses of repetitive DNA sequences have been often performed by hybridization. Thus, multiple telomeric chromatin structure analyses have been performed by hybridizing immunoprecipitated DNA with a telomeric probe. However, these analyses displayed simultaneously the chromatin structures of telomeres and ITSs. High throughput sequencing analyses of the immunoprecipitated DNA might help overcome this problem. Nevertheless, since the reads obtained with these techniques at present are short, it is still difficult to ascertain whether the enrichment of immunoprecipitated telomeric sequences corresponds to telomeres or to ITSs. Third-generation long-read accurate technologies and new algorithms that discriminate between telomeres and ITSs should solve the problem.In principle, the combination of immunocytolocalization and ChIP experiments should help to differentiate between telomeres and ITSs. However, since subtelomeric regions are known to influence telomere function and contain degenerated ITSs, at least in some organisms like humans or Arabidopsis, this may not be necessarily true.⁶ A specific epigenetic mark might be required for telomere function, found associated with telomeric repeats by ChIP and with the end of chromosomes by immunocytolocalization and still not associate with true telomeres but with subtelomeric regions and ITSs or just with subtelomeric ITSs.An alternative way to analyze the chromatin structure of telomeres by ChIP involves the use of frequently cutting restriction enzymes. The chromatin structures of Arabidopsis telomeres and ITSs have been independently studied by using Tru9I, a restriction enzyme that recognizes the sequence TTAA.²⁴ Since telomeres in Arabidopsis and in other model systems are composed of perfect telomeric repeat arrays, they remain uncut after digestion with Tru9I.²⁵ In contrast, Arabidopsis ITSs are frequently cut because they are composed of short arrays of perfect telomeric repeats interspersed with degenerated repeats.^25–28 Thus, when Arabidopsis genomic DNA is digested with Tru9I and hybridized with a telomeric probe, most of the signals corresponding to ITSs disappear.²⁵ The use of Tru9I has made possible to discover that Arabidopsis telomeres exhibit euchromatic features. In contrast, Arabidopsis ITSs and subtelomeric regions are heterochromatic.²⁴ In Arabidopsis, heterochromatin is characterized by cytosine methylation, which can be targeted at CpG, CpNpG or CpNpN residues (where N is any nucleotide), and by H3K9me1,2, H3K27me1,2 and H4K20me1. In turn, Arabidopsis euchromatin is characterized by H3K4me1,2,3, H3K36me1,2,3, H4K20me2,3 and by histones acetylation.²⁹ ChIP experiments processed with Tru9I have revealed that Arabidopsis telomeres have high levels of euchromatic marks (H3K4me2, H3K9 and H4K16 acetylation) and low levels of heterochromatic marks (H3K9me2, H3K27me1 and DNA methylation).²⁴ Therefore, Arabidopsis telomeres exhibit epigenetic modifications characteristic of euchromatin.Different studies in mice, humans or Arabidopsis have reported that telomeres are heterochromatic based on the existence of siRNAs containing telomeric sequences, on the association of telomeric sequences with telomeric and with heterochromatin proteins, on the methylation of telomeric sequences or on the histones modifications associated with telomeric sequences.^30–34 However, the experiments presented in those studies addressed simultaneously the chromatin organizations of telomeres and subtelomeric regions or of telomeres and ITSs. Telomeres have also been reported to be heterochromatic based on the existence of the so-called TElomeric Repeat containing RNAs (TERRA), which are present in different eukaryotes.³⁵ At telomeric regions, TERRA are transcribed from subtelomeric promoters towards chromosome ends. Since human subtelomeric TERRA are mostly composed of subtelomeric sequences, with only about 200 bp of telomeric sequences at their 3′ ends, they might be related to subtelomeric heterochromatin formation rather than to the formation of telomeric chromatin. Nevertheless, TERRA interact with human telomeric proteins and influence telomere function. In addition, TERRA might also be related to ITSs heterochromatinization.^34,35We believe that the scenario found in Arabidopsis could also be found in other model systems if the chromatin structures of telomeres, subtelomeric regions and ITSs are independently analyzed. Several reports have described the presence of histone H3.3 at mice telomeres.^36–39 Since this histone variant has been previously associated with active chromatin, these studies are compatible with a euchromatic organization of telomeres. However, again in these reports, the experiments shown addressed simultaneously the chromatin organization of telomeres and subtelomeric regions or of telomeres and ITSs. In general terms, we believe that a clear distinction between telomeres and ITSs should be established when future ChIP experiments are analyzed. The use of third generation high throughput sequencing technologies or of frequently cutting restriction enzymes might help in this task.As mentioned above, the epigenetic modifications associated with telomeric regions are known to be important for telomere function. These modifications are required to provide genome stability.³³ In this context, it will be relevant to ascertain how the function of Arabidopsis telomeres is influenced by their euchromatic marks and by the presence of heterochromatin at subtelomeric regions.     

Telomeres act autonomously in maize to organize the meiotic bouquet from a semipolarized chromosome orientation

During meiosis, chromosomes undergo large-scale reorganization to allow pairing between homologues, which is necessary for recombination and segregation. In many organisms, pairing of homologous chromosomes is accompanied, and possibly facilitated, by the bouquet, the clustering of telomeres in a small region of the nuclear periphery. Taking advantage of the cytological accessibility of meiosis in maize, we have characterized the organization of centromeres and telomeres throughout meiotic prophase. Our results demonstrate that meiotic centromeres are polarized prior to the bouquet stage, but that this polarization does not contribute to bouquet formation. By examining telocentric and ring chromosomes, we have tested the cis-acting requirements for participation in the bouquet. We find that: (a) the healed ends of broken chromosomes, which contain telomere repeats, can enter the bouquet; (b) ring chromosomes enter the bouquet, indicating that terminal position on a chromosome is not necessary for telomere sequences to localize to the bouquet; and (c) beginning at zygotene, the behavior of telomeres is dominant over any centromere-mediated chromosome behavior. The results of this study indicate that specific chromosome regions are acted upon to determine the organization of meiotic chromosomes, enabling the bouquet to form despite large-scale changes in chromosome architecture.     

New telomere formation during the process of chromatin diminution in Ascaris suum

Chromatin diminution in the parasitic nematode Ascaris suum represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. At the molecular level, it is a rather complex event including chromosome breakage, new telomere formation and DNA degradation. Analysis of a cloned somatic telomere (pTel1) revealed that it has been newly created during the process of chromatin diminution by the addition of telomeric repeats (TTAGGC)n to a chromosomal breakage site (Müller et al., 1991). However, telomere addition does not occur at a single chromosomal locus, but at many different sites within a short chromosomal region, termed CBR1 (chromosomal breakage region 1). Here we present the cloning and the analysis of 83 different PCR amplified telomere addition sites from the region of CBR1. The lack of any obvious sequence homology shared among them argues for a telomerase-mediated healing process, rather than for a recombinational event. This hypothesis is strongly supported by the existence of 1-6 nucleotides corresponding to and being in frame with the newly added telomeric repeats at almost all of the telomere addition sites. Furthermore, we show that telomeres are not only added to the ends of the retained chromosomal portions, but also to the eliminated part of the chromosomes, which later on become degraded in the cytoplasm. This result suggests that de novo telomere formation during the process of chromatin diminution represents a non-specific process which can heal any broken DNA end.     

On the chromatin structure of eukaryotic telomeres

Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed.     

Modulation of telomere length dynamics by the subtelomeric region of tetrahymena telomeres

Tetrahymena telomeres usually consist of approximately 250 base pairs of T(2)G(4) repeats, but they can grow to reach a new length set point of up to 900 base pairs when kept in log culture at 30 degrees C. We have examined the growth profile of individual macronuclear telomeres and have found that the rate and extent of telomere growth are affected by the subtelomeric region. When the sequence of the rDNA subtelomeric region was altered, we observed a decrease in telomere growth regardless of whether the GC content was increased or decreased. In both cases, the ordered structure of the subtelomeric chromatin was disrupted, but the effect on the telomeric complex was relatively minor. Examination of the telomeres from non-rDNA chromosomes showed that each telomere exhibited a unique and characteristic growth profile. The subtelomeric regions from individual chromosome ends did not share common sequence elements, and they each had a different chromatin structure. Thus, telomere growth is likely to be regulated by the organization of the subtelomeric chromatin rather than by a specific DNA element. Our findings suggest that at each telomere the telomeric complex and subtelomeric chromatin cooperate to form a unique higher order chromatin structure that controls telomere length.     

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.     

HOT1 is a mammalian direct telomere repeat‐binding protein contributing to telomerase recruitment

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase–telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC‐based DNA–protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere‐binding protein 1 (HOT1). HOT1 directly and specifically binds double‐stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase‐dependent telomere elongation.     

Closed chromatin loops at the ends of chromosomes

The termini of eukaryotic chromosomes contain specialized protective structures, the telomeres, composed of TTAGGG repeats and associated proteins which, together with telomerase, control telomere length. Telomere shortening is associated with senescence and inappropriate telomerase activity may lead to cancer. Little is known about the chromatin context of telomeres, because, in most cells, telomere chromatin is tightly anchored within the nucleus. We now report the successful release of telomere chromatin from chicken erythrocyte and mouse lymphocyte nuclei, both of which have a reduced karyoskeleton. Electron microscopy reveals telomere chromatin fibers in the form of closed terminal loops, which correspond to the "t-loop" structures adopted by telomere DNA. The ability to recognize isolated telomeres in their native chromatin conformation opens the way for detailed structural and compositional studies.     

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.     

Telomere binding of the Rap1 protein is required for meiosis in fission yeast.

Telomeres are essential for chromosome integrity, protecting the ends of eukaryotic linear chromosomes during cell proliferation. Telomeres also function in meiosis; a characteristic clustering of telomeres beneath the nuclear membrane is observed during meiotic prophase in many organisms from yeasts to plants and humans, and the role of the telomeres in meiotic pairing and the recombination of homologous chromosomes has been demonstrated in the fission yeast Schizosaccharomyces pombe and in the budding yeast Saccharomyces cerevisiae. Here we report that S. pombe Rap1 is a telomeric protein essential for meiosis. While Rap1 is conserved in budding yeast and humans, schemes for telomere binding vary among species: human RAP1 binds to the telomere through interaction with the telomere binding protein TRF2; S. cerevisiae Rap1, however, binds telomeric DNA directly, and no orthologs of TRF proteins have been identified in this organism. In S. pombe, unlike in S. cerevisiae, an ortholog of human TRF has been identified. This ortholog, Taz1, binds directly to telomere repeats [18] and is necessary for telomere clustering in meiotic prophase. Our results demonstrate that S. pombe Rap1 binds to telomeres through interaction with Taz1, similar to human Rap1-TRF2, and that Taz1-mediated telomere localization of Rap1 is necessary for telomere clustering and for the successful completion of meiosis. Moreover, in taz1-disrupted cells, molecular fusion of Rap1 with the Taz1 DNA binding domain recovers telomere clustering and largely complements defects in meiosis, indicating that telomere localization of Rap1 is a key requirement for meiosis.     

Contribution of telomerase RNA retrotranscription to DNA double-strand break repair during mammalian genome evolution

Background  

In vertebrates, tandem arrays of TTAGGG hexamers are present at both telomeres and intrachromosomal sites (interstitial telomeric sequences (ITSs)). We previously showed that, in primates, ITSs were inserted during the repair of DNA double-strand breaks and proposed that they could arise from either the capture of telomeric fragments or the action of telomerase.     

Extent and variability of interstitial telomeric sequences and their effects on estimates of telomere length

Telomeres often shorten with time, although this varies between tissues, individuals and species, and their length and/or rate of change may reflect fitness and rate of senescence. Measurement of telomeres is increasingly important to ecologists, yet the relative merits of different methods for estimating telomere length are not clear. In particular the extent to which interstitial telomere sequences (ITSs), telomere repeats located away from chromosomes ends, confound estimates of telomere length is unknown. Here we present a method to estimate the extent of ITS within a species and variation among individuals. We estimated the extent of ITS by comparing the amount of label hybridized to in‐gel telomere restriction fragments (TRF) before and after the TRFs were denatured. This protocol produced robust and repeatable estimates of the extent of ITS in birds. In five species, the amount of ITS was substantial, ranging from 15% to 40% of total telomeric sequence DNA. In addition, the amount of ITS can vary significantly among individuals within a species. Including ITSs in telomere length calculations always underestimated telomere length because most ITSs are shorter than most telomeres. The magnitude of that error varies with telomere length and is larger for longer telomeres. Estimating telomere length using methods that incorporate ITSs, such as Southern blot TRF and quantitative PCR analyses reduces an investigator's power to detect difference in telomere dynamics between individuals or over time within an individual.     

Induction of telomere-mediated chromosomal truncation and stability of truncated chromosomes in Arabidopsis thaliana

Minichromosomes possess functional centromeres and telomeres and thus should be stably inherited. They offer an enormous opportunity to plant biotechnology as they have the potential to simultaneously transfer and stably express multiple genes. Segregating independently of host chromosomes, they provide a platform for accelerating plant breeding. Following a top‐down approach, we truncated endogenous chromosomes in Arabidopsis thaliana by Agrobacterium‐mediated transfer of T‐DNA constructs containing telomere sequences. Blocks of A. thaliana telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into the natural tetraploid A. thaliana accession Wa‐1, chromosome truncation by T‐DNA‐induced de novo formation of telomeres could be confirmed by DNA gel blot analysis, PCR (polymerase chain reaction), and fluorescence in situ hybridisation. The addition of new telomere repeats in this process could start alternatively from within the T‐DNA‐derived telomere repeats or from adjacent sequences close to the right border of the T‐DNA. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than expected according to Mendelian rules.     

Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment

Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization. A telomere-binding protein complex from human sperm (hSTBP) has been isolated by detergent treatment and was partially purified. hSTBP specifically binds double-stranded telomeric DNA and does not contain known somatic telomere proteins TRF1, TRF2, and Ku. Surprisingly, the essential component of this complex has been identified as a specific variant of histone H2B. Indirect immunofluorescence shows punctate localization of H2B in sperm nuclei, which in part coincides with telomeric DNA localization established by fluorescent in situ hybridization. Anti-H2B antibodies block interactions of hSTBP with telomere DNA, and spH2B forms specific complex with this DNA in vitro, indicating that this protein plays a role in telomere DNA recognition. We propose that hSTBP participates in the membrane attachment of telomeres that may be important for ordered chromosome withdrawal after fertilization.     

A complex network of interactions governs DNA methylation at telomeric regions

DNA methylation modulates telomere function. In Arabidopsis thaliana, telomeric regions have a bimodal chromatin organization with unmethylated telomeres and methylated subtelomeres. To gain insight into this organization we have generated TAIR10-Tel, a modified version of the Arabidopsis reference genome with additional sequences at most chromosome ends. TAIR10-Tel has allowed us to analyse DNA methylation at nucleotide resolution level in telomeric regions. We have analysed the wild-type strain and mutants that encode inactive versions of all currently known relevant methyltransferases involved in cytosine methylation. These analyses have revealed that subtelomeric DNA methylation extends 1 to 2 kbp from Interstitial Telomeric Sequences (ITSs) that abut or are very near to telomeres. However, DNA methylation drops at the telomeric side of the telomere-subtelomere boundaries and disappears at the inner part of telomeres. We present a comprehensive and integrative model for subtelomeric DNA methylation that should help to decipher the mechanisms that govern the epigenetic regulation of telomeres. This model involves a complex network of interactions between methyltransferases and subtelomeric DNA sequences.