Instability of interstitial telomeric sequences in the human genome

The length variability of four human interstitial telomeric sequences (ITs) is described. Three of the ITs contain short telomeric stretches ranging between 53 and 84 bp and are localized in 21q22, 2q31, and 7q36; the fourth IT derives from the subtelomeric domain of chromosome 6p and contains a tract of a few hundred basepairs of exact and degenerate repeats. Using primers flanking the repeats, we amplified the genomic DNA from unrelated individuals and from family members, and we found that all the loci are polymorphic. At the 21q22 IT locus, two equally frequent alleles were found, while the number of alleles at the 2q31, 7q36, and 6pter IT loci was 8, 6, and 4, respectively. Sequence analysis revealed that in the three loci containing short ITs the alleles differ from one another for multiples of the hexanucleotide; it is likely that the mechanism leading to the polymorphism is DNA polymerase slippage. These loci were also unstable in gastric tumor cells characterized by microsatellite instability. At the 6pter IT locus, the four alleles range in length from about 500 to about 700 bp; this variability is probably due to unequal exchange or gene conversion. Our data indicate that stretches of exact internal telomeric repeats can be highly unstable, like microsatellites with shorter units, and that they can be useful polymorphic markers for linkage analysis, for forensic applications, and for the detection of genetic instability in tumors.     

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.     

Endings in the middle: current knowledge of interstitial telomeric sequences

Interstitial telomeric sequences (ITSs) consist of tandem repeats of the canonical telomeric repeat and are common in mammals. They are localized at intrachromosomal sites, including those repeats located close to the centromeres and those found at interstitial sites, i.e., between the centromeres and the telomeres. ITSs might originate from ancestral intrachromosomal rearrangements (inversions and fusions), from differential crossing-over or from the repair of double-strand break during evolution. Three classes of ITSs have been described in the human genome, namely, short ITSs, long subtelomeric ITSs and fusion ITSs. The fourth class of ITSs, pericentromeric ITSs, has been found in other species. The function of ITSs can be inferred from the association of heritable diseases with ITS polymorphic variants, both in copy number and sequence. This is one of the most attractive aspects of ITS studies because it leads to new and useful markers for genetic linkage studies, forensic applications, and detection of genetic instability in tumors. Some ITSs also might be hotspots of chromosome breakage, rearrangement and amplification sites, based on the type of clastogens and the nature of ITSs. This study will contribute new knowledge with respect to ITSs' biology and mechanism, prevalence of diseases, risk evaluation and prevention of related diseases, thus facilitates the design of early detection markers for diseases caused by genomic instability.     

A TRF1-controlled common fragile site containing interstitial telomeric sequences

Mouse telomeres have been suggested to resemble common fragile sites (CFS), showing disrupted TTAGGG fluorescent in situ hybridization signals after aphidicolin treatment. This “fragile” telomere phenotype is induced by deletion of TRF1, a shelterin protein that binds telomeric DNA and promotes efficient replication of the telomeric ds[TTAGGG]n tracts. Here we show that the chromosome-internal TTAGGG repeats present at human chromosome 2q14 form an aphidicolin-induced CFS. TRF1 binds to and stabilizes CFS 2q14 but does not affect other CFS, establishing 2q14 as the first CFS controlled by a sequence-specific DNA binding protein. The data show that telomeric DNA is inherently fragile regardless of its genomic position and imply that CFS can be caused by a specific DNA sequence.     

Isolation and characterization of salmonid telomeric and centromeric satellite DNA sequences

Satellite DNA clones with a 37 bp repeat unit were obtained from BglII-digested genomic DNA of Masu salmon (Oncorhynchus masou) and Chum salmon (O. keta). Fluorescence in situ hybridization (FISH) analysis with the isolated clones as a probe showed that these repetitive sequences were localized in the telomeric regions of chromosomes in both species. Southern and dot blot analyses suggested conservation of homologous sequences with similar repeat unit in other salmonids including the species of the genus Oncorhynchus and Salvelinus, but lack or scarcity of such sequences in the genus Hucho and Salmo. Similarly, polymerase chain reaction (PCR)-based cloning of satellite DNA referring to a reported Rainbow trout (O. mykiss) centromeric sequence was successful for the Oncorhynchus, Salvelinus and Hucho species. The obtained satellite DNA clones were localized with FISH in the centromeric regions of chromosomes of the species from these three genera. Although PCR cloning of the centromeric satellite DNA had failed in the Salmo species due to some base changes in the priming sites, dot blot hybridization analysis suggested conservation of homologous satellite DNA in the genus Salmo as in the other three genera. In the neighbor-joining tree of cloned centromeric satellite DNA sequences, the genus Oncorhynchus and Salvelinus formed adjacent clades, and the clade of the genus Hucho included the reported centromeric sequence of the genus Salmo. Conservation pattern and molecular phylogeny of the telomeric and centromeric satellite DNA sequences isolated herein support a close phylogenetic relationship between the genus Oncorhynchus and Salvelinus and between the Salmo and Hucho.     

Bleomycin induces delayed instability of interstitial telomeric sequences in Chinese hamster ovary cells

We analyzed the behavior of interstitial telomeric sequences (ITSs) in the progeny of Chinese Hamster Ovary (CHO) cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if ITSs play some role in the long-term clastogenic effect of this antibiotic. To this end, CHO cells were treated with a single concentration of BLM (2.5μg/ml), and the frequency of unstable chromosomal aberrations was determined at several times after treatment (18h, and 6, 15 and 34/36 days) by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations decreased on average five times 6 days after treatment compared with the one induced 18h after treatment. Moreover, no significant differences in the frequency of aberrations were observed between untreated and BLM-exposed cells at 15 or 34/36 days after treatment. These data indicate that, in terms of unstable aberrations, the in vitro clastogenic effect of BLM on CHO cells persists for at least 6 days but less than 15 days after exposure. In addition, we found that BLM induces ITSs instability, cytogenetically detectable as acentric fragments (18h after treatment) or additional (new) FISH signals (6 days after treatment). We propose that the delayed effect of BLM on ITSs mainly results from breakage of heterochromatic ITSs blocks and further insertion of these sequences at the sites of monochromatid breaks occurring at G2 phase of the cell cycle, since most of the additional FISH signals were present as single dots and located at interstitial sites of the involved chromosomes.     

Different DNA-PKcs functions in the repair of radiation-induced and spontaneous DSBs within interstitial telomeric sequences

Interstitial telomeric sequences (ITSs) in hamster cells are hot spots for spontaneous and induced chromosome aberrations (CAs). Most data on ITS instability to date have been obtained in DNA repair-proficient cells. The classical non-homologous end joining repair pathway (C-NHEJ), which is the principal double strand break (DSB) repair mechanism in mammalian cells, is thought to restore the morphologically correct chromosome structure. The production of CAs thus involves DNA-PKcs-independent repair pathways. In our current study, we investigated the participation of DNA-PKcs from the C-NHEJ pathway in the repair of spontaneous or radiation-induced DSBs in ITSs using wild-type and DNA-PKcs mutant Chinese hamster ovary cells. Our data demonstrate that DNA-PKcs stabilizes spontaneous DSBs within ITSs from the chromosome 9 long arm, leading to the formation of terminal deletions. In addition, we show that DNA-PKcs-dependent C-NHEJ is employed following radiation-induced DSBs in other ITSs and restores morphologically correct chromosomes, whereas DNA-PKcs independent mechanisms co-exist in DNA-PKcs proficient cells leading to an excess of CAs within ITSs.     

Heterochromatin and interstitial telomeric DNA homology

The purpose of this investigation was twofold. The first objective was to demonstrate that, in most of ten mammalian species commonly used in biomedical research, not all constitutive heterochromatin (C-bands) represents telomeric DNA. For example, the C-bands in human chromosomes, the long arm of the X and the entire Y chromosome of Chinese hamster, and most of the short arms of Peromyscus and Syrian hamster chromosomes are not telomeric DNA. In addition to the usual terminal telomeric DNA in the chromosomes of these mammalian species, the pericentromeric regions of seven or eight Syrian hamster chromosomes and all Chinese hamster chromosomes except pair one have pericentromeric regions that hybridize with telomeric DNA, some in C-bands and some not. The second objective was to describe a simple fluorescence in situ hybridization (FISH) reverse-printing procedure to produce black-and-white microphotographs of metaphase and interphase cells showing locations of telomeric DNA with no loss of resolution. Thus, at least three different types of heterochromatin (telomeric heterochromatin, nontelomeric heterochromatin and a combination of both) are present in these mammalian species, and this simple black-and-white reverse printing of telomeric FISH preparations can depict them economically without sacrificing clarity.     

Streptonigrin induces delayed chromosomal instability involving interstitial telomeric sequences in Chinese hamster ovary cells

We analyzed the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells exposed to the radiomimetic compound streptonigrin (SN), in order to determine whether interstitial telomeric sequences (ITSs) are involved in the long-term clastogenic effect of this antibiotic. CHO cells were treated with a single concentration of SN (100ng/ml), and the frequency of unstable chromosomal aberrations was determined at three times after treatment (18h, and 6 and 15 days) by using PNA-FISH with a pan-telomeric probe. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in SN-exposed cultures vs. untreated cultures. The percentage of damaged cells and the yield of SN-induced aberrations at 6 days after treatment increased on average twofold compared with the ones at 18h after treatment. Moreover, a significant decrease in the frequency of aberrations was observed in SN-exposed cells at 15 days after treatment, resulting in a frequency of aberrations significantly lower than the frequency of aberrations observed in the corresponding control cultures. These data indicate that SN induces delayed chromosomal instability in CHO cells, and that the in vitro clastogenic effect of this compound persists for at least 6 days but less than 15 days after treatment. In addition, we found that SN induces delayed ITSs instability, cytogenetically detectable as additional FISH signals and centromeric breaks involving dissociation of the telomeric signal 6 days after treatment. We propose that the delayed effect of SN on ITSs results from breakage of heterochromatic centromeric ITSs blocks and further insertion of these sequences at the sites of mono- or isochromatid breaks occurring at G2 or G1-S phases of the cell cycle, respectively, since most of the additional FISH signals were present as single or double dots, and located at interstitial sites of the involved chromosomes.     

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 of (TTAGG)(n) telomeric sequences among ants (Hymenoptera,Formicidae)

To determine the telomere sequence in Tapinoma nigerrimum, we carried out in situ hybridization using TTAGGG and TTAGG repeat polymerase chain reaction (PCR)-generated probes. No hybridization signals were found when TTAGGG was used as a probe. However, strong signals were observed at the end of the chromosomes with the TTAGG probe. Southern blot analysis carried out on genomic DNA using TTAGG as a probe showed a strong hybridization signal even under highly stringent conditions. Similar results were obtained in Southern blot analysis carried out on genomic DNA of 19 species of ants belonging to three different subfamilies. In accordance with all the results shown in this article, the TTAGG repeat seems to be the major component of the telomere sequence in the majority of ant species.     

DNA methylation at tobacco telomeric sequences

Majerová et al. (Plant Mol Biol, 2011) have recently reported that a considerable fraction of cytosines at tobacco telomeres is methylated. Although the data presented in this report indicate that tobacco telomeric sequences undergo certain levels of DNA methylation, it is not clear whether the methylated sequences are at telomeres, at internal chromosomal loci or at both.     

Spontaneous and radiation-induced chromosomal breakage at interstitial telomeric sites

The Chinese hamster genome contains a total of 18 cytologically detectable arrays of interstitial telometic sequences. A combination of G-banding and twocolour fluorescence in situ hybridization revealed that 25 out of 27 (93%) breakpoints of spontaneously occurring terminal deletions in four immortalized Chinese hamster cell lines were located in chromosomal regions containing interstitial telomeric sequences. Each of the four immortalized Chinese hamster cell lines expressed telomerase. Radiation experiments revealed the sensitivity of interstitial telomeric sequences to radiation-induced chromosomal breakage in all telomerase-positive cell lines. However, radiation-induced chromosomal breakage at interstitial telomeric sites in non-transformed, primary Chinese hamster cells was almost non-existent. Telomerase activity in primary Chinese hamster cells was not detected. These results indirectly suggest that interstitial telomeric sites represent a favourable substrate for chromosomal healing.     

Detection of interstitial telomeric sequences in the Arctic charr (Salvelinus alpinus) (Teleostei: Salmonidae)

Highly polymorphic Arctic charr ( Salvelinus alpinus Linnaeus, 1758) chromosomes were studied using conventional and molecular methods. The diploid chromosome number in the studied individuals was 2n = 81 or 2n = 82, with a fundamental arm number (NF) = 100. These differences are due to Robertsonian fusions. Interindividual variation in the number and size of DAPI and CMA(3) positively stained chromatin sites was observed in studied specimens. In the case of two individuals, the subtelomeric region of the long arm (q) of the largest acrocentric chromosome (chromosome number 10) was positively stained by CMA(3) fluorochrome. Both primed in situ labelling (PRINS) and fluorescence in situ hybridization (FISH) revealed that this CMA(3)-positive region was flanked by telomeric sequences. Previously, the subterminal position of interstitial telomeric sequences located in the vicinity of the CMA(3)-positive guanine-rich chromatin have been described in two other Salvelinus species, brook trout ( Salvelinus fontinalis ) and lake trout ( Salvelinus namaycush ). Moreover, multichromosomal location and variation in size of CMA(3) bands have been observed in various Salvelinus taxa, including fishes with internally located telomeric sequences. These results suggest that relocation of CMA(3)-positive chromatin segments in these species may be facilitated by flanking interstitial telomeric sequences (ITSs).     

Mapping using unique sequences

Theoretical predictions are given for the progress expected, when mapping DNA by identifying clones containing specific unique sequences. Progress is measured in three ways; however, all results depend on (dimensionless counterparts of) the number of clones and the number of unique sequences used. Furthermore, the effects of clone length dispersion are included in the theoretical predictions. Both the clones in the library and the unique sequences are assumed to be generated randomly, with uniform probability of originating at any base in the region to be mapped. The first measure of progress is the expected length fraction of the region to be mapped covered by at least one clone, when clones containing at least one unique sequence are included in the map. The second measure of progress is the expected length fraction of the region to be mapped in "covered intervals", an interval being the region between adjacent unique sequences. Alternative definitions for clones covering an interval are analyzed. The third measure of progress is the expected number of clone islands generated; an island covers successive intervals. Finally, using these measures of progress, we compare the efficiency of this new mapping strategy with conventional clone mapping strategies.     

Origin-dependent initiation of DNA replication within telomeric sequences

Replication of telomeres requires the action of telomerase, the semi-conservative replication machinery and the stabilization of the replication fork during passage through telomeric DNA. Whether vertebrate telomeres support initiation of replication has not been experimentally addressed. Using Xenopus cell free extracts we established a system to study replication initiation within linear telomeric DNA substrates. We show binding of TRF2 to telomeric DNA, indicating that exogenous DNA exclusively composed of telomeric repeats is recognized by shelterin components. Interaction with telomere binding proteins is not sufficient to prevent a DNA damage response. Notably, we observe regulated assembly of the pre-replicative complex proteins ORC2, MCM6 and Cdc6 to telomeric DNA. Most importantly, we detect origin-dependent replication of telomeric substrates under conditions that inhibit checkpoint activation. These results indicate that pre-replicative complexes assemble within telomeric DNA and can be converted into functional origins.     

Hairpin and parallel quartet structures for telomeric sequences.

The role of thymine residues in the formation of G-quartet structures for telomeric sequences has been investigated using model oligonucleotides of the type d(G4TnG4), with n = 1-4. Sequences d(G4T3G4) and d(G4T4G4) adopt a G-quartet structure formed by hairpin dimerization in 70 mM NaCl as judged by a characteristic circular dichroism signature with a 295 nm positive and 265 nm negative bands while d(G4TG4) adopts a parallel G-quartet structure like d(G12) which exhibits a strong positive band at 260 nm and a negative band at 240 nm. The sequence d(G4T2G4) exhibits a mixture of both conformations. The stability of hairpin G-quartet structures decreases with decrease in the number of intervening thymine residues. Potassium permanganate, a single strand specific probe has been used to establish the presence of loops composed of T residues in the hairpin G quartet structures formed by the oligonucleotides d(G4TnG4) with n = 2-4 in 70 mM NaCl. The formation of hairpin G quartet structure for the above sequences is further supported by the enhanced electrophoretic mobility observed on non-denaturing polyacrylamide gels. Human telomeric sequence d(TTAGGG)4 which showed enhanced electrophoretic mobility like Tetrahymena telomeric sequence d(T2G4)4 also exhibited a characteristic CD spectrum for a folded-back G-quartet structure. A detailed model for G-quartet structure involving hairpin dimer with alternating syn-anti-syn-anti conformation for the guanine residues both along the chain as well as around the G tetrad with at least two thymine residues in the loop is proposed. Intermolecular association of short telomeric sequences reported here provides a possible model for chromosomal pairing.     

Differential maintenance of DNA sequences in telomeric and centromeric heterochromatin

Repeated DNA in heterochromatin presents enormous difficulties for whole-genome sequencing; hence, sequence organization in a significant portion of the genomes of multicellular organisms is relatively unknown. Two sequenced BACs now allow us to compare telomeric retrotransposon arrays from Drosophila melanogaster telomeres with an array of telomeric retrotransposons that transposed into the centromeric region of the Y chromosome >13 MYA, providing a unique opportunity to compare the structural evolution of this retrotransposon in two contexts. We find that these retrotransposon arrays, both heterochromatic, are maintained quite differently, resulting in sequence organizations that apparently reflect different roles in the two chromosomal environments. The telomere array has grown only by transposition of new elements to the chromosome end; the centromeric array instead has grown by repeated amplifications of segments of the original telomere array. Many elements in the telomere have been variably 5'-truncated apparently by gradual erosion and irregular deletions of the chromosome end; however, a significant fraction (4 and possibly 5 or 6 of 15 elements examined) remain complete and capable of further retrotransposition. In contrast, each element in the centromere region has lost ≥ 40% of its sequence by internal, rather than terminal, deletions, and no element retains a significant part of the original coding region. Thus the centromeric array has been restructured to resemble the highly repetitive satellite sequences typical of centromeres in multicellular organisms, whereas, over a similar or longer time period, the telomere array has maintained its ability to provide retrotransposons competent to extend telomere ends.     

Guanine-rich telomeric sequences stimulate DNA polymerase activity in vitro

Guanine-rich oligonucleotides and short telomeric DNA sequences can self-associate into G-quartet stabilized complexes. We discovered that this self-association can occur in sequencing reactions and that higher-order structures stimulate DNA polymerase to synthesize extended DNA strands. Base analogues were used to identify Hoogsteen base pairings as stabilizing forces in these stimulatory DNA structures. Scanning force microscopy confirmed that quartet-DNA was formed from these oligomers and that these extended, four-stranded structures could be bound by DNA polymerase. Since guanine quartet-stabilized structures are proposed to exist in vivo, such structures may stimulate DNA polymerization in vivo.     

High-resolution mapping of interstitial telomeric repeats in Syrian hamster metaphase chromosomes

Karyotype analysis of the Syrian hamster (Mesocricetus auratus) was performed after DAPI-banding of metaphase chromosomes obtained from cultivated skin fibroblasts of a newborn animal. Fluorescence in situ hybridization with telomeric FITC-conjugated peptide nucleic acid probe was applied to map interstitial blocks of (TTAGGG)(n) repeats. Strong fluorescence in situ hybridization signals corresponded to interstitial telomeric repeats in pericentromeric chromatin bands of chromosomes 2, 4, 14, 20, and X. High-resolution DAPI-banding allowed specifying the arrangement of bands in the pericentromeric regions of these chromosomes.