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.     

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.     

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.     

Hypomethylating drugs efficiently decrease cytosine methylation in telomeric DNA and activate telomerase without affecting telomere lengths in tobacco cells

Telomere homeostasis is regulated at multiple levels, including the local chromatin structure of telomeres and subtelomeres. Recent reports demonstrated that a decrease in repressive chromatin marks, such as levels of cytosine methylation in subtelomeric regions, results in telomere elongation in mouse cells. Here we show that a considerable fraction of cytosines is methylated not only in subtelomeric, but also in telomeric DNA of tobacco BY-2 cells. Drug-induced hypomethylation (demonstrated at subtelomeric, telomeric, and global DNA levels) results in activation of telomerase. However, in contrast to mouse cells, the decrease in 5-methylcytosine levels and upregulation of telomerase do not result in any changes of telomere lengths. These results demonstrate the involvement of epigenetic mechanisms in the multilevel process of regulation of telomerase activity in plant cells and, at the same time, they indicate that changes in telomerase activity can be overridden by other factors governing telomere length stability.     

Non-symmetrical cytosine methylation in tobacco pollen DNA

We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region, 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1×m⁵CpTpC, 1×m⁵CpGpT, 1×m⁵CpCpT, 2×m⁵CpTpT, 2×m⁵CpGpG, and 3×m⁵CpApT of which only m⁵CpGpG and m⁵CpGpT fitted the consensus sequence for symmetrical methylation in plants.     

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.     

Protection of DNA ends by telomeric 3' G-tail sequences

The extreme ends of eukaryotic chromosomes contain 3' extensions in the form of single-stranded G-rich repeats, referred to as telomeric 3' G-tails or overhangs. Increasing evidence has suggested that telomeric 3' G-tails can adopt a G-quadruplex conformation both in vitro and in vivo. However, the role of G-quadruplexes on the structure and function of telomeric 3' G-tails remains unclear. In the current study, we showed that the human telomeric 3' G-tail sequence protected the duplex DNA ends in cis from being recognized as double strand breaks. This protection is dependent on the G-quadruplex conformation of the 3' G-tail sequence. These results suggest that the ability of telomeric 3' G-tails to adopt the endprotecting G-quadruplex conformation may be one of the reasons for the existence of the evolutionarily conserved G-stretch motifs in telomeric DNA sequences.     

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.     

Pericentromeric location of the telomeric DNA sequences on the European grayling chromosomes

The chromosomal characteristics, locations and variations of the C-band positive heterochromatin and telomeric DNA sequences were studied in the European grayling karyotype (Thymallus thymallus, Salmonidae) using conventional C-banding, endonucleases digestion banding, silver nitrate (AgNO₃), chromomycin A₃ and 4′,6-diamidino-2-phenylindole staining techniques as well as fluorescence in situ hybridization (FISH) and primed in situ labelling. Original data on the chromosomal distribution of segments resistant to AluI restriction endonuclease and identification of the C-banded heterochromatin presented here have been used to characterize the grayling karyotype polymorphism. Structural and length polymorphism of the chromosome 21 showing a conspicuous heterochromatin block adjacent to the centromere seems to be the result of the deletion and inversion. Two pairs of nuclear organizer regions (NOR)-bearing chromosomes were found to be polymorphic in size and displaying several distinct forms. FISH with telomeric peptide nucleic acid probe enabled recognition of the conservative telomeric DNA sequences. The karyotype of the thymallid fish is thought to experienced numerous pericentric inversions and internal telomeric sites (ITSs) observed at the pericentromeric regions of the six European grayling metacentric chromosomes are likely relics of the these rearrangements. None of the ITS sites matched either chromosome 21 or NOR bearing chromosomes.     

Analysis and location of a rice BAG clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG) n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescence in situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.     

Analysis and location of a rice BAG clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)_n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG)_n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescencein situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.     

Analysis and location of a rice BAC clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)_n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG)_n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescencein situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.     

Tissue specific methylation of human Y chromosomal DNA sequences

This report describes two moderately repetitive human Y chromosomal DNA sequences isolated from a flow sorted Y chromosonal library. These sequences are present in XY male and XY female DNAs but absent in XX male and XX female DNAs. Genomic Southern blot analysis against DNAs isolated from different tissues showed tissue specific DNA methylation patterns. In contrast to the 2.1 kb Hae III repeats which are hypomethylated in sperm DNA, the moderately repetitive sequences used in this study are highly methylated in sperm, less methylated in blood and brain and least methylated in placental DNA.     

Interstitial telomeric sequences at the junction site of a jumping translocation

The mechanism(s) for the origin of jumping translocations (JTs) are unknown. To assess the possible involvement of telomeric sequences in the jumping process, metaphases of a patient with hydrops fetalis having a JT were analyzed for the presence of interstitial telomeres. Telomere DNA sequences were detected at the junction sites of the donor and the recipient chromosomes. Interstitial telomeric sequences have so far only been detected in JTs involving chromosome 15q in patients with Prader-Willi syndrome. Our finding of interstitial telomeric sequences in a JT with a chromosome different from chromosome arm 15q in a patient without Prader-Willi syndrome implies that telomere sequences may be common to all telomeric JTs. The possible role of telomeric sequences as a cause of the observed chromosomal mosaicism is discussed. Received: 24 September 1996 / Revised: 15 December 1996     

The DNA sequence specificity of bleomycin cleavage in telomeric sequences in human cells

Bleomycin is an antibiotic drug that is widely used in cancer chemotherapy. Telomeres are located at the ends of chromosomes and comprise the tandemly repeated DNA sequence (GGGTTA) _n in humans. Since bleomycin cleaves DNA at 5??-GT dinucleotide sequences, telomeres are expected to be a major target for bleomycin cleavage. In this work, we determined the DNA sequence specificity of bleomycin cleavage in telomeric sequences in human cells. This was accomplished using a linear amplification procedure, a fluorescently labelled oligonucleotide primer and capillary gel electrophoresis with laser-induced fluorescence detection. This represents the first occasion that the DNA sequence specificity of bleomycin cleavage in telomeric DNA sequences in human cells has been reported. The bleomycin DNA sequence selectivity was mainly at 5??-GT dinucleotides, with lesser amounts at 5??-GG dinucleotides. The cellular bleomycin telomeric DNA damage was also compared with bleomycin telomeric damage in purified human genomic DNA and was found to be very similar. The implications of these results for the understanding of bleomycin??s mechanism of action in human cells are discussed.