Preferential binding of a G-quadruplex ligand to human chromosome ends

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, ³H-360A (2,6-N,N′-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-³H]. We verified the in vitro selectivity of ³H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that ³H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3′-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with ³H-360A. We found that ³H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.     

Major cutbacks at chromosome ends

To distinguish a telomere from a double-strand break, a minimum number of telomere repeats must 'cap' each chromosome end. The length of each repeat array will reflect a unique history of addition and losses. Telomere losses are predicted to occur slowly but surely with every replication cycle (referred to as 'typical' telomere loss) in addition to intermittently and, potentially, rapidly ('sporadic'). Recent studies have shown that sporadic telomere losses can result from failure to properly repair (oxidative) damage to telomeric DNA, from failure to properly process higher-order structures of G-rich DNA and from homologous recombination reactions. Differences in telomere-erosion pathways between normal and malignant cells provide novel targets for the prevention and therapy of disease.     

Acentric chromosome ends are prone to fusion with functional chromosome ends through a homology-directed rearrangement

The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR.     

Equine FISH mapping of 36 genes known to locate on human chromosome ends

The INRA and the CHORI-241 horse BAC libraries were screened by hybridization with DNA probes and/or directly by PCR with primers designed in consensus sequences of genes localized at the end of each human chromosome. BAC clones were retrieved and 36 could be FISH mapped after the expected gene was confirmed in each BAC by sequencing. Our results show that 16 BACs can be considered to be at telomeric or centromeric positions in the horse and 15 were found at the boundary of actually defined conserved segments even-though often located within conserved syntenic fragments between horse and human. There is no straightforward relation between the end position of a marker in human and its end position in the horse. A gene was first anchored to ECA27 by FISH mapping. The localization of these markers expands the cytogenetic map of the horse and will serve as anchors for the integrated and future physical maps. It should also help to better understand the different chromosomal rearrangements that occurred during evolution of genomes derived from a common ancestral karyotype.     

In vitro and in vivo reconstitution and stability of vertebrate chromosome ends.

Telomeres are essential repetitive sequences at the ends of chromosomes that prevent chromosome fusion and degradation. We have successfully recapitulated these two protective functions in vivo and in vitro by incubating blunt-end DNA constructs having vertebrate telomeric ends in Xenopus eggs and egg extracts. Constructs with telomeric ends are stable as linear molecules; constructs with non-telomeric ends undergo intramolecular fusion. In extracts, 99.8% of the telomeric constructs from 78 to 700 bp in length are assembled into 'model telomeres' in <5 min and have an extra-polated half-life of >3.5 years. Non-telomeric constructs circularize with first order kinetics and a half-life of 4 h. In living eggs the telomeric constructs are protected from fusion and degradation. The stability of the telomeric constructs is not due to covalent processing. Extract can protect approximately 100 pM telomeric ends (equivalent to 1.7 x 10(7) ends/egg) even in the presence of a 20-fold excess of double-stranded telomeric DNA, suggesting that protection requires end-specific factors. Constructs with (TTGGGG) n repeats are unstable, suggesting that short tracts of this and other telomere-like sequences found within human telomeres could lead to genome instability if exposed by partial telomere erosion during aging.     

The Saccharomyces retrotransposon Ty5 influences the organization of chromosome ends.

Retrotransposons are ubiquitous components of eukaryotic genomes suggesting that they have played a significant role in genome organization. In Saccharomyces cerevisiae, eight of 10 endogenous insertions of the Ty5 retrotransposon family are located within 15 kb of chromosome ends, and two are located near the subtelomeric HMR locus. This genomic organization is the consequence of targeted transposition, as 14 of 15 newly transposed Ty5 elements map to telomeric regions on 10 different chromosomes. Nine of these insertions are within 0.8 kb and three are within 1.5 kb of the autonomously replicating consensus sequence in the subtelomeric X repeat. This suggests that the X repeat plays an important role in directing Ty5 integration. Analysis of endogenous insertions from S.cerevisiae and its close relative S.paradoxus revealed that only one of 12 insertions has target site duplications, indicating that recombination occurs between elements. This is further supported by the observation that Ty5 insertions mark boundaries of sequence duplications and rearrangements in these species. These data suggest that transposable elements like Ty5 can shape the organization of chromosome ends through both transposition and recombination.     

Role of the telomeric DNA-binding protein TRF2 in the stability of human chromosome ends

A major issue in telomere research is to understand how the integrity of chromosome ends is preserved. A recent study shows that expression of a dominant-negative form of the human telomeric protein TRF2 increases the number of chromosome fusions in immortalized cells and decreases the quantity of G-rich telomeric DNA 3' overhang, the G tail.⁽¹⁾ Consequently, TRF2 appears to control the structure of the very end of the chromosomal DNA molecule and to prevent recombination between two telomeres. Remarkably, the same study reveals a potential role of TRF2 in cell division control. BioEssays 20:879–883, 1998. © 1998 John Wiley & Sons, Inc.     

Telomeric chromatin: replicating and wrapping up chromosome ends

Recent advances in our understanding of the specialized chromatin structure at telomeres, the ends of eukaryotic chromosomes, have focused on three separate areas: replication of telomeres through the coordinated action of conventional DNA polymerases and the telomerase enzyme, protection of the chromosome end from DNA damage checkpoint sensors and DNA-repair processes, and the discovery of a novel deacetylase enzyme (Sir2p) required for the establishment and maintenance of telomeric heterochromatin. Although the number of proteins and the complexity of their interactions at telomeres continues to grow, a picture of at least some of the major players and mechanisms underlying telomere replication, end 'capping' and chromatin assembly is beginning to emerge.     

Structural variability of human chromosome 9 in relation to its evolution

Summary Human chromosome 9 shows a high susceptibility for structural rearrangements, particularly pericentric inversions, which often are transmitted. Three types of pericentric inversions can be observed on No. 9: 1) Type I, showing the total constitutive heterochromatin in the short arm. 2) Type II with part of the C heterochromatin on the short arm, the rest located on the long arm proximal to the centromere. 3) Type III: a subtelocentric chromosome with part of the C heterochromatin in the very short arm and the rest located interstitially on the long arm. With these inversions as well as with other structural rearrangements, e.g. translocations, the break-points are located preferentially within the C heterochromatin or close to the heterochromatic-euchromatic junctions. These findings are in contrast to the findings in lymphocytes from 5 patients with Fanconi's anemia and after irradiation in vitro, reported in the literature. In lymphocytes break-points seem to be distributed more or less by chance. These observations together led us to speculate that human chromosome 9 primarily was an acrocentric chromosome; in morphology and at least in some functions similar to D-and G-group chromosomes. During evolution this acrocentric chromosome changed to a submetacentric one due to a pericentric inversion.The author is sponsored by the Deutsche Forschungsgemeinschaft.     

Physical mapping of duplicated genomic regions of two chromosome ends in rice.

Two genomic regions duplicated in distal ends of the short arms of chromosomes 11 and 12 in rice (Oryza sativa L.) were characterized by YAC ordering with 46 genetic markers. Physical maps covering most of the duplicated regions were generated. Thirty-five markers, including 21 rice cDNA clones, showed the duplicated loci arrayed strictly in the same order along the two specific genomic regions. Regardless of their different genetic distances, the two duplicated segments may have a similar and minimum physical size with an expected length of about 2.5 Mb. However, differences of RFLP frequency for the duplicated DNA copies and recombination frequency for a given homoeologous area between the two regions were observed, indicating that these changes in genome organization occurred after the duplication. Our results establish a good model system for resolving the relationships between gene duplication, expression of duplicated genes, and the frequency of meiotic recombination in small chromosomal regions.     

Structure and dynamics of human interphase chromosome territories in vivo

A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5–10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture. Received: 10 November 1997 / Accepted: 24 November 1997     

Structure and variability of human chromosomes analyzed by recent techniques

Summary Besides the AT-specific fluorochromes, GC-specific fluorescent antibiotics are now available for chromosomal analysis. Chromosomal bands represent large accumulation of DNA sequences with similar AT:GC ratio. These uniform differences from the mean AT:GC ratio in the bands can be explained only by at least partial repetition of short DNA sequences in these regions. By comparison of various staining techniques more information also on the constitutive heterochromatin in man becomes available. The human NOR region exhibits a complex organization when studied by various basespecific fluorochromes and silver staining. The DNA-specific fluorochromes are also useful tools in cytophotometric DNA measurements.     

Frequent transpositions of Drosophila melanogaster HeT-A transposable elements to receding chromosome ends.

HeT-A elements are a new family of transposable elements in Drosophila that are found exclusively in telomeric regions and in the pericentric heterochromatin. Transposition of these elements onto broken chromosome ends has been implicated in chromosome healing. To monitor the fate of HeT-A elements that had attached to broken ends of the X chromosome, we examined individual X chromosomes from a defined population over a period of 17 generations. The ends of the X chromosomes with new HeT-A additions receded at the same rate as the broken ends before the HeT-A elements attached. In addition, some chromosomes, approximately 1% per generation, had acquired new HeT-A sequences of an average of 6 kb at their ends with oligo(A) tails at the junctions. Thus, the rate of addition of new material per generation matches the observed rate of terminal loss (70-75 bp) caused by incomplete replication at the end of the DNA molecule. One such recently transposed HeT-A element which is at least 12 kb in length has been examined in detail. It contains a single open reading frame of 2.8 kb which codes for a gag-like protein.