The plasticity of human telomeres demonstrated by a hypervariable telomere repeat array that is located on some copies of 16p and 16q.

Human telomeres are composed of tandem arrays of TTAGGG repeats with many variant repeats at the proximal ends. Comparison of the interspersion of variant and TTAGGG repeats between alleles can be used to study telomere instability, but the difficulty in identifying chromosome-specific sequences close to the start of autosomal telomeres has hampered such investigations. A chromosome end, including a telomere and adjacent sequence, that is polymorphic for its presence or absence in unrelated individuals has been identified. The telomere-adjacent DNA shows strong homology (92-99%) to sequences, including two expressed sequence tags, that are usually located in subterminal regions of human chromosomes but not adjacent to telomeres. Since this chromosome end arose, it has relocated at least once. In Caucasians, it forms the telomere of approximately 6% of 16q and 2% of 16p chromosome arms. The mechanism of relocation is unknown but must have involved the telomere-adjacent DNA rather than the telomere itself, as copies on 16p and 16q share the same telomere-adjacent sequence. The interspersion patterns of TTAGGG with TGAGGG, TTGGGG and non-amplifying repeat sequences revealed extensive allelic variation, such that 47 different alleles were observed among the 50 alleles mapped. Closely related alleles differ by small changes in copy number at blocks of adjacent like repeats, as seen at the Xp/Yp pseudoautosomal telomere. Such differences are compatible with a model in which the majority of mutations arise by intra-allelic mechanisms, in individuals hemizygous for a single copy of the chromosome end.     

Comparative sequencing of a multicopy subtelomeric region containing olfactory receptor genes reveals multiple interactions between non-homologous chromosomes.

In this study, we assess the evolutionary relationships among different chromosomal copies of a subtelomeric block of sequence. This block contains homology to three olfactory receptor genes and is dispersed on at least 14 different chromosome ends in humans. It is single-copy in non-human primates. We analyzed single nucleotide polymorphisms in two 1 kb subregions and a polymorphic Alu insertion within 181 copies of this block from 12 chromosome ends and found evidence for recent interactions between the subtelomeric regions of non-homologous chromosomes. First, several sequence haplotypes are each present on multiple chromosomes, and several chromosomes each have multiple alleles with divergent haplotypes. Secondly, the observed variation clearly indicates that chromosomes 5q, 8p, 11p and/or 15q have each received the block from at least two different sources by non-homologous exchange. In addition, we observe at least one ectopic gene conversion event. Awareness of such exchange among sequences on non-homologous chromosomes is critical for accurate analysis of these complex and dynamic regions of the genome.     

Cytogenetic mapping of common bean chromosomes reveals a less compartmentalized small-genome plant species

Cytogenetic maps of common bean chromosomes 3, 4 and 7 were constructed by fluorescence in-situ hybridization (FISH) of BAC and a few other genomic clones. Although all clones were selected with genetically mapped markers, mostly with single-copy RFLPs, a large subset of BACs, from 13 different genomic regions, contained repetitive sequences, as concluded from the regional distribution patterns of multiple FISH signals on chromosomes: pericentromeric, subtelomeric and dispersed. Pericentromeric repeats were present in all 11 chromosome pairs with different intensities, whereas subtelomeric repeats were present in several chromosome ends, but with different signal intensities depending on the BAC, suggesting that the terminal heterochromatin fraction of this species may be composed of different repeats. The correlation of genetic and physical distances along the three studied chromosomes was obtained for 23 clones. This correlation suggests suppression of recombination around extended pericentromeric regions in a similar way to that previously reported for plant species with larger genomes. These results indicate that a relatively small plant genome may also possess a large proportion of repeats interspersed with single-copy sequences in regions other than the pericentromeric heterochromatin and, nevertheless, exhibit lower recombination around the pericentromeric fraction of the genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interchromosomal repeat array interactions between chromosomes 4 and 10: a model for subtelomeric plasticity

Chromosomal rearrangements occur more frequently in subtelomeric domains than in other regions of the genome and are often associated with human pathology. To further elucidate the plasticity of subtelomeric domains, we examined the 3.3 kb D4Z4 repeat array on chromosome 4 and its homologue on chromosome 10 in 208 Dutch blood donors by pulsed field gel electrophoresis. These subtelomeric repeats are known to rearrange and partial deletions of this polymorphic array on chromosome 4 are associated with facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant myopathy. Our results show that mitotic rearrangements occur frequently as 3% of individuals display somatic mosaicism for a repeat expansion or contraction explaining the high variability of subtelomeric repeat array sizes. Translocated 4-type repeat arrays on chromosome 10 and the reverse configuration of 10-type repeat arrays on chromosome 4 are observed in 21% of individuals. The translocated repeat arrays on chromosome 4 tend to be more heterogeneous than 4-type repeats on chromosome 10. The repeat length on chromosome 4 is on average larger than on chromosome 10. But on both chromosomes we observe a multi-modal repeat length distribution with equidistant peaks at intervals of 65 kb, possibly reflecting a higher-order chromatin structure. Interestingly, in as many as six random blood donors (3%) we identified FSHD-sized 4-type repeat arrays. Assuming that these individuals are clinically unaffected, these results imply an incomplete penetrance in the upper range of FSHD alleles. Overall, the observed dynamic characteristics of these homologous domains may serve as a model for subtelomeric plasticity.     

Genomic structure and evolution of the ancestral chromosome fusion site in 2q13-2q14.1 and paralogous regions on other human chromosomes

Human chromosome 2 was formed by the head-to-head fusion of two ancestral chromosomes that remained separate in other primates. Sequences that once resided near the ends of the ancestral chromosomes are now interstitially located in 2q13-2q14.1. Portions of these sequences had duplicated to other locations prior to the fusion. Here we present analyses of the genomic structure and evolutionary history of >600 kb surrounding the fusion site and closely related sequences on other human chromosomes. Sequence blocks that closely flank the inverted arrays of degenerate telomere repeats marking the fusion site are duplicated at many, primarily subtelomeric, locations. In addition, large portions of a 168-kb centromere-proximal block are duplicated at 9pter, 9p11.2, and 9q13, with 98%-99% average sequence identity. A 67-kb block on the distal side of the fusion site is highly homologous to sequences at 22qter. A third ~100-kb segment is 96% identical to a region in 2q11.2. By integrating data on the extent and similarity of these paralogous blocks, including the presence of phylogenetically informative repetitive elements, with observations of their chromosomal distribution in nonhuman primates, we infer the order of the duplications that led to their current arrangement. Several of these duplicated blocks may be associated with breakpoints of inversions that occurred during primate evolution and of recurrent chromosome rearrangements in humans.     

Canonical TTAGG-repeat telomeres and telomerase in the honey bee, Apis mellifera

The draft assembly of the honey bee Apis mellifera genome sequence reveals that the 17 centromeric-distal telomeres are of a simple, shared, and canonical structure, with 3-4 kb of a unique subtelomeric sequence, followed by several kilobases of TTAGG or variant telomeric repeats. This simple subtelomeric structure differs from the centromeric-proximal telomeres on the short arms of the 15 acrocentric chromosomes, which are apparently composed primarily of the 176-bp AluI tandem repeat. This dichotomy between the distal and proximal telomeres may involve differential participation of the telomeres of the 15 acrocentric chromosomes in the Rabl configuration after mitosis and the chromosome bouquet in meiotic prophase I. As expected from the presence of canonical TTAGG telomeric repeats, we identified a candidate telomerase gene in the bee, as well as the silkmoth Bombyx mori and the flour beetle Tribolium castaneum.     

Diverse mutational mechanisms cause pathogenic subtelomeric rearrangements

Chromosome rearrangements are a significant cause of intellectual disability and birth defects. Subtelomeric rearrangements, including deletions, duplications and translocations of chromosome ends, were first discovered over 40 years ago and are now recognized as being responsible for several genetic syndromes. Unlike the deletions and duplications that cause some genomic disorders, subtelomeric rearrangements do not typically have recurrent breakpoints and involve many different chromosome ends. To capture the molecular mechanisms responsible for this heterogeneous class of chromosome abnormality, we coupled high-resolution array CGH with breakpoint junction sequencing of a diverse collection of subtelomeric rearrangements. We analyzed 102 breakpoints corresponding to 78 rearrangements involving 28 chromosome ends. Sequencing 21 breakpoint junctions revealed signatures of non-homologous end-joining, non-allelic homologous recombination between interspersed repeats and DNA replication processes. Thus, subtelomeric rearrangements arise from diverse mutational mechanisms. In addition, we find hotspots of subtelomeric breakage at the end of chromosomes 9q and 22q; these sites may correspond to genomic regions that are particularly susceptible to double-strand breaks. Finally, fine-mapping the smallest subtelomeric rearrangements has narrowed the critical regions for some chromosomal disorders.     

Analysis of the CMT1A-REP repeat: mapping crossover breakpoints in CMT1A and HNPP

The CMT1A-REP repeat sequence flanks a 1.5 megabase pair (Mb)segment of chromosome 17p11.2–12 which is duplicated inCharcot—Marie—Tooth neuropathy type 1A (CMT1A) anddeleted in hereditary neuropathy with liability to pressurepalsies (HNPP). The CMT1A-REP repeat is proposed to mediatemisalignment and unequal crossover resulting in reciprocal chromosomalrearrangements in CMT1A and HNPP. We have constructed a physicalmap of the proximal and distal CMT1A-REP repeats. Cloned fragmentsfrom CMT1A-REP repeat regions were used to determine the sizeof the repeats and to assess regions of homology. The crossoverbreakpoints were mapped in a series of 30 unrelated CMT1A patientsand 22 unrelated HNPP patients. The CMT1A-REP repeat spans approximately27 kilobase pairs and appears to be continuous. Locations ofrestriction enzyme sites are highly conserved for the proximaland distal CMT1A-REP repeats. All crossovers mapped within theCMT1A-REP repeat sequence and heterogeneity for breakpoint locationwas demonstrated. Seventy-seven percent (40 of 52) of CMT1Aand HNPP chromosomes contained breakpoints which mapped withina 7.9 kb interval, suggesting the presence of a possible ‘hotspot’for recombination in CMT1A-REP. DNA sequence analysis for 4kb of the interval containing the majority of crossovers revealedover 98% sequence identity between proximal and distal CMT1A-REPrepeat sequences. Probes useful for molecular-based diagnosisof CMT1A and HNPP are described.     

A human interstitial telomere associates in vivo with specific TRF2 and TIN2 proteins

Mammalian telomeres are composed of long arrays of TTAGGG repeats that form a nucleoprotein complex which protects the chromosome ends. Human telomere function is known to require two TTAGGG repeat factors, TRF1 and TRF2, and several interacting proteins, but the mechanism by which the DNA/protein complex prevents end to end fusion in vivo has not been elucidated. In order to better understand the role of specific telomere-associated proteins in the organisation of chromosome ends, we have studied a patient with a rare chromosome rearrangement that has given rise to an interstitial telomere. Using specific antibodies and immuno-FISH on unfixed metaphase chromosomes, we show that the proteins TRF2 and TIN2 (TIN2 interacts with TRF1) co-localise with the interstitial TTAGGG repeats. Our results demonstrate, for the first time in humans, that TRF2 and TIN2 proteins associate with interstitial duplex TTAGGG repeats, in vivo. They confirm that double stranded-telomeric repeats, even when complexed with specific proteins, are not sufficient to create a functional telomere. Finally, they suggest a possible role for proteins in stabilising interstitial TTAGGG repeats.     

Del(18p) shown to be a cryptic translocation using a multiprobe FISH assay for subtelomeric chromosome rearrangements.

We have previously described a fluorescence in situ hybridisation (FISH) assay for the simultaneous analysis of all human subtelomeric regions using a single microscope slide. Here we report the use of this multiprobe FISH assay in the study of a patient whose karyotype was reported by G banding analysis as 46,XX,del(18)(p11.2). Although the proband had some features suggestive of a chromosomal abnormality, relatively few of the specific features of del(18p) were present. She was a 37 year old female with mild distal spinal muscular atrophy (SMA), arthritis of the hands, an abnormal chest shape (pectus excavatum), and an unusual skin condition (keratosis pilaris). Reverse chromosome painting with degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) amplified del(18p) chromosomes as a probe confirmed the abnormality as del(18p), with no evidence of any other chromosome involvement. Subsequently, the multiprobe FISH assay confirmed deletion of 18p subtelomeric sequence. However, the assay also showed that sequences corresponding to the 2p subtelomeric probe were present on the tip of the shortened 18p. The patient is therefore monosomic for 18p11.2-pter and trisomic for 2p25-pter, and the revised karyotype is 46,XX,der(18)t(2;18)(p25; p11.2). We believe that a proportion of all cases reported as telomeric deletions may be cryptic translocations involving other chromosome subtelomeric regions. Further studies such as this are necessary to define accurately the clinical characteristics associated with pure monosomy in chromosomal deletion syndromes.     

Differentially regulated and evolved genes in the fully sequenced Xq/Yq pseudoautosomal region

Human sex chromosomes, which are morphologically and genetically different, share few regions of homology. Among them, only pseudoautosomal regions (PARs) pair and recombine during meiosis. To better address the complex biology of these regions, we sequenced the telomeric 400 kb of the long arm of the human X chromosome, including 330 kb of the human Xq/YqPAR and the telomere. Sequencing reveals subregions with distinctive regulatory and evolutionary features. The proximal 295 kb contains two genes inactivated on both the inactive X and Y chromosomes [ SYBL1 and a novel homologue ( HSPRY3 ) of Drosophila sprouty ]. The GC-rich distal 35 kb, added in stages and much later in evolution, contains the X/Y expressed gene IL9R and a novel gene, CXYorf1, only 5 kb from the Xq telomere. These properties make Xq/YqPAR a model for studies of region-specific gene inactivation, telomere evolution, and involvement in sex-limited conditions.     

Sequence organization of the human chromosome 2q telomere

The terminal 240 kb of a human 2q telomere region was clonedin two overlapping yeast artificial-chromosomes (YACs). ThisDNA contains a region of low-copy subtelomeric repeats (within50 kb of the 2q telomere), a segment of DNA duplicated on distal8p23 (100 kb from the 2q telomere), and a region of single-copyDNA (230 kb from the 2q telomere). Two CpG islands are presentin the DNA segment duplicated on distal 8p23. RecA-assistedrestriction endonuclease cleavage of genomic DNA samples revealeda potential 55 kb chromosome length polymorphism at the 2q telomere.This work provides telomeric closure of maps for human chromosome2q, demonstrates a novel, subtelomere-specific DNA duplication,and will permit detailed molecular and cytological studies ofthis human telomere region.     

Controlled exchange of chromosomal arms reveals principles driving telomere interactions in yeast

The 32 telomeres in the budding yeast genome cluster in three to seven perinuclear foci. Although individual telomeres and telomeric foci are in constant motion, preferential juxtaposition of some telomeres has been scored. To examine the principles that guide such long-range interactions, we differentially tagged pairs of chromosome ends and developed an automated three-dimensional measuring tool that determines distances between two telomeres. In yeast, all chromosomal ends terminate in TG(1-3) and middle repetitive elements, yet subgroups of telomeres also share extensive homology in subtelomeric coding domains. We find that up to 21 kb of >90% sequence identity does not promote telomere pairing in interphase cells. To test whether unique sequence elements, arm length, or chromosome territories influence juxtaposition, we reciprocally swapped terminal domains or entire chromosomal arms from one chromosome to another. We find that the distal 10 kb of Tel6R promotes interaction with Tel6L, yet only when the two telomeres are present on the same chromosome. By manipulating the length and sequence composition of the right arm of chr 5, we confirm that contact between telomeres on opposite chromatid arms of equal length is favored. These results can be explained by the polarized Rabl arrangement of yeast centromeres and telomeres, which promote to telomere pairing by allowing contact between chromosome arms of equal length in anaphase.     

Submicroscopic deletion 9(q34.3) and duplication 19(p13.3): identified by subtelomere specific FISH probes

Submicroscopic rearrangements involving chromosome ends are responsible for the unexplained mental retardation and multiple congenital anomalies observed in a number of patients. We have studied a patient with mental retardation, significant microcephaly, alopecia universalis, and other anomalies who carries an unbalanced segregant from a cryptic reciprocal translocation involving chromosomes 9 and 19. FISH studies using subtelomere specific probes revealed a derivative chromosome 9 in which the 9q subtelomeric sequence has been replaced by 19p subtelomeric sequence. As a result, the patient has partial monosomy 9q and partial trisomy 19p. The patient inherited the derivative 9 from his father, who carries a cryptic apparently balanced reciprocal translocation involving the terminal regions of 9q and 19p. This case is exceptional in that reports of rearrangements involving distal chromosome 9q and 19p are rare. This study demonstrates the utility of subtelomere specific FISH probes for detecting cryptic subtelomeric rearrangements in patients with idiopathic mental retardation and normal appearing karyotypes.     

Shelterin: the protein complex that shapes and safeguards human telomeres

Added by telomerase, arrays of TTAGGG repeats specify the ends of human chromosomes. A complex formed by six telomere-specific proteins associates with this sequence and protects chromosome ends. By analogy to other chromosomal protein complexes such as condensin and cohesin, I will refer to this complex as shelterin. Three shelterin subunits, TRF1, TRF2, and POT1 directly recognize TTAGGG repeats. They are interconnected by three additional shelterin proteins, TIN2, TPP1, and Rap1, forming a complex that allows cells to distinguish telomeres from sites of DNA damage. Without the protective activity of shelterin, telomeres are no longer hidden from the DNA damage surveillance and chromosome ends are inappropriately processed by DNA repair pathways. How does shelterin avert these events? The current data argue that shelterin is not a static structural component of the telomere. Instead, shelterin is emerging as a protein complex with DNA remodeling activity that acts together with several associated DNA repair factors to change the structure of the telomeric DNA, thereby protecting chromosome ends. Six shelterin subunits: TRF1, TRF2, TIN2, Rap1, TPP1, and POT1.     

Segmental polymorphisms in the proterminal regions of a subset of human chromosomes

The subtelomeric domains of chromosomes are probably the most rapidly evolving structures of the human genome. The highly variable distribution of large duplicated subtelomeric segments has indicated that frequent exchanges between nonhomologous chromosomes may have been taking place during recent genome evolution. We have studied the extent and variability of such duplications using in situ hybridization techniques and a set of well-defined subtelomeric cosmid probes that identify discrete regions within the subtelomeric domain. In addition to reciprocal translocation and illegitimate recombination events that could explain the observed mosaic pattern of subtelomeric regions, it is likely that homology-based recombination mechanisms have also contributed to the spread of distal subtelomeric sequences among particular groups of nonhomologous chromosome arms. The frequency and distribution of large-scale subtelomeric polymorphisms may have direct implications for the design of chromosome-specific probes that are aimed at the identification of cryptic subtelomeric deletions. Furthermore, our results indicate that the relevance of some of the telomere closures proposed within the present Human Genome Sequence draft are restricted to specific allelic variants of unknown frequencies.     

Molecular organization of terminal repetitive DNA in Beta species

We have isolated families of subtelomeric satellite DNA sequences from species of four sections of the genus Beta and from spinach, a related Chenopodiaceae. Twenty-five clones were sequenced and representative repeats of each family were characterized by Southern blotting and FISH. The families of ApaI restriction satellite repeats were designated pAv34, pAc34, the families of RsaI repeats pRp34, pRn34 and pRs34. The repeating units are 344–362 bp long and 45.7–98.8% homologous with a clear species-specific divergence. Each satellite monomer consists of two subrepeats SR1 and SR2 of 165–184 bp, respectively. The repeats of each subrepeat group are highly identical across species, but share only a homology of 40.8–54.8% with members of the other subrepeat group. Two evolutionary steps could be supposed in the phylogeny of the subtelomeric satellite family: the diversification of an ancestor satellite into groups representing SR1 and SR2 in the progenitor of Beta and Spinacea species, followed by the dimerization and diversification of the resulting 360 bp repeats into section-specific satellite DNA families during species radiation. The chromosomal localization of telomeric, subtelomeric and rDNA tandem repeats was investigated by multi-colour FISH. High-resolution analysis by fibre FISH revealed a unique physical organization of B. vulgaris chromosome ends with telomeric DNA and subtelomeric satellites extending over a maximum of 63 kb and 125 kb, respectively.