Two distinct domains in Drosophila melanogaster telomeres

Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila melanogaster contains two distinct subdomains: a subtelomeric region of repetitive DNA, termed TAS, and a terminal array of retrotransposons, which perform the elongation function instead of telomerase. We have identified several P-element insertions into this retrotransposon array and compared expression levels of transgenes with similar integrations into TAS and euchromatic regions. In contrast to insertions in TAS, which are silenced, reporter genes in the terminal HeT-A, TAHRE, or TART retroelements did not exhibit repressed expression in comparison with the same transgene construct in euchromatin. These data, in combination with cytological studies, provide evidence that the subtelomeric TAS region exhibits features resembling heterochromatin, while the terminal retrotransposon array exhibits euchromatic characteristics.     

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.     

Drosophila telomeres: A variation on the telomerase theme

In Drosophila, the role of telomerase is carried out by three specialized retrotransposable elements, HeT-A, TART and TAHRE. Telomeres contain long tandem head-to-tail arrays of these elements. Within each array, the three elements occur in random, but polarized, order. Some are truncated at the 5' end, giving the telomere an enriched content of the large 3' untranslated regions, which distinguish these telomeric elements from other retrotransposons. Thus, Drosophila telomeres resemble other telomeres because they are long arrays of repeated sequences, albeit more irregular arrays than those produced by telomerase. The telomeric retrotransposons are reverse-transcribed directly onto the end of the chromosome, extending the end by successive transpositions. Their transposition uses exactly the same method by which telomerase extends chromosome ends--copying an RNA template. In addition to these similarities in structure and maintenance, Drosophila telomeres have strong functional similarities to other telomeres and, as variants, provide an important model for understanding general principles of telomere function and evolution.     

Genomic analysis of Drosophila melanogaster telomeres: full-length copies of HeT-A and TART elements at telomeres

The repetitive nature of heterochromatin hampers its analysis in general genome-sequencing projects. Specific studies are needed to extend the sequence into telomeric and centromeric heterochromatin. Drosophila telomeres lack the telomerase-generated repeats that are characteristic of other eukaryotic chromosomes. Instead, they consist of tandem arrays of HeT-A and TART elements. Herein, we present the genomic organization of the telomeres in the isogenic strain (y; cn bw sp) that was used for the Drosophila melanogaster sequencing project. The data indicate that the canonical features of telomere organization are widely conserved in evolution. In addition, we have identified full-length elements, likely competent elements, for HeT-A and TART.     

Telomere fusion in Drosophila: The role of subtelomeric chromatin

Drosophila telomeres are maintained by transposition to chromosome ends of the HeT-A, TART and TAHRE retrotransposons, collectively designated as HTT. Although all Drosophila telomeres terminate with HTT arrays and are capped by the terminin complex, they differ in the type of subtelomeric chromatin. The HTT sequences of YS, YL, XR, and 4L are juxtaposed to constitutive heterochromatin, while the HTTs of the other telomeres are linked to either the TAS repeat-associated chromatin (XL, 2L, 2R, 3L, 3R) or to the specialized 4R chromatin. We found that mutations in pendolino (peo) cause (telomeric fusions) that preferentially involve the heterochromatin-associated telomeres (Ha-telomeres), a telomeric fusion pattern never observed in the other 10 telomere-capping mutants characterized so far. Peo, is homologous to the E2 variant ubiquitin-conjugating enzymes and is required for DNA replication. Our analyses lead us to hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in Ha-telomeres. These data provide the first demonstration that subtelomeres can affect telomere fusion.     

Telomere fusion in Drosophila: The role of subtelomeric chromatin

Drosophila telomeres are maintained by transposition to chromosome ends of the HeT-A, TART and TAHRE retrotransposons, collectively designated as HTT. Although all Drosophila telomeres terminate with HTT arrays and are capped by the terminin complex, they differ in the type of subtelomeric chromatin. The HTT sequences of YS, YL, XR, and 4L are juxtaposed to constitutive heterochromatin, while the HTTs of the other telomeres are linked to either the TAS repeat-associated chromatin (XL, 2L, 2R, 3L, 3R) or to the specialized 4R chromatin. We found that mutations in pendolino (peo) cause (telomeric fusions) that preferentially involve the heterochromatin-associated telomeres (Ha-telomeres), a telomeric fusion pattern never observed in the other 10 telomere-capping mutants characterized so far. Peo, is homologous to the E2 variant ubiquitin-conjugating enzymes and is required for DNA replication. Our analyses lead us to hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in Ha-telomeres. These data provide the first demonstration that subtelomeres can affect telomere fusion.     

Telomere maintenance in Drosophila: Rapid transposon evolution at chromosome ends

The maintenance of terminal sequences is an important role of the telomere, since it prevents the loss of internal regions that encode essential genes. In most eukaryotes, this is accomplished by the telomerase. However, telomere length can also be maintained by other mechanisms, such as homologous recombination and transposition of telomeric retrotransposons to the chromosome ends. A remarkable situation is the case of Drosophila, where telomerase was lost, and thus telomeres managed to be maintained by occasional retrotransposition of telomeric elements to the receding ends. In the recent analysis of 12 Drosophila genomes, ¬¬the multiplicity of autonomous and non-autonomous telomere-specific retrotransposons has revealed extensive and rapid evolution of telomeric DNA. The phylogenetic relationship among these telomeric retrotransposons is congruent with the species phylogeny, suggesting that they have been vertically transmitted from a common ancestor. In this review, we also suggest that the formation of a non-canonical DNA structure at Drosophila telomeres could be the way to protect the ends.     

Drosophila Telomeres: A Variation on the Telomerase Theme

In Drosophila, the role of telomerase is carried out by three specialized retrotransposable elements, HeT?A, TART and Tahre. Telomeres contain long tandem head?to?tail arrays of these elements. Within each array, the three elements occur in random, but polarized, order. Some are truncated at the 5' end, giving the telomere an enriched content of the large 3' untranslated regions, which distinguish these telomeric elements from other retrotransposons. Thus, Drosophila telomeres resemble other telomeres because they are long arrays of repeated sequences, albeit more irregular arrays than those produced by telomerase. The telomeric retrotransposons are reverse?transcribed directly onto the end of the chromosome, extending the end by successive transpositions. Their transposition uses exactly the same method by which telomerase extends chromosome ends—copying an RNA template. In addition to these similarities in structure and maintenance, Drosophila telomeres have strong functional similarities to other telomeres and, as variants, provide an important model for understanding general principles of telomere function and evolution.

Reprinted from: Origin and Evolution of Telomeres, edited by Jozef Nosek and ?ubomír Tomá?ka. ©2008 Landes Bioscience.     

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.     

Arabidopsis thaliana telomeres exhibit euchromatic features

Telomere function is influenced by chromatin structure and organization, which usually involves epigenetic modifications. We describe here the chromatin structure of Arabidopsis thaliana telomeres. Based on the study of six different epigenetic marks we show that Arabidopsis telomeres exhibit euchromatic features. In contrast, subtelomeric regions and telomeric sequences present at interstitial chromosomal loci are heterochromatic. Histone methyltransferases and the chromatin remodeling protein DDM1 control subtelomeric heterochromatin formation. Whereas histone methyltransferases are required for histone H3K9(2Me) and non-CpG DNA methylation, DDM1 directs CpG methylation but not H3K9(2Me) or non-CpG methylation. These results argue that both kinds of proteins participate in different pathways to reinforce subtelomeric heterochromatin formation.     

Characterization of Arabidopsis thaliana telomeres isolated in yeast.

In an effort to learn more about the genomic organization of chromosomal termini in plants we employed a functional complementation strategy to isolate Arabidopsis thaliana telomeres in the yeast, Saccharomyces cerevisiae. Eight yeast episomes carrying A. thaliana telomeric sequences were obtained. The plant sequences carried on two episomes, YpAtT1 and YpAtT7, were characterized in detail. The telomeric origins of YpAtT1 and YpAtT7 insert DNAs were confirmed by demonstrating that corresponding genomic sequences are preferentially degraded during exonucleolytic digestion. The isolated telomeric restriction fragments contain G-rich repeat arrays characteristic of A. thaliana telomeres, as well as subterminal telomere-associated sequences (TASs). DNA sequence analysis revealed the presence of variant telomeric repeats at the centromere-proximal border of the terminal block of telomere repeats. The TAS flanking the telomeric G-rich repeat in YpAtT7 corresponds to a repetitive element present at other A. thaliana telomeres, while more proximal sequences are unique to one telomere. The YpAtT1 TAS is unique in the Landsberg strain of A. thaliana from which the clone originated; however, the Landsberg TAS cross-hybridizes weakly to a second telomere in the strain Columbia. Restriction analysis with cytosine methylation-sensitive endonucleases indicated that both TASs are highly methylated in the genome.     

Intracellular targeting of Gag proteins of the Drosophila telomeric retrotransposons

Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.     

Cis- and trans-acting influences on telomeric position effect in Drosophila melanogaster detected with a subterminal transgene

One model of telomeric position effect (TPE) in Drosophila melanogaster proposes that reporter genes in the vicinity of telomeres are repressed by subterminal telomere-associated sequences (TAS) and that variegation of these genes is the result of competition between the repressive effects of TAS and the stimulating effects of promoters in the terminal HeT-A transposon array. The data presented here support this model, but also suggest that TPE is more complex. Activity of a telomeric white reporter gene increases in response to deletion of some or all of the TAS on the homolog. Only transgenes next to fairly long HeT-A arrays respond to this trans-interaction. HeT-A arrays of 6-18 kb respond by increasing the number of dark spots on the eye, while longer arrays increase the background eye color or increase the number of spots sufficiently to cause them to merge. Thus, expression of a subtelomeric reporter gene is influenced by the telomere structure in cis and trans. We propose that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as TPE. In the wild-type telomere TAS may play an important role in controlling telomere elongation by repressing HeT-A promoter activity. Modulation of this repression by the homolog may thus regulate telomere elongation.     

HeT-A and TART, two Drosophila retrotransposons with a bona fide role in chromosome structure for more than 60 million years

Drosophila telomeres have been maintained by retrotransposition for at least 60 MY, which predates the separation of extant species of this genus. Studies of D. melanogaster, D. yakuba, and D. virilis show that, in Drosophila, telomeres are composed of two non-LTR retrotransposons, HeT-A and TART. Far from being static, HeT-A and TART evolve faster than Drosophila euchromatic genes. In spite of their high rate of sequence change, HeT-A and TART maintain their basic structures and unusual individual features. The maintenance of their separate identities suggests that HeT-A and TART cooperate either in the process of retrotransposition onto the chromosome end, or in the formation of telomere chromatin by transposed DNA copies. The telomeric retrotransposons and the Drosophila genome constitute an example of a robust symbiotic relationship between mobile elements and the genome.