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.     

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.     

MTV sings jubilation for telomere biology in Drosophila

Telomere protects the ends of linear chromosomes. Telomere dysfunction fuels genome instability that can lead to diseases such as cancer. For over 30 years, Drosophila has fascinated the field as the only major model organism that does not rely on the conserved telomerase enzyme for end protection. Instead of short DNA repeats at chromosome ends, Drosophila has domesticated retrotransposons. In addition, telomere protection can be entirely sequence-independent under normal laboratory conditions, again dissimilar to what has been established for telomerase-maintained systems. Despite these major differences, recent studies from us and others have revealed remarkable similarities between the 2 systems. In particular, with the identification of the MTV complex as an ssDNA binding complex essential for telomere integrity in Drosophila (Zhang et al. 2016 Plos Genetics), we have now established several universal principles that are intrinsic to chromosome extremities but independent of the underlying DNA sequences or the telomerase enzyme. Telomere studies in Drosophila will continue to yield fundamental insights that are instrumental to the understanding of the evolution of telomere and telomeric functions.     

Structure of telomeric chromatin in <Emphasis Type="Italic">Drosophila</Emphasis>

The telomeric nucleoprotein complex protects linear chromosome ends from degradation. In contrast to most eukaryotes in which telomerase is responsible for telomere elongation by adding short DNA repeats synthesized using an RNA template, the telomere elongation in Drosophila involves transposition of specialized telomeric retroelements onto chromosome ends. Proteins that bind telomeric and subtelomeric sequences form specific telomeric chromatin, and its components are highly conserved among organisms employing different mechanisms of telomere elongation. This review is focused on the analysis of components of the Drosophila telomeric complex and its comparison with telomeric proteins in telomerase-encoded organisms. Structural and functional analysis of Drosophila telomeres suggests that there are three distinct chromatin regions: protective structure at the very end of chromosome (cap), subtelomeric region which is characterized by condensed chromatin structure, and the terminal retrotransposon array whose expression is under the control of an RNAi (RNA interference)-based mechanism. The link between RNAi and telomeric chromatin formation in germinal tissues is discussed.     

Euchromatic and heterochromatic domains at Drosophila telomeres.

Noncoding repetitive sequences make up a large portion of eukaryotic genomes, but their function is not well understood. Large blocks of repetitive DNA-forming heterochromatin around the centromeres are required for this region to function properly, but are difficult to analyze. The smaller regions of heterochromatin at the telomeres provide an opportunity to study their DNA and protein composition. Drosophila telomere length is maintained through the targeted transposition of specific non-long terminal repeat retrotransposons to chromosome ends, where they form long tandem arrays. A subterminal telomere-associated sequence (TAS) lies immediately proximal to the terminal-retrotransposon array. Here, we review the experimental support for the heterochromatic features of Drosophila telomeres, and provide evidence that telomeric regions contain 2 distinct chromatin subdomains: TAS, which exhibits features that resemble beta heterochromatin; and the terminal array of retrotransposons, which appears euchromatic. This organization is significantly different from the telomeric organization of other eukaryotes, where the terminal telomerase-generated repeats are often folded in a t-loop structure and become part of the heterochromatin protein complex.     

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.     

Control of telomere length by a trimming mechanism that involves generation of t-circles

Telomere lengths are maintained in many cancer cells by the ribonucleoprotein enzyme telomerase but can be further elongated by increasing telomerase activity through the overexpression of telomerase components. We report here that increased telomerase activity results in increased telomere length that eventually reaches a plateau, accompanied by the generation of telomere length heterogeneity and the accumulation of extrachromosomal telomeric repeat DNA, principally in the form of telomeric circles (t-circles). Telomeric DNA was observed in promyelocytic leukemia bodies, but no intertelomeric copying or telomere exchange events were identified, and there was no increase in telomere dysfunction-induced foci. These data indicate that human cells possess a mechanism to negatively regulate telomere length by trimming telomeric DNA from the chromosome ends, most likely by t-loop resolution to form t-circles. Additionally, these results indicate that some phenotypic characteristics attributed to alternative lengthening of telomeres (ALT) result from increased mean telomere length, rather than from the ALT mechanism itself.     

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.     

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.     

Length regulation and dynamics of individual telomere tracts in wild-type Arabidopsis

Although length of the telomeric DNA tract varies widely across evolution, a species-specific set point is established and maintained by unknown mechanisms. To investigate how telomere length is controlled in Arabidopsis thaliana, we analyzed bulk telomere length in 14 wild-type accessions. We found that telomere tracts in Arabidopsis are fairly uniformly distributed throughout a size range of 2 to 9 kb. Unexpectedly, telomeres in plants of the Wassilewskija ecotype displayed a bimodal size distribution, with some individuals harboring telomeres of 2 to 5 kb and others telomeres of 4 to 9 kb. F1 and F2 progeny of a cross between long and short telomere parents had intermediate telomeres, implying that telomere length in Arabidopsis is not controlled by a single genetic factor. We provide evidence that although global telomere length is strictly regulated within an ecotype-specific range, telomere tracts on individual chromosome ends do not occupy a predetermined length territory. We also demonstrate that individual telomere tracts on homologous chromosomes are coordinately regulated throughout development and that telomerase acts preferentially on the shortest telomeres. We propose that an optimal size for telomere tracts is established and maintained for each Arabidopsis ecotype.     

How will telomeric complex be further contributed to our longevity? — The potential novel biomarkers of telomere complex counteracting both aging and cancer

With the smooth move towards the coming expected clinical reports of anticancer pharmaceutical molecules targeting telomeres and telomerase, and also with the exciting success in the extension of lifespan by regulating telomerase activity without increased onset of oncogenesis in laboratory mouse models (Garcia-Cao et al., 2006; Jaskelioff et al., 2011), we are convinced that targeting telomeres based on telomerase will be a potential approach to conquer both aging and cancer and the idea of longevity seems to be no more mysterious. More interestingly, emerging evidences from clinical research reveal that other telomeric factors, like specifi c telomeric binding proteins and nonspecific telomere associated proteins also show crucial importance in aging and oncogenesis. This stems from their roles in the stability of telomere structure and in the inhibition of DNA damage response at telomeres. Uncapping these proteins from chromosome ends leads to dramatic telomere loss and telomere dysfunction which is more abrupt than those induced by telomerase inactivation. Abnormal expression of these factors results in developmental failure, aging and even oncogenesis evidenced by several experimental models and clinical cases, indicating telomere specifi c proteins and its associated proteins have complimentary roles to telomerase in telomere protection and controlling cellular fate. Thus, these telomeric factors might be potential clinical biomarkers for early detection or even therapeutic targets of aging and cancer. Future studies to elucidate how these proteins function in telomere protection might benefit patients suffering aging or cancer who are not sensitive to telomerase mediation.     

How telomerase reaches its end: mechanism of telomerase regulation by the telomeric complex

The telomerase enzyme, which synthesizes telomeric DNA repeats, is regulated in cis at individual chromosome ends by the telomeric protein/DNA complex in a manner dependent on telomere repeat-array length. A dynamic interplay between telomerase-inhibiting factors bound at duplex DNA repeats and telomerase-promoting ones bound at single-stranded terminal DNA overhangs appears to modulate telomerase activity and to be directly related to the transient deprotection of telomeres. We discuss recent advances on the mechanism of telomerase regulation at chromosome ends in both yeast and mammalian systems.