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.     

Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster

Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Their transposition to broken chromosome ends has been implicated in chromosome healing and telomere elongation. We have developed a genetic system which enables the determination of the frequency of telomere elongation events and their mechanism. The frequency differs among lines with different genotypes, suggesting that several genes are in control. Here we show that the Su(var)2-5 gene encoding heterochromatin protein 1 (HP1) is involved in regulation of telomere length. Different Su(var)2-5 mutations in the heterozygous state increase the frequency of HeT-A and TART attachment to the broken chromosome end by more than a hundred times. The attachment occurs through either HeT-A/TART transposition or recombination with other telomeres. Terminal DNA elongation by gene conversion is greatly enhanced by Su(var)2-5 mutations only if the template for DNA synthesis is on the same chromosome but not on the homologous chromosome. The Drosophila lines bearing the Su(var)2-5 mutations maintain extremely long telomeres consisting of HeT-A and TART for many generations. Thus, HP1 plays an important role in the control of telomere elongation in D. melanogaster.     

HeT-A_pi1, a piRNA target sequence in the Drosophila telomeric retrotransposon HeT-A, is extremely conserved across copies and species

The maintenance of the telomeres in Drosophila species depends on the transposition of the non-LTR retrotransposons HeT-A, TAHRE and TART. HeT-A and TART elements have been found in all studied species of Drosophila suggesting that their function has been maintained for more than 60 million years. Of the three elements, HeT-A is by far the main component of D. melanogaster telomeres and, unexpectedly for an element with an essential role in telomere elongation, the conservation of the nucleotide sequence of HeT-A is very low. In order to better understand the function of this telomeric retrotransposon, we studied the degree of conservation along HeT-A copies. We identified a small sequence within the 3' UTR of the element that is extremely conserved among copies of the element both, within D. melanogaster and related species from the melanogaster group. The sequence corresponds to a piRNA target in D. melanogaster that we named HeT-A_pi1. Comparison with piRNA target sequences from other Drosophila retrotransposons showed that HeT-A_pi1 is the piRNA target in the Drosophila genome with the highest degree of conservation among species from the melanogaster group. The high conservation of this piRNA target in contrast with the surrounding sequence, suggests an important function of the HeT-A_pi1 sequence in the co-evolution of the HeT-A retrotransposon and the Drosophila genome.     

HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster.

Eight terminally deleted Drosophila melanogaster chromosomes have now been found to be "healed." In each case, the healed chromosome end had acquired sequence from the HeT DNA family, a complex family of repeated sequences found only in telomeric and pericentric heterochromatin. The sequences were apparently added by transposition events involving no sequence homology. We now report that the sequences transposed in healing these chromosomes identify a novel transposable element, HeT-A, which makes up a subset of the HeT DNA family. Addition of HeT-A elements to broken chromosome ends appears to be polar. The proximal junction between each element and the broken chromosome end is an oligo(A) tract beginning 54 nucleotides downstream from a conserved AATAAA sequence on the strand running 5' to 3' from the chromosome end. The distal (telomeric) ends of HeT-A elements are variably truncated; however, we have not yet been able to determine the extreme distal sequence of a complete element. Our analysis covers approximately 2,600 nucleotides of the HeT-A element, beginning with the oligo(A) tract at one end. Sequence homology is strong (greater than 75% between all elements studied). Sequence may be conserved for DNA structure rather than for protein coding; even the most recently transposed HeT-A elements lack significant open reading frames in the region studied. Instead, the elements exhibit conserved short-range sequence repeats and periodic long-range variation in base composition. These conserved features suggest that HeT-A elements, although transposable elements, may have a structural role in telomere organization or maintenance.     

Attachment of HeT-A sequences to chromosomal termini in Drosophila melanogaster may occur by different mechanisms

Drosophila telomeres contain arrays of the retrotransposonlike elements HeT-A and TART. Their transposition to broken chromosomal termini has been implicated in chromosome healing and telomere elongation. The HeT-A element is attached by its 3' end, which contains the promoter. To monitor the behavior of HeT-A elements, we used the yellow gene with terminal deficiencies consisting of breaks in the yellow promoter region that result in the y-null phenotype. Attachment of the HeT-A element provides the promoterless yellow gene with a promoter that activates yellow expression in bristles. The frequency of HeT-A transpositions to the yellow terminal deficiency depends on the genotype of the line and varies from 2 x 10(-3) to less than 2 x 10(-5). Loss of the attached HeT-A due to incomplete replication at the telomere leads to inactivation of yellow expression, which is restored by attachment of a new HeT-A element upstream of yellow. New HeT-A additions occur at a frequency of about 1.2 x 10(-3). Short DNA attachments are generated by gene conversion using the homologous telomeric sequences as templates. Longer DNA attachments are generated either by conventional transposition of an HeT-A element to the chromosomal terminus or by recombination between the 3' terminus of telomeric HeT-A elements and the receding end of HeT-A attached to the yellow gene.     

The genomic organization of HeT-A retroposons inDrosophila melanogaster

Members of theDrosophila HeT-A family of transposable elements are LINE-like retroposons that are found at telomeres and in centric heterochromatin. We recently characterized an active HeT-A element that had transposed to a broken chromosome end fewer than mine generations before it was isolated. The sequence arerangement of this element, called 9D4, most likely represents the organization of an actively transposing member of the HeT-A family. Here we assess the degree of divergence among members of the HeT-A family and test a model of telomere length maintenance based on HeT-A transposition. The region containing the single open reading frame of this element appears to be more highly conserved than the non-coding regions. The HeT-A element has been implicated in theDrosophila telomere elongation process, because frequent transpositions to chromosome ends are sufficient to counter-balance nucleotide loss due to incomplete DNA replication. The proposed elongation model and the hypothetical mechanism of HeT-A transposition predict a predominant orientation of HeT-A elements with their oligo (A) tails facing proximally at chromosome ends, as well as the existence of irregular tandem arrays of HeT-A elements at chromosome ends resulting from transposition of new HeT-A elements onto chromosome ends with existing elements. Twenty-nine different HeT-A fragments were isolated from directional libraries that were enriched in terminal DNA fragments. Sequence analyses of these fragments and comparisons with the organization of the HeT-A element, 9D4, fit these two predictions and support the model ofDrosophila telomere elongation by transposition of HeT-A elements.     

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.     

Enhancer of terminal gene conversion,a new mutation in Drosophila melanogaster that induces telomere elongation by gene conversion

Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Terminally deleted chromosomes can be maintained for many generations. Thus, broken chromosome ends behave as real telomeres. It was previously shown that gene conversion may extend the broken ends. Here we found that the frequency of terminal DNA elongation by gene conversion strongly depends on the genotype. A dominant E(tc) (Enhancer of terminal gene conversion) mutation markedly increases the frequency of this event but does not significantly influence the frequency of HeT-A and TART attachment to the broken chromosome end and recombination between directly repeated sequences at the end of the truncated chromosome. The E(tc) mutation was mapped to the 91-93 region on chromosome 3. Drosophila lines that bear the E(tc) mutation for many generations have telomeres, consisting of HeT-A and TART elements, that are longer than those found in wild-type lines. Thus, the E(tc) mutation plays a significant role in the control of telomere elongation in D. melanogaster.     

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.     

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.