Regulation of telomere length in Drosophila

Abstract

Telomeres in all organisms must perform the same vital functions to ensure cell viability: to act as a protective chromosome cap that distinguishes natural chromosome ends from DNA double strand breaks, and to balance the loss of DNA from the chromosome end due to incomplete DNA replication. Most eukaryotes rely on a specialized reverse transcriptase, telomerase, to generate short repeats at the chromosome end to maintain chromosome length. Drosophila, however, uses retrotransposons that target telomeres. Transposition of these elements may be controlled by small RNAs and spreading of silent chromatin from the telomere associated sequence, both of which limit the retrotransposon expression level. Proteins binding to the retrotransposon array, such as HP1 and PROD, may also modulate transcription. It is not clear however, that simply increasing transcript levels of the telomeric retrotransposons is sufficient to increase transposition. The chromosome cap may control the ability of the telomere-specific elements to attach to chromosome ends. As in other organisms, chromosomes can be elongated by gene conversion. Although the mechanism is not known, HP1, a component of the cap, and the Ku proteins are key components in this pathway.

Fig. 1

(A) Telomeric non-LTR retrotransposons. The GAG and RT open reading frames are indicated. All three elements carry long 3′ UTRs of at least 3 kb. The 3′ oligo(A) tail used to attach to chromosome ends is indicated by AAAAAA. (B) Drosophila telomere structure. Starting at the distal end, the chromosome carries a protein complex that binds to the end independently of DNA sequence and stabilizes the terminus. Most telomeres include a tandem mixed array of variably 5′ truncated retrotransposons. The “A” at each junction indicates the 3′ oligo(A) tail. Proximal to the retrotransposons is a complex subterminal repeat array, termed telomere associated sequence (TAS), indicated by repeated disks, followed by unique sequence chromosomal DNA.

Fig. 2

Two modes of Drosophila telomere elongation. Gene conversion, on the left, proposes that the 3′ strand of a chromosome end invades another chromosome, possibly a sister or homologue. The invading strand is extended using the host sequence as a template, then is used as a template in second strand synthesis. Ligation of the newly replicated fragment results in an extended chromosome. Transposition, on the right, proposes that transcripts are generated from a telomeric retrotransposon using promoter activity located in the 3′ UTR of an upstream HeT-A or TAHRE element. The transcripts leave the nucleus to serve as mRNA for the element-encoded GAG-like protein and possibly reverse transcriptase (ovals). GAG-like proteins bind the RNA and facilitate re-entry into the nucleus. After docking to a chromosome end, perhaps mediated by a protein-protein interaction between the GAG-like protein and the chromosome cap, a reverse transcriptase uses the free 3′ hydroxyl group at the chromosome end as primer to copy the RNA intermediate into the first DNA strand. Second strand synthesis completes the addition of a new retrotransposon. While it is possible that HTT transcripts that have not left the nucleus may also be used a templates for reverse transcription at the chromosome ends, sequence analyses of very recently transposed HeT-A elements and of several in native telomeric arrays suggest that there is a selection for the incorporation of elements with a functional GAG-like ORF.

Fig. 3

Two representative genetic assays for transposition onto chromosome ends. (A) P{w^var) is a P element containing a complete white (w) gene that has inserted into TAS of the left arm of chromosome 2 associated with loss of the HTT array and part of TAS distal to the insertion (Golubovsky et al., 2001). Some, but not all, variants have some HTT sequence at the end of the chromosome. Those variants with little or no HTT show pale to light orange eyes, depending on the length of the w sequence remaining. Transposition of a HeT-A to the chromosome end allows the HeT-A 3′ promoter to initiate transcription into the w gene. This can be seen as a change increased pigment in the eye. (B) The yellow (y) gene is normally located 250 kb from the XL TAS. Terminal deficiencies can be induced that bring the end of the chromosome close to the y promoter and inactivate transcription (Kahn et al., 2000). Transposition of a HeT-A element to the chromosome end allows read-through transcription from the HeT-A promoter into the y gene, resulting in a change in the color of the cuticle and wings. Many of the assays for HTT addition upstream of y at the chromosome end incorporate the y ac deficiency that removes the y gene from the homologous chromosome. Thus, there is only one y gene present. In the presence of a full length homologue in which the y gene is complete, gene conversion can transfer upstream y sequences to the truncated chromosome, as in Figure 2.