The Interactive Fly

Zygotically transcribed genes


Telomeres capping function is essential for genome integrity

Telomeres consist of retrotransposon array of HeT-A, TAHRE, and TART sequences

Two distinct domains in Drosophila melanogaster telomeres

Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila 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. Several P-element insertions into this retrotransposon array have been identified and expression levels of transgenes with similar integrations into TAS and euchromatic regions were compared. 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 (Biessmann, 2005).

Noncoding repetitive sequences make up a large portion of eukaryotic genomes. Large blocks of repetitive DNA are mostly packaged into heterochromatin around centromeres, but their organization and structure has been difficult to analyze. By contrast, the smaller regions of heterochromatin at the telomeres provide an opportunity to study their DNA and protein composition. EM data provide the first clear evidence that two distinct chromatin subdomains exist within a telomeric region: the terminal retrotransposon array is diffuse and morphologically resembles interband regions or puffs, while the subterminal TAS region resembles regular bands. These results show that the terminal retrotransposon array at Drosophila telomeres is not refractory to the integration of P elements, but reporter genes inserted into these two domains of the Drosophila telomere are affected differently. Except for the P-element integrations described in this study, the insertion of a full-length roo element into the otherwise stereotypical HeT-A/TAHRE/TART array is the only documented insertion of a transposon into the telomeric retrotransposon region and demonstrates that transposable elements are capable of inserting into the telomeric array (Biessmann, 2005).

Analyzing the structure and composition of the telomeric retrotransposon array can provide information about the dynamic events of new transpositions and terminal erosion that shape the organization of chromosome ends in Drosophila. HeT-A, TAHRE, and TART sequences are predominantly found at telomeres, but tandem arrays of relatively short HeT-A segments also occur in autosomal centromeric heterochromatin and in interstitial regions of the heterochromatic Y chromosome. Thus, isolation of HeT-A sequences from genomic DNA libraries does not ensure that the cloned fragments originated from a telomere. By walking from TAS into the terminal retrotransposon array, two normal telomeres have been analyzed, defining the junction between the proximalmost HeT-A element and the subtelomeric TAS. Directional cloning of chromosome ends demonstrated that the oligo(A) tails of HeT-A elements face toward the centromere (Biessmann, 2005 and references therein).

The results presented in this study confirm and extend these observations. The telomeric retrotransposon arrays are highly polymorphic. HeT-A, TAHRE, and TART elements are intermingled, and the elements are often truncated at the 5' end, although full-length elements were also found. These results are consistent with analyses of BACs that span the TAS regions and extend into the terminal arrays and support the idea of a dynamic Drosophila telomere. The abundance of 5'-truncated retroelements is striking. While these incomplete elements will not produce full-length transcripts of the element, they may provide additional promoters for transcription of proximally located elements (Biessmann, 2005).

Heterochromatin in Drosophila is distinct from euchromatin by several criteria, including cytological staining, timing of replication, a propensity for ectopic pairing, underreplication in polytene chromosomes, and ability to repress gene activity. Most heterochromatin is found around the centromeres, but smaller regions are present at the telomeres and scattered around the genome as intercalary heterochromatin. It has been inferred that telomeres exist in a heterochromatic configuration. While this may be true in part, most studies lack the resolution to distinguish between subdomains within the telomeric region. For instance, earlier observations showed that some but not all telomeres are replicated late; however, they are not among the last sequences to be replicated during S phase (Biessmann, 2005).

Ectopic pairing is a feature often associated with heterochromatin. Telomere-telomere interactions have been well documented and shown to vary widely between strains and over time. While the nature of these ectopic contacts is not known, threads connecting the telomeres, at least in some cases, hybridize with HeT-A and TAS probes. The observation that telomere interactions are dramatically increased in the Tel strain, which has extremely long telomeric retrotransposon arrays, suggests that these interactions are mediated by the retrotransposons or proteins associated with them. These interactions are resolved in diploid brain cells in mitosis, arguing against covalent DNA-DNA bonds (Biessmann, 2005).

Direct comparison of copy number of TAS and HeT-A sequences in diploid vs. polytene tissues to determine possible underreplication is not possible, because these sequences are also found in other genomic locations. Therefore, P-element insertions into the subtelomeric TAS and the pericentric heterochromatin have been used as tags to address this question. These measurements reflect vast differences according to the insertion locations, but telomeric insertions into TAS exhibit very modest, if any, underrepresentation in polytene chromosomes (Biessmann, 2005).

Transcriptional silencing is a sensitive criterion for defining heterochromatin. TAS is likely to be directly involved in silencing telomeric transgenes, suggesting a heterochromatic character. Indeed, a 6-kb 2L TAS array exhibits array-length-dependent and orientation-dependent repression of a w reporter gene, and a single 1.2-kb region derived from the 1.8-kb X TAS repeat induces pairing-sensitive repression of a reporter gene. Telomeric silencing is different from silencing that occurs in closely linked copies of mini-white genes, because TPE on the major autosomes does not respond to mutations in Su(var)205, the gene that encodes HP1 (Biessmann, 2005).

P-element insertions allowed detection of telomeric subdomains by their different ability to silence integrated transgenes. In agreement with the previous studies, it was found that reporter genes surrounded by TAS are repressed. The same P-element constructs inserted into the terminal retrotransposon array, however, generally resemble euchromatic insertions in their level of reporter gene expression, except when they are located close to TAS. These observations support a model for TPE that proposes that variegated expression of reporter genes at telomeres is the result of competition between the repressive effects of TAS and the stimulating effects of the HeT-A promoters. This interaction between HeT-A and TAS might constitute a mechanism by which TAS regulate telomere elongation by controlling HeT-A promoter activity (Biessmann, 2005).

The HP1 protein has been reported to play a role in telomere capping, elongation, and HeT-A transcription . The mechanism by which HP1 might act to promote HeT-A transcription and elongation is unclear, since it is not possible to estimate the number of transcripts per genomic HeT-A copy number, because both increase in the presence of a mutation in Su(var)205. Further, mutations in Su(var)205 do not affect TPE , and HP1 does not bind to the long terminal retrotransposon arrays carried by Tel mutants, except at the cap region (Biessmann, 2005).

The relative position of heterochromatic telomeric domains in Drosophila appears to be reversed from that in telomeres of other eukaryotes. In yeast, the terminal-most telomeric repeats are heterochromatic by virtue of their nonnucleosomal chromatin packaging and their gene silencing ability even in nontelomeric locations, while insertions of reporters into the subtelomeric Y' elements are generally subjected to very little, if any, repression. This difference between Drosophila and yeast may reflect the fundamental difference in how the terminal DNA structures are generated. In yeast and most other eukaryotes, simple repeats are added by telomerase onto the chromosome end, where they bind a number of proteins and assume a heterochromatin-like state called the telosome. In contrast, the terminal retrotransposon arrays in Drosophila are themselves the source of RNA transcripts that are essential components in telomere elongation by serving as mRNA for the synthesis of proteins necessary for transposition and as templates for reverse transcription. The fact that HeT-A and TART elements are actively transcribed would not necessarily require that they be embedded in a euchromatic structure, because a number of active genes transcribed from Pol II promoters are known to be located in centric heterochromatin . These promoters appear well adapted to their heterochromatic environment and display PEV when moved to euchromatic locations. It has been proposed that the HeT-A promoter may belong to this category. However, the findings suggest that the HeT-A promoter is more likely a euchromatic promoter, consistent with the observation that it functions normally when moved to other euchromatic positions and that HeT-A elements placed upstream of a telomeric white or yellow gene have an activating, not a repressing, influence on gene expression (Biessmann, 2005).

Three distinct chromatin domains in telomere ends of polytene chromosomes in Drosophila melanogaster Tel mutants

Drosophila telomeric DNA is known to comprise two domains: the terminal tract of retrotransposons (HeT-A, TART and TAHRE) and telomere-associated sequences (TAS). Chromosome tips are capped by a protein complex, which is assembled on the chromosome ends independently of the underlying terminal DNA sequences. To investigate the properties of these domains in salivary gland polytene chromosomes, use was made of Tel mutants. Telomeres in this background are elongated owing to the amplification of a block of terminal retroelements. Supercompact heterochromatin is absent from the telomeres of polytene chromosomes: electron microscopy analysis identifies the telomeric cap and the tract of retroelements as a reticular material, having no discernible banding pattern, whereas TAS repeats appear as faint bands. According to the pattern of bound proteins, the cap, tract of retroelements and TAS constitute three distinct and non-overlapping domains in telomeres. SUUR, HP2, SU(VAR)3-7 and H3Me3K27 localize to the cap region, as has been demonstrated for HP1. All these proteins are also found in pericentric heterochromatin. The tract of retroelements is associated with proteins characteristic for both heterochromatin (H3Me3K9) and euchromatin (H3Me3K4, JIL-1, Z4). The TAS region is enriched for H3Me3K27. PC and E(Z) are detected both in TAS and many intercalary heterochromatin regions. Telomeres complete replication earlier than heterochromatic regions. The frequency of telomeric associations in salivary gland polytene chromosomes does not depend on the SuUR gene dosage, rather it appears to be defined by the telomere length (Andreyeva, 2005).

Molecular and genetic analyses provide the evidence for existence of three distinct domains in distal regions of chromosomes: cap complex, which is assembled on the terminal DNA in a sequence-independent manner, the array of HeT-A/TAHRE/TART elements, and TAS repeats. The size of the HeT-A/TAHRE/TART tract varies in different chromosome arms, totaling up to 147 kb in the X, 0-50 kb in 2L, 90 kb in 2R, 26 kb in 3L, and 43 kb in 3R. The HeT-A/TAHRE/TART array length is significantly increased on Su(var)205 backgrounds. Electron microscopy analysis demonstrates that distal regions of chromosomes in Tel mutants appear as a decompacted reticular-like material which, according to the FISH data, corresponds to the amplified HeT-A/TAHRE/TART repeats. Cap complex is estimated to span 4-6 kb of terminal DNA (Savitsky, 2002), and in Tel chromosomes cap region cannot be distinguished from the neighboring domain by morphology. In general, both the cap and the chromatin comprising HeT-A/TAHRE/TART arrays do not resemble typical intercalary and pericentric heterochromatin, and look more similar to the decompacted ß-heterochromatin, which also displays reticular structure. Reticular morphology probably originates from the repeated nature of the DNA in this region, which leads to homologous pairing between the fragments of the same strand (Andreyeva, 2005 and references therein).

As visualized by electron microscopy, the HeT-A/TAHRE/TART array is bordered with faint bands, which correspond to the localization sites of TAS repeats, according to FISH analysis. The total length of the TAS domain in telomeric regions is known to be small, approximately 10-25 kb. A middle-sized band normally contains about 30 kb DNA. Analysis of bands formed from the DNA of transposons having a known amount of DNA showed that 5 kb is the minimal size necessary for creating a band discernible under the electron microscope. The size of TAS repeats in D. melanogaster telomeres is at the resolution threshold at the electron microscopy level, in contrast to the IH regions, which often form very large and dense bands spanning up to 200-300 kb. Thus, at the level of cytology, faint bands formed by TAS are distinct from typical heterochromatin (Andreyeva, 2005).

The cap region binds a number of proteins that are known to be localized to the silenced pericentric heterochromatin regions. These are HP2, SU(VAR)3-7, SUUR and H3Me3K27. Association of HP1 and HOAP (Caravaggio) with the cap region has been demonstrated (Andreyeva, 2005 and references therein).

It is possible that HP1 targeting to the cap region occurs via interactions with other proteins. One candidate is HOAP, which forms a complex with HP1 and is present in cap regions of Su(var)205 null mutants. A number of additional proteins appear to contribute to the stability of the HOAP/HP1 complex since, in tefu (ATM), mre11 and rad50 mutants, HP1 and HOAP fail to accumulate in cap regions in polytene chromosomes (Andreyeva, 2005 and references therein).

In polytene chromosomes, the region of HeT-A/TAHRE/TART repeats also associates with a striking combination of proteins: H3Me3K9, characteristic of heterochromatin, and a euchromatin-specific histone isoform H3Me3K4, Z4 and JIL-1. None of these proteins localizes to the cap region (Andreyeva, 2005).

There are several lines of evidence indicating that the chromatin in the HeT-A/TAHRE/TART region in polytene chromosomes might exist in a state that is poised for activation. First, according to electron microscopy data, in salivary gland cells the HeT-A/TAHRE/TART domain does not show a high degree of DNA compaction. Second, this domain has a histone H3 lysine 4 tri-methylation mark, which is associated with actively transcribed genes. However, no actively elongating RNA polymerase isoform (with CTD phosphorylated at serine 5) is detected in this region, nor are the transcripts of HeT-A and TART transposons produced in salivary glands (Andreyeva, 2005).

TAS region is distinct from other telomeric domains, recruiting specific proteins, such as PC and E(Z), that are known to be subunits of the PRC1 and ESC/E(Z) complexes respectively. In vitro E(z) displays histonemethyltransferase activity towards histone H3 lysine residues 9 and 27. Strong enrichment of H3Me3K27 isoform is found in TAS repeat regions (Andreyeva, 2005).

Further support comes from the correlation of TAS presence and localization of PC and E(Z) proteins, which was demonstrated in the current work for all but one telomere. Previous studies found no significant correlation between the TAS repeats and localization of the Pc-G (PC, PH, PSC, and SCM) proteins, which might be attributable to the polymorphism for TAS repeats in the stocks used. SCM was reported to be recruited to the 2R telomere in some cases. It is possible that the 2R telomere recruits a third silencing complex, distinct from PRC1 and ESC/E(Z), which contains the SCM protein. Why different TAS might recruit distinct complexes of Pc-G proteins is currently unknown and this requires further investigation (Andreyeva, 2005).

When an X-chromosome TAS 1.8 kb fragment is placed in a transgenic construct, it displays properties analogous to those of Polycomb response elements (PRE): it contributes to pairing sensitive repression of the adjacent reporter gene and mediates targeting of Pc-G proteins to the transposon insertion site. Strong correlation of PC and E(Z) localization sites with the presence of TAS repeats in the telomeres of chromosome arms 2L and 3L thus suggests that these TAS elements should also possess PRE-like properties (Andreyeva, 2005).

Similar to PRE, TAS repeats cause reporter gene inactivation in transgenic assays. When the reporter is integrated within TAS or immediately adjacent in the context of telomere, the same effect is also observed, which is generally referred to as TPE. Taking into account the parallels between PRE and TAS, and the fact that both PRE and TAS bind repressive Pc-G complexes of proteins, TAS appear to represent the regions of Pc-G-mediated silencing. Recent evidence further supports this idea: the only established TPE modifier, grappa (gpp), codes for a protein with an H3Me2K79 histonemethyltransferase activity, and shows genetic interactions with the Pc-G genes (Shanower, 2005). However, no data are available to prove a direct effect, since H3Me2K79 is not present at the telomeres (Shanower, 2005), whereas the tri-methyl isoform is absent from Drosophila. The effects of many other described TPE modifiers require thorough reassessment, since the early screenings for TPE modifiers did not account for the possible influence of the genetic background. To summarize, the only feature that appears common for TAS regions and IH is that both of them appear to be subject to Pc-G-dependent silencing (Andreyeva, 2005).

The distinct localization pattern observed for a number of chromatin proteins in the most distal regions of polytene chromosomes in the Tel stock is not unique to this mutant background. Thus far, HP1 and Pc-G proteins were localized to the distinct telomere domains in a stock with short HeT-A/TAHRE/TART tracts. According to the data, HP1 did not co-localize with H3Me3K9 in Tel and y w stocks, which differ in HeT-A/TAHRE/TART array length. Finally, very similar protein localization patterns (most notably JIL-1 and Z4) have been described in chromosomes of wild-type stocks (Andreyeva, 2005).

Telomeres in polytene chromosomes, as well as intercalary and pericentric heterochromatin regions, are capable of forming contacts with each other. Nevertheless, the nature of telomeric associations (TAs) and the mechanism of ectopic pairing of heterochromatic regions are obviously different, because the TA frequency is independent of the amount of SUUR protein, remaining unchanged whether SuUR gene is mutant or overexpressed. This contrasts with the observation that ectopic pairing of heterochromatic regions is completely undetectable in SuUR mutants and increases greatly with higher SUUR protein levels, concomitant with the increase in underreplication extent. Since DNA underreplication is a prerequisite for ectopic pairing, then either the telomeres are not underreplicated, or underreplication in telomeres is SuUR-independent. There is no late replication in the region of cap and of the HeT-A/TAHRE/TART array in telomeres of Tel mutants, and therefore these regions might be undergoing complete replication. By contrast, underreplication has been demonstrated for the TAS repeats in the minichromosome Dp1187 and for the w+ reporter inserted into the TAS clusters of 2R and 3R chromosomes, ranging from 1.4- to 2.6-fold in extent. Nevertheless, TAs and ectopic pairing of heterochromatic regions in polytene chromosomes of salivary glands represent fundamentally distinct phenomena, because TAs appear to be mainly dependent on the size of the HeT-A/TAHRE/TART array. The removal of Tel and Su(var)205 mutant alleles from the genome did not modify the frequencies of TAs of chromosomes that were elongated in the mutant stock, whereas the newly introduced chromosomes with short telomeres displayed consistently low frequency of forming TAs in polytene tissue. Therefore, in both Su(var)205 and Tel mutants, the TA frequency in polytene chromosomes largely depends on the length of the HeT-A/TAHRE/TART arrays, independently of whether associations of telomeres are resolved in diploid tissue. In mutants, the lack of proteins encoded by the genes Su(var)205, tefu (ATM), mre11 and rad50 leads to a dramatic increase in frequency of telomeric fusions in diploid dividing cells. Since these associations of telomeres do not break in mitotic anaphase, this observation suggests that these proteins play an important role in protecting the telomeres from fusions. The important differences observed between the polytene and the mitotically dividing cells are most probably due to the fact that salivary gland differentiation takes place in early embryogenesis. Transition of mitotic divisions to endocycles occurs in 8-9-hour-old embryos. At this time, the maternally contributed HP1 obtained from heterozygous Su(var)205/Balancer mothers is still sufficient to suppress telomeric fusions. If formed in the interphase of the last mitosis, associations of telomeres persist through the endocycles, and the polytene nucleus represents a relic of the pre-formed telomeric associations. In this situation, the key factor is the length of the HeT-A/TAHRE/TART array, whereas the deficit of maternal HP1 in mutant third instar larvae provides the explanation for the dependence of telomeric fusion frequency on HP1 level in mitotically dividing neuroblasts and imaginal disks cells (Andreyeva, 2005).

This paper has established that the three telomeric regions - cap, HeT-A/TAHRE/TART and TAS repeats - target specific sets of proteins and thus form distinct non-overlapping domains. The heterochromatin characteristics widely attributed to telomeres in salivary gland polytene chromosomes, such as formation of dense bands, late completion of replication, formation of swellings upon SUUR overexpression, ectopic contacts with intercalary and pericentric heterochromatin regions, involve not the telomeres but the subtelomeric regions, which in the chromosome arms X and 2R are typical intercalary heterochromatin (IH) regions. In chromosomes with normal, short telomeres, these regions appear to be located on the chromosome tips, and are misidentified as telomeric heterochromatin. Although cap and TAS regions resemble intercalary and pericentric heterochromatin in the protein repertoires bound, neither displays features of heterochromatin. This can be partly explained by the small sizes of cap and TAS regions: they are significantly smaller than the huge IH blocks that encompass hundreds of kilobase pairs of DNA. The short DNA sequences that form TAS repeats and cap complex can complete replication early and, therefore, replicate completely. Ectopic contacts in the IH largely depend on the degree of underreplication in these regions. Absence of detectable underreplication appears to lead to the inability of the telomeric regions to associate with other regions in heterochromatin. Formation of telomeric associations is possibly based on homologous pairing, which would be dependent on the copy number of HeT-A/TAHRE/TART and TAS repeats (Andreyeva, 2005).

However, the small size of telomeric DNA is not the only factor that makes these regions unique. Although cap and TAS appear similar to heterochromatic regions, these domains are nevertheless distinct from heterochromatin, since they lack a typical heterochromatic protein marker, H3Me3K9. More striking is the overlapping localization of H3Me3K9 and of a number of typical euchromatic proteins within HeT-A/TAHRE/TART arrays. These findings argue that telomeric domains in polytene chromosomes should not be viewed as classic heterochromatin. The organization of telomeric domains is probably defined by the specific functions of these structures and requires further investigation, especially in diploid tissues and in the wild-type background (Andreyeva, 2005).

Telomere elongation is under the control of the RNAi-based mechanism in the Drosophila germline; mutations in the spn-E and aub cause an increase in the frequency of telomeric element retrotransposition to a broken chromosome end

Telomeres in Drosophila (for a review see Pardue, 2005) are maintained by transposition of specialized telomeric retroelements HeT-A, TAHRE, and TART instead of the short DNA repeats generated by telomerase in other eukaryotes. This study implicates the RNA interference machinery in the control of Drosophila telomere length in ovaries. The abundance of telomeric retroelement transcripts is up-regulated owing to mutations in the spn-E and aub genes, encoding a putative RNA helicase and protein of the Argonaute family, respectively, which are related to the RNA interference (RNAi) machinery. These mutations cause an increase in the frequency of telomeric element retrotransposition to a broken chromosome end. spn-E mutations eliminate HeT-A and TART short RNAs in ovaries, suggesting an RNAi-based mechanism in the control of telomere maintenance in the Drosophila germline. Enhanced frequency of TART, but not HeT-A, attachments in individuals carrying one dose of mutant spn-E or aub alleles suggests that TART is a primary target of the RNAi machinery. At the same time, enhanced HeT-A attachments to broken chromosome ends were detected in oocytes from homozygous spn-E mutants. Double-stranded RNA (dsRNA)-mediated control of telomeric retroelement transposition may occur at premeiotic stages, resulting in the maintenance of appropriate telomere length in gamete precursors (Savitsky, 2006).

The problems of end-under-replication and stability of linear chromosomes are resolved by telomeres. The lengthening of terminal regions of linear eukaryotic chromosomes is often provided by RNA-templated addition of repeated DNA by reverse transcriptase enzyme, telomerase. In most eukaryotes, telomeric DNA is maintained by the action of telomerase, which is responsible for the synthesis of short 6-8-nucleotide (nt) arrays using an RNA component as a template. In contrast, telomeres of Drosophila are maintained as a result of retrotranspositions of specialized telomeric non-long-terminal repeat (LTR) HeT-A, TAHRE, and TART retrotranspositions (Biessmann, 1992b; Levis, 1993; for review, see Pardue, 2003; Abad, 2004b). Retrotransposons are also found in telomeric regions of such diverse organisms as Bombyx mori, Chlorella and Giardia lamblia. HeT-A, TAHRE, and TART are found at Drosophila telomeres in tandem arrays. HeT-A, the most abundant Drosophila telomeric element, contains a single ORF encoding a Gag-like RNA-binding protein, but lacks reverse transcriptase (RT). It is proposed that the RT necessary for its transposition might be provided in trans, perhaps by TART (Rashkova, 2002). TART ORF2 encodes a reverse transcriptase related to the catalytic subunit of telomerase. The recently discovered TAHRE element shows extensive similarity to HeT-A, but contains a second ORF, which encodes a reverse transcriptase (Abad, 2004b). A HeT-A promoter located in the 3' region of the element directs synthesis of a downstream neighbor (Danilevskaya, 1997). The TART element was shown to be transcribed bidirectionally using a putative internal sense promoter and antisense one that was localized within the 1-kb region of the TART 3' end (Danilevskaya, 1999). Maintenance of Drosophila telomere length is mediated by HeT-A and TART transpositions to chromosome ends as well as by terminal recombination/gene conversion (Mikhailovsky, 1999; Kahn, 2000). Most of the observed spontaneous attachments to telomeres are HeT-A transpositions (Biessmann, 1992a; Kahn, 2000; Golubovsky, 2001), but TART attachments (Sheen, 1994) were also detected (Savitsky, 2006 and references therein).

The spn-E and aub genes, encoding an RNA helicase and a protein of Argonaute family, respectively, are involved in double-stranded RNA (dsRNA)-triggered RNA interference (RNAi) in embryos, in transcriptional silencing of transgenes, and in the control of Drosophila retrotransposon transcript abundance in the germline, especially in ovaries. No effects of RNAi gene mutations on HeT-A and TART expression and telomere structure were observed in somatic tissues (Perrini, 2004). This study shows that increased HeT-A and TART transcript abundance in ovaries, owing to RNAi mutations, is correlated with a high frequency of telomeric element attachments to broken chromosome ends. Addition of HeT-A or TART to a truncated X chromosome, with a break in the upstream regulatory region of yellow, activates yellow expression in aristae, which enables monitoring of the elongation events (Kahn, 2000; Savitsky, 2002). Using this genetic system, the effects of RNAi mutations were studied on the frequency and molecular nature of telomeric attachments. A high frequency of TART but not HeT-A attachments in heterozygous RNAi mutants suggests that TART may be the primary target of the RNAi-based silencing mechanism. These results highlight for the first time the importance of TART, but not the more abundant HeT-A element, in Drosophila telomere maintenance. The disappearance of short TART and HeT-A RNAs was found in spn-E mutant ovaries, strongly suggesting an RNAi-based pathway in the control of telomere maintenance in the Drosophila germline (Savitsky, 2006).

An RNAi-based mechanism has been proposed to evolve in order to immobilize transposable elements and was found to control expression of endogenous transposable elements and their mobility in different species. Drosophila telomeres are maintained by successive transpositions of specialized telomeric retroelements HeT-A and TART. This study shows that transposition of both telomeric elements is under the control of the spn-E and aub genes, known to be related to the RNAi machinery. Hence, an RNAi-based mechanism may be considered not only as a defense against retrotransposon expansion, but also as a regulatory system responsible for proper telomere length maintenance in Drosophila (Savitsky, 2006).

spn-E is required for appropriate localization of mRNA and proteins involved in the establishment of axis formation in the embryo and encodes a member of the DEAD/DE-H protein family possessing RNA-binding and RNA helicase activity. aub encodes a protein of the Argonaute family that was shown to be a component of the RNAi effector complex RISC. aub and spn-E mutations strongly diminished effects of the injected dsRNA into mature oocytes. Both genes are implicated in small interfering RNA (siRNA)-dependent silencing of testis-expressed Stellate genes. Thus, spn-E and aub are components of RNAi-based silencing pathways in Drosophila. Mutations in these genes result in the derepression of a wide spectrum of retrotransposons in the germline, including the HeT-A telomeric element (Aravin, 2001; Stapleton, 2001; Kogan, 2003). This study demonstrates that spn-E and aub mutations increase the frequency of telomeric element retrotranspositions to broken chromosome termini, suggesting that the RNAi machinery controls telomere length in Drosophila (Savitsky, 2006).

Both telomeric elements are shown to be the targets of RNAi. The present results emphasize the differences in the response of HeT-A and TART elements to RNAi mutations. Surprisingly, two different spn-E mutant alleles and an aub mutation in the heterozygous state increase considerably TART mobility, whereas attachments of HeT-A to broken chromosome ends were detected much more rarely in spn-E1/+ ovaries and are not observed in ovaries of spn-Ehls3987/+ and aubQC42/+ flies. One copy of a spn-E mutation is sufficient to increase TART transcript abundance. Strong accumulation of HeT-A transcripts is found only in homozygous mutants, correlating with a high frequency of HeT-A attachments to the broken chromosome ends in the developing oocytes. This observation argues that TART is a primary target of the RNAi machinery in ovaries (Savitsky, 2006).

TART and HeT-A, in spite of sharing the region of integration, are dissimilar in their structure and expression strategy. While both sense and antisense TART transcription has been demonstrated, antisense transcripts are more abundant. In situ RNA analysis detected sense and antisense TART transcripts in the cytoplasm of nurse cells in the late-stage egg chambers, suggesting a possibility of dsRNA formation. However, it was found that the level of antisense TART transcripts is not affected in RNAi mutants. Only sense HeT-A transcription was observed by Northern or by in situ RNA analyses. Nevertheless, HeT-A- and TART-specific siRNAs were revealed among the cloned short RNA species in Drosophila, and short RNAs corresponding to both HeT-A and TART elements are detected by Northern analysis. Antisense HeT-A RNA is probably transcribed at a low level from an unidentified promoter, possibly, from the HeT-A internal region. Actually, a low level of antisense activity of the HeT-A 3' end has been observed . While TART transcripts were observed only in the nurse cells, HeT-A transcripts were detected both in the growing oocyte and nurse cells. It is proposed that TART is a primary target of the RNAi controlling system, since one dose of an RNAi mutation causes preferential TART, but not HeT-A, attachments to broken chromosome ends in ovaries. In contrast, one dose of a mutant Su(var)205 gene (HP1) considerably increasess the frequency of HeT-A rather than TART attachments to the chromosome ends (Savitsky, 2002). Thus, a specific effect of RNAi components on telomeric element expression is observed . Although TART copies are much less abundant in the genome than HeT-A and no TART elements are detected in some telomeres, TART is a conserved component of telomeres in distant Drosophila species. TART was considered as a source of RT production, thus ensuring retrotranspositions of both TART and HeT-A elements. One may propose that TART supplies an RNAi-regulated template for RT production, thus providing telomere-specific transpositions of both elements (Savitsky, 2006).

Drosophila telomeres contain a multisubunit protein complex forming a chromosome cap protecting chromosomes from DNA repair and end-to-end fusions. However, no HeT-A or TART sequences were detected at the stably maintained broken chromosome end, which is protected from telomere fusions. Thus, a sequence-independent system performs telomere capping functions. The capping complex contains HP1, HOAP (HP1/ORC associated protein), as well as ATM-kinase and DNA repair MRN complex and the Ku70/Ku80 heterodimer. HP1 and the Ku heterodimer act also as negative regulators of telomere elongation by retrotransposition of telomeric elements. Deficiencies that remove either the Ku70 or the Ku80 gene increase the transposition rate of HeT-A and TART elements but exert no effect on the HeT-A expression, suggesting that Ku proteins control the accessibility of the telomere to transposition events. At the same time, mutations in the Su(var)205 gene increase both transcript abundance of HeT-A and TART and the frequency of their attachments to chromosome ends. RNAi affects both telomeric retrotransposon expression and the rate of transposition to the telomere. Probably, this effect is mediated through HP1 recruitment and silencing of HeT-A and/or TART chromatin (Savitsky, 2006).

siRNAs produced from telomeric elements TART and HeT-A belong to the long size class (25-29 nt) in contrast to 21-22-nt RNAs guiding post-transcriptional RNAi. In plants, long siRNAs are associated with RNA-directed DNA methylation and play an essential role in the transcriptional retrotransposon silencing. dsRNA and proteins of the RNAi machinery can direct chromatin alteration to homologous DNA sequences and induce transcriptional silencing. RNAi mutations cause delocalization of HP1 in yeast and Drosophila. Actually, the increase in accessibility of HeT-A chromatin and its enrichment in K9-acetylated H3 histone were revealed in ovaries of spn-E mutants. It is also possible that TART and/or HeT-A short RNAs can be targeted to telomeric repeats in a transcriptional silencing complex (Savitsky, 2006).

RNAi disruption affects neither HeT-A and TART expression, nor telomere fusions in somatic cells. No effect was observed of spn-E mutations on HeT-A expression, even in actively dividing cells of imaginal discs, where HeT-A expression was found. The data indicate a crucial role of the RNAi machinery in the regulation of telomere elongation in germinal cells. The appearance of a cluster of individuals with identical retroelement attachments indicates that dsRNA-mediated control of terminal elongation may occur at premeiotic stages of oogenesis (Savitsky, 2006).

This study has demonstrated that expression and retrotransposition of specific telomeric repeats is under control of an RNAi-based system in the Drosophila germline. In this case, the telomerase-dependent mechanism of telomere stability is substituted by retrotranspositions. Interestingly, telomerase-dependent telomere functioning during meiosis in the yeasts Schizosaccharomyces pombe and Tetrahymena is also under the control of RNAi machinery. These observations and the current data indicate that dsRNA-mediated regulation of telomere dynamics in the germline may be a general phenomenon independent of a mode of telomere maintenance (Savitsky, 2006).

Genomic organization of the Drosophila telomere retrotransposable elements

The emerging sequence of the heterochromatic portion of the Drosophila melanogaster genome, with the most recent update of euchromatic sequence, gives the first genome-wide view of the chromosomal distribution of the telomeric retrotransposons, HeT-A, TART, and Tahre. As expected, these elements are entirely excluded from euchromatin, although sequence fragments of HeT-A and TART 3' untranslated regions are found in nontelomeric heterochromatin on the Y chromosome. The proximal ends of HeT-A/TART arrays appear to be a transition zone because only here do other transposable elements mix in the array. The sharp distinction between the distribution of telomeric elements and that of other transposable elements suggests that chromatin structure is important in telomere element localization. Measurements reported in this study show (1) D. melanogaster telomeres are very long, in the size range reported for inbred mouse strains (averaging 46 kb per chromosome end in Drosophila stock 2057). As in organisms with telomerase, their length varies depending on genotype. There is also slight under-replication in polytene nuclei. (2) Surprisingly, the relationship between the number of HeT-A and TART elements is not stochastic but is strongly correlated across stocks, supporting the idea that the two elements are interdependent. Although currently assembled portions of the HeT-A/TART arrays are from the most-proximal part of long arrays, ~61% of the total HeT-A sequence in these regions consists of intact, potentially active elements with little evidence of sequence decay, making it likely that the content of the telomere arrays turns over more extensively than has been thought (George, 2006).

A surprising finding of this study has been the number of apparently functional HeT-A elements deep within the telomere arrays. If addition of telomere repeats serves only to replace eroded sequence on the chromosome end, one would expect sequences deep inside the arrays to decay because, once added to the end, there should be little constraint to maintain function if their only function is to buffer a chromosome end. Instead, the full-length sequences here have maintained ORFs and other regions needed for function. The existence of functional elements in proximal regions of these long telomere arrays suggests that these interior sequences may be renewed more frequently than has been thought and that turnover in these arrays does not simply replace terminal sequence lost in DNA replication. A likely possibility is that telomeres sometimes undergo drastic shortening, perhaps by a mechanism such as that Non-LTR elements are frequently 5'-truncated, presumably because reverse transcription, which begins at the 3' end, is incomplete. In their analysis of sequence from the euchromatic parts of the D. melanogaster genome, it has been found that 79% of the non-LTR retroelements identified were partial elements. It was expected that HeT-A and TART would be as likely to undergo incomplete reverse transcription as other non-LTR elements and, in addition, to suffer perhaps significant erosion during the time when each element forms the end of a telomere (George, 2006).

The data do not support the expectation that significantly more telomere elements would be truncated; 70% (14 of 20) of HeT-A and 71% (5 of 7) of TART elements are truncated, slightly less than the 79% seen for elements not subject to end erosion. For this calculation, the tiny 'tags,' which are believed are byproducts of the unusual HeT-A promoter, were omitted (George, 2006).

This observation that a significant fraction of HeT-A elements in the array shows little, if any, terminal erosion suggests that ends are protected from degradation or that transpositions frequently occur in rapid succession before erosional loss. These possibilities are not mutually exclusive. Protection could be provided by terminal structures like the t-loops seen on chromosomes in other organisms; however, it is not yet known whether Drosophila telomeres have such structures (George, 2006).

Quantitative Southern hybridization analyses give a reasonably accurate measurement of the number of HeT-A and TART ORF equivalents in the female genomes of several stocks and, with the sequence analysis reported above, provides a basis for estimating the total length of HeT-A and TART sequence in telomeres (George, 2006).

That estimate has several uncertainties. Apparently intact elements can differ by indels that add up to several hundred base pairs; the 5' end of TART presents technical problems because of its Perfect Non-Terminal Repeats (PNTRs) and may not be completely defined; also, telomere arrays have severely 5' truncated elements (without any ORF) not detected by Southern hybridizations. By using data from the assembled sequence on chromosomes XL and 4R, it is possible to correct for truncated elements. Although the most distal element in the 4R sequence is a 5' truncated TART, which could be the true end of the chromosome, it is treated like the most distal, 5' truncated, HeT-A in XL, which is clearly truncated by cloning, and, in order not to bias estimation of the results, exclude both. From these measurements it was determined that

  • The average length of the complete HeT-A element is 5893 +/- 169 bp (SD)
  • ~61% of the total HeT-A sequence in the 2057 stock is in complete elements.
  • ~80% of the total HeT-A coding sequence in 2057 is in complete coding sequences.
  • The consensus length of the TART element of 11,734 bp (subfamilies A, B, and C) is consistent with the new data.
  • ~89% of the total TART sequence in 2057 is in complete elements.
  • ~100% of the total TART coding sequence in 2057 is in complete coding sequences.

Using these numbers, it is calculated from hybridization results that the 2057 genome contains polarized HeT-A/TART arrays with ~29 complete HeT-A elements and approximately seven complete TART elements. Correcting for partial elements, ~365 kb of total HeT-A and TART sequence was calculated on eight telomeres, an average of ~45.6 kb of HeT-A and TART sequence per telomere. Perforce, the same correction factors were used for estimates of other genomes (George, 2006).

Although most eukaryotes have very similar telomere sequences, multicellular eukaryotes have much longer telomere arrays than do unicellular eukaryotes. Among the longest studied telomeres are those of inbred strains of laboratory mice. These telomeres range from 30-150 kb, approximately the length of D. melanogaster HeT-A/TART arrays. In contrast, wild-derived inbred mouse strains have telomeres in the 4-15 kb range, approximately the size of human telomeres (George, 2006).

It is interesting that mice and flies, the two organisms known to have unusually long telomeres, are also unusual because they have been kept in small isolated laboratory populations for many years, suggesting that something about the population structure or relatively luxurious laboratory conditions may affect telomere length. It will be interesting to see whether wild-derived D. melanogaster have shorter telomeres, like wild-derived mice (George, 2006).

Studies of several organisms have shown that, although telomere length varies, these variations are held within a relatively narrow range and the center of this range can be changed by external conditions or by changes in genotype. For example, a recent study identified ~150 nonessential genes in Saccharomyces cerevisiae that changed the average around which telomere length fluctuated. Loss of some of these genes led to longer telomeres; conversely, loss of other genes led to shorter telomeres. These studies show that addition and loss of telomere sequence is under complex control (George, 2006).

The retrotransposon telomeres of Drosophila, similar to those maintained by telomerase, have genetically modulated length control. It has been reported that three stocks carrying different mutant alleles of Su(var)205 have high levels of telomeric DNA. However, stocks from a different laboratory but carrying the same alleles have lower amounts of telomeric DNA. The Su(var)2054 stock was found to have a lesser amount of telomeric DNA than reported for the other Su(var)205 stocks. Comparison of these two sets of mutant stocks suggests that different genetic backgrounds can modify the effect of the Su(var)205 mutation on telomere length. Tel-1 mutant flies have significantly more telomeric DNA than the other stocks, and the amounts are influenced by genetic background (George, 2006).

Analysis of the assembled sequence suggested that Drosophila telomeres occasionally undergo large deletions of the type reported in yeast and humans. In contrast, DNA measurements show that stocks and cell lines maintain relatively constant equilibrium telomere lengths, under some genetic controls, so deleted material must be rapidly replaced (while maintaining significant correlation of the numbers of HeT-A and TART elements) (George, 2006).

HP1 is distributed within distinct chromatin domains at Drosophila telomeres

Telomeric regions in Drosophila are composed of three subdomains. A chromosome cap distinguishes the chromosome end from a DNA double-strand break; an array of retrotransposons, HeT-A, TART, and TAHRE (HTT), maintains telomere length by targeted transposition to chromosome ends; and telomere-associated sequence (TAS), which consists of a mosaic of complex repeated sequences, has been identified as a source of gene silencing. Heterochromatin protein 1 (HP1) and HP1-ORC-associated protein (HOAP) are major protein components of the telomere cap in Drosophila and are required for telomere stability. Besides the chromosome cap, HP1 is also localized along the HTT array and in TAS. Mutants for Su(var)205, the gene encoding HP1, have decreased the HP1 level in the HTT array and increased transcription of individual HeT-A elements. This suggests that HP1 levels directly affect HeT-A activity along the HTT array, although they have little or no effect on transcription of a white reporter gene in the HTT. Chromatin immunoprecipitation to identify other heterochromatic proteins indicates that TAS and the HTT array may be distinct from either heterochromatin or euchromatin (Frydrychova, 2008).

On the basis of expression of telomeric white and yellow transgenes Drosophila telomeres have been proposed to have two distinct domains: TAS, which resembles heterochromatin and the HTT array, which behaves like euchromatin. According to the pattern of chromatin proteins revealed by immunostaining of extended polytene chromosomes in a Tel mutant, telomeres consist of three distinct and nonoverlapping domains: the chromosome cap, the HTT array, and TAS. The immunostaining results indicate that HP1 in telomeres is restricted to the cap region (Frydrychova, 2008).

Using ChIP, this study has shown that HP1 is also present along the HTT array outside of the cap as well as in TAS. The difference between these observations and previous reports might be due to a higher abundance of HP1 in the telomere cap than in the internal HTT region or better accessibility of antibodies to the telomere cap, and thus the difference in the reports may be explained by higher sensitivity of ChIP compared to immmnostaining of polytene chromosomes. The difference may be caused also by different properties of long telomeres of a Tel mutant or different biological properties of polytene salivary chromosomes compared to diploid or other polyploid cells. In any case, ChIP data on whole animals are more likely to be generalizable than immunostaining data on a specific cell type (Frydrychova, 2008).

Su(var)205 belongs to a group of suppressor of variegation [Su(var)] genes, many of which encode chromosomal proteins or modifiers of chromosomal proteins. Mutations in Su(var) genes lead to suppression of position-effect variegation (PEV), which is repressed and variegated expression of genes placed in or near pericentric heterochromatin. Despite phenotypic similarities between PEV and telomere position effect (TPE), TPE does not respond to Su(var) mutations. Although TAS was identified as a source of telomeric silencing, and the retrotransposon array genetically resembles euchromatin, comparable levels of HP1 were found at transgenes inserted in these two telomeric domains. The levels of other marks for silent chromatin, such as histone H2A.v and MeK9H3, however, did vary between these two regions in a manner consistent with proposals in previous reports that HTT is associated with open chromatin and TAS is associated with closed chromatin. TPE may thus be caused by a silencing system different from HP1-mediated heterochromatin. One candidate is Polycomb silencing; Polycomb group proteins were found associated with TAS. Since levels of the chromatin markers in all tested regions, including euchromatin and pericentric heterochromatin, showed significant differences, interpretation of HTT and TAS as either heterochromatin or euchromatin is rather difficult. It may suggest that HTT and TAS are in a category of some transitional type of chromatin between euchromatin and heterochromatin, such as closed/inactive euchromatin, or it suggests the existence of additional chromatin types (Frydrychova, 2008).

The relatively high level of HP1 on a transgene inserted into pericentric heterochromatin compared with transgenes in either HTT or TAS may suggest that failure of telomeric HP1 to silence telomeric transgenes is caused by its relative paucity. HP1, however, is a negative regulator of telomere length; its mutations lead to an increase in the transcriptional activity of HeT-A and TART, as well as an accumulation of these elements at the chromosome end. The promoter activity of a telomeric w transgene inserted between the HTT array and TAS significantly exceeds the activity of a single HeT-A promoter. This study shows that that Su(var)205 mutations lead to a severalfold increase in the transcriptional activity of HeT-A, however no increase is seen in transcription of a w gene inserted into the HTT array. In particular, using HeT-A/P-element readthrough transcripts in three P-element insertion lines, it was found that Su(var)205 mutations lead to stimulation of HeT-A elements along the HTT array in all regions assayed. With regard to the low level of HP1 in telomeric regions compared to pericentric heterochromatin, as observed by ChIP experiments, it is conceivable that the relatively weak HeT-A promoter is more sensitive to HP1 concentration than the more robust w promoter. However, HP1 per se cannot be considered as a signal for silencing. An analysis of genomewide correlations between the HP1 binding pattern and the pattern of gene expression revealed that recruitment of the protein is not sufficient to repress transcription completely. Moreover, some euchromatic genes in Drosophila are activated by the presence of HP1. With respect to these observations, it is difficult to predict the effect of HP1 recruitment on the transcription pattern in any specific region (Frydrychova, 2008).

HP1, by interaction with HOAP, forms capping complexes at the ends of Drosophila chromosomes. Formation or maintenance of the HP1-HOAP capping complex requires ATM. Loss of ATM reduces localization of HP1 and HOAP at telomeres and leads to frequent telomeric fusions. tefu and cav mutations, however, did not lead to a profound increase in HeT-A transcription, as was observed in Su(var)205 mutants. This suggests that HP1 presence in the cap does not significantly participate in overall HeT-A transcriptional activity, and that HeT-A transcription is regulated mainly by HP1 in the HTT array outside the cap. The data are consistent with previous studies that suggested two distinct mechanisms for HP1 control of telomere capping and telomere elongation by retroelement transcription. It was proposed that the capping function of HP1 is due to its direct binding to telomeric DNA, while the silencing of telomeric sequences and control of transcription of telomeric retroelements is due to interaction of HP1 with MeK9H3 and spreading of HP1 and repressive chromatin along the telomere (Frydrychova, 2008).

Collectively, these data show that HP1 is present along the HTT array as well as in TAS and plays a role as a negative regulator of transcription of telomeric retroelements. The present data also support the observation that the HeT-A promoter is relatively weak compared with a mini-w promoter and more sensitive to local HP1 concentration and suggest that telomeric chromatin in Drosophila may be distinct from either euchromatin or heterochromatin (Frydrychova, 2008).

HipHop interacts with HOAP and HP1 to protect Drosophila telomeres in a sequence-independent manner

Telomeres prevent chromosome ends from being repaired as double-strand breaks (DSBs). Telomere identity in Drosophila is determined epigenetically with no sequence either necessary or sufficient. To better understand this sequence-independent capping mechanism, proteins were isolated that interact with the HP1/ORC-associated protein (HOAP) capping protein, and HipHop was identified as a subunit of the complex. Loss of one protein destabilizes the other and renders telomeres susceptible to fusion. Both HipHop and HOAP are enriched at telomeres, where they also interact with the conserved HP1 protein. A model telomere lacking repetitive sequences was developed to study the distribution of HipHop, HOAP and HP1 using chromatin immunoprecipitation (ChIP). It was discovered that they occupy a broad region >10 kb from the chromosome end and their binding is independent of the underlying DNA sequence. HipHop and HOAP are both rapidly evolving proteins yet their telomeric deposition is under the control of the conserved ATM and Mre11-Rad50-Nbs (MRN) proteins that modulate DNA structures at telomeres and at DSBs (Gao, 2009). This characterization of HipHop and HOAP reveals functional analogies between the Drosophila proteins and subunits of the yeast and mammalian capping complexes, implicating conservation in epigenetic capping mechanisms (Gao, 2010).

Telomeres shield the ends of linear chromosomes from DNA repair activities. This capping function is essential for genome integrity, as uncapping can lead to chromosome fusions. Telomeres also facilitate the elongation of chromosome ends, a function performed by the telomerase enzyme in most eukaryotic organisms studied. Loss of telomerase function does not impair genome stability immediately, but only does so when telomeric repeats become critically short after several generations. However, loss of the capping function can have immediate effects on genome integrity, suggesting that the presence of telomeric repeats is not sufficient for maintaining telomere identity. Furthermore, specialized yeast and plant cells can be immortalized in the absence of telomeric repeats with protected telomeres, suggesting that the presence of the repeats is also not necessary for capping. These results suggest that sequence-independent capping might serve as a backup mechanism in telomerase-maintained organisms (Gao, 2010 and references therein).

The understanding of this mechanism requires a clear picture of chromatin structure at telomeres. In lower eukaryotes, telomeric repeats are not packaged into regular nucleosomes, while the bulk of telomeric repeats in mammalian cells are packaged into nucleosome arrays. Partly due to the repetitive nature of telomeric sequences, it has been difficult to study how duplex-binding proteins are distributed over telomeric chromatin in most organisms. The Rap1 protein from budding yeast binds telomeric repeats to serve its functions in telomere elongation and capping regulation. Interestingly, Rap1 from budding and fission yeast and Taz1 from fission yeast have been localized to subtelomeric regions, suggesting that the binding of capping proteins need not be limited to the extreme end of a chromosome (Gao, 2010 and references therein).

In Drosophila, telomere identity is determined epigenetically. Although telomeres are elongated by the transposition of telomere-specific retrotransposons, these elements are neither necessary nor sufficient for capping. In particular, terminally deleted chromosomes that lack telomeric retrotransposons are stable, hence capped, for many generations. In addition, population studies uncovered frequent occurrences of such terminally deleted chromosomes in natural populations (Gao, 2010 and references therein).

Despite using a telomerase-independent mechanism for elongating chromosome ends, Drosophila use highly conserved factors to regulate capping. The ATM and ATR checkpoint kinases, along with the Mre11-Rad50-Nbs (MRN) complex and the ATRIP protein, respectively, control redundant pathways for capping regulation that are conserved in other organisms. Several other proteins serving capping function in Drosophila have homologs in other organisms: HP1, UbcD1, Woc and the H2A.Z histone variant. Epigenetic capping mechanisms that might be conserved in other organisms can be effectively studied in the unique system of Drosophila due to the natural uncoupling of the end capping function from the end elongation function (Gao, 2010).

Telomeres in yeast and mammals are capped by multi-subunit protein complexes that protect both the duplex and single-stranded regions of the telomere. In Drosophila, the structural constituents of the 'cap' remain poorly defined. The HP1/ORC-associated protein (HOAP) is cytologically present at telomeres, and loss of HOAP leads to telomere fusions. This study isolated HOAP-interacting proteins by affinity immunoprecipitation and identified the HP1-HOAP-interacting protein (HipHop) as a new component of the Drosophila capping complex. Using chromatin immunoprecipitation (ChIP) performed on a model telomere devoid of telomeric transposons, a large domain of telomeric chromatin was discovered enriched with HipHop, HOAP and HP1, suggesting that this capping complex prevents end fusion by maintaining a chromatin state that is independent of its underlying DNA sequence. Both HipHop and HOAP are fast-evolving proteins highlighting a common feature among telomeric-binding proteins in other organisms. On the basis of functional similarity and analogies in distribution patterns, it is suggested that HipHop and HOAP serve similar function as subunits of the capping complex that bind the duplex region of telomeric DNA in other organisms (Gao, 2010).

This study identified HipHop based on its ability to associate with HOAP through biochemical purification. Such an approach could be useful for future studies in Drosophila telomere biology. The biochemical approach was aided by an ability to epitope-tag the endogenous caravaggio cav locus, eliminating potential artifacts associated with the overproduction of bait proteins. With the recent development of the SIRT targeting method in Drosophila, biochemical purification using endogenous tags could be efficiently applied in the study of other biological processes in Drosophila (Gao, 2010).

Several lines of evidence suggest that HipHop and HOAP likely function as a complex. First, HipHop was abundantly present in HOAP IPs, suggesting a strong interaction between the two proteins. Second, bacteria expressed HipHop was able to interact with HOAP in fly extracts. Third, the changes of HOAP and HipHop levels showed inter-dependency. Fourth, the loading of both HipHop and HOAP to telomeres was under the same genetic controls of MRN and ATM. Finally, the two proteins had very similar distribution patterns on the model telomere and co-localized precisely in immunostaining experiments. On the basis of some of the same criteria, HP1 is likely to be a part of the complex. The Modigliani(Moi)/DTL protein was recently identified as another capping protein that is enriched at telomeres and interacts with both HOAP and HP1. No Moi/DTL peptides in were detected in HOAP IPs (Gao, 2010).

The model telomere D4ATD has allowed an unprecedented view of the chromatin landscape in the vicinity of a Drosophila telomere. HipHop, HOAP and HP1 were located essentially at the very end of a chromosome, strengthening earlier results from immunolocalization experiments. Remarkably, HipHop, HOAP and HP1 seem to bind to a much larger region than the immediate vicinity of the chromosome end. One possible mechanism is envisioned that could lead to such a binding pattern. After the initial recruitment of the HipHop-HOAP complex to the chromosome end, the complex 'spreads' internally to cover a larger region. It is tempting to speculate that this 'spreading' might be mediated by HP1, since a binding pattern of HP1 was observed essentially identical to those of HipHop and HOAP on D4ATD. However, results from ChIP experiments using HeT-A primers suggest that HP1 occupies a larger region than HipHop or HOAP on transposon-capped telomeres, which implies that the mere presence of HP1 on chromatin is not sufficient for HipHop or HOAP binding. In addition, HOAP can be localized to telomeres in su(var)205/hp1 mutants, suggesting that HP1 is not necessary for HOAP and possibly HipHop binding to telomeres. Whether HP1 affects the extent of HipHop-HOAP spreading requires ChIP localization of HipHop and HOAP on the model telomere in a su(var)205 mutant background (Gao, 2010).

It is suggested that the binding patterns of HipHop and HOAP on the model telomere is a qualitative reflection of their patterns on natural telomeres, since very similar binding intensity of HipHop on D4ATD versus its homologous telomere is observed in immunostaining experiments. Similar observations were documented for HP1 on polytene and HOAP on mitotic telomeres using TDs (Gao. 2010).

HipHop and HOAP share functional characteristics with capping proteins in other eukaryotes. First, they bind to the double-stranded region of the telomere in vivo. Second, they occupy a large domain on telomeric chromatin. Third, they are continuously present at the telomeres. Finally, the loss of these proteins leads to frequent telomere fusions. It is suggested that HipHop and HOAP behave similarly and might serve similar functions as the Rap1 protein in S. cerevisiae, Taz1 in S. pombe, and TRF2 in mammals. Further dissection of HipHop and HOAP's molecule function would be needed to confirm this suggestion (Gao, 2010).

The telomere loading of HipHop and HOAP is under the control of ATM and MRN. The same set of proteins mediate the loading of various telomeric factors including telomerase activity, and the Cdc13 capping protein in yeast. This high degree of functional conservation suggest that it is unlikely that these factors directly act on capping proteins, which are generally divergent at the sequence level. It is more likely that these proteins modulate a common DNA/chromatin structure at telomeres of eukaryotic cells. One conceivable candidate for this 'universal' structure is the terminal 3' overhang (reviewed in Lydall, 2009). The reduced occupancy of HipHop, HOAP and HP1 at the extreme end of the model telomere, suggests that Drosophila chromosomes might also terminate as a 3' overhang (Gao, 2010).

HipHop and HOAP seem to evolve faster than typical proteins. An interesting proposition is that this faster rate of evolution is driven by the fast-evolving telomeric retrotransposons (Villasante, 2008), to which the HipHop-HOAP complex binds. HOAP was implicated in binding DNA (Shareef, 2001). Whether HipHop is capable of binding DNA directly is currently under investigation. Under the limited resolution of immunostaining, no change was detected in HipHop-HOAP binding efficiency to telomeres with different levels of retrotransposons. Nor were observed any phenotypic effects of having a 'retrotransposon-free' telomere. Although TDs can be efficiently maintained under laboratory conditions, it remains undetermined whether there is any fitness cost for animals with a TD irrespective of the loss of essential genes. Therefore, further studies are required to identify the driving force for the fast evolution of HipHop and HOAP (Gao, 2010).

Interestingly, telomeric proteins from other systems are generally less conserved at the sequence level and show signs of fast evolution. Further investigation into the functional relationship between HipHop-HOAP and the telomeric retrotransposons in Drosophila might reveal the significance for this fast evolution of telomeric proteins in general (Gao, 2010).

Drosophila telomere capping protein HOAP interacts with DSB sensor proteins Mre11 and Nbs

In eukaryotes, specific DNA-protein structures called telomeres exist at linear chromosome ends. Telomere stability is maintained by a specific capping protein complex. This capping complex is essential for the inhibition of the DNA damage response (DDR) at telomeres and contributes to genome integrity. In Drosophila the central factors of telomere capping complex are HOAP and HipHop. Furthermore, a DDR protein complex Mre11-Rad50-Nbs (MRN) is known to be important for the telomere association of HOAP and HipHop. However, whether MRN interacts with HOAP and HipHop, and the telomere recognition mechanisms of HOAP and HipHop are poorly understood. This study shows that Nbs interacts with Mre11 and transports the Mre11-Rad50 complex from the cytoplasm to the nucleus. In addition, this study reports that HOAP interacts with both Mre11 and Nbs. The N-terminal region of HOAP is essential for its co-localization with HipHop. Finally, it is revealed that Nbs interacts with the N-terminal region of HOAP (On, 2021).

Gag proteins of Drosophila telomeric retrotransposons: collaborative targeting to chromosome ends

TAHRE, the least abundant of the three retrotransposons forming telomeres in Drosophila melanogaster, has high sequence similarity to the gag gene and untranslated regions of HeT-A, the most abundant telomere-specific retrotransposon. Despite TAHRE's apparent evolutionary relationship to HeT-A, TAHRE Gag cannot locate to telomere-associated 'Het dots' unless collaborating with HeT-A Gag. TAHRE Gag is carried into nuclei by HeT-A or TART Gag, but both TART and TAHRE Gags need HeT-A Gag to localize to Het dots. When coexpressed with the appropriate fragment of HeT-A and/or TART Gags, TAHRE Gag multimerizes with either protein. HeT-A and TART Gags form homo- and heteromultimers using a region containing major homology region (MHR) and zinc knuckle (CCHC) motifs, separated by a pre_C2HC motif (motifs common to other retroelements). This region's sequence is strongly conserved among the three telomeric Gags, with precise spacing of conserved residues. Nontelomeric Gags neither interact with the telomeric Gags nor have this conserved spacing. TAHRE Gag is much less able to enter the nucleus by itself than HeT-A or TART Gags. The overall telomeric localization efficiency for each of the three telomeric Gag proteins correlates with the relative abundance of that element in telomere arrays, suggesting an explanation for the relative rarity of TAHRE elements in telomere arrays and supporting the hypothesis that Gag targeting to telomeres is important for the telomere-specific transposition of these elements (Fuller, 2010).

Drosophila telomeres are maintained by a remarkable variant of the telomerase mechanism that maintains telomeres in almost all organisms. As in other organisms, Drosophila telomeres are elongated by tandem repeats that are reverse transcribed onto the ends of the chromosomes. What makes Drosophila telomeres unusual is the RNA template that is reverse transcribed to produce these repeats: Drosophila telomere repeats are copied from full-length retrotransposons (HeT-A, TART, and TAHRE; see Drosophila telomere retrotransposons), rather than from a short segment of the RNA molecule that makes up part of the telomerase holoenzyme (Fuller, 2010).

Although clearly related to other retrotransposons in the Drosophila melanogaster genome, the three retrotransposons that make up telomeres have several characteristics that set them apart from the more typical retrotransposable elements. One of these characteristics is their localization to telomere arrays. The euchromatic regions of the D. melanogaster genome have been completely sequenced. Analysis of these gene-rich regions reveals no sequence from any of the three telomeric elements, although these euchromatic regions are littered with other retrotransposons. Conversely, the long arrays of telomeric retrotransposons do not contain their nontelomeric relatives. Thus, the telomeric and nontelomeric elements have distinctly different genomic distributions, except for small 'transition zones' at the proximal ends of telomere arrays where fragments of both kinds of elements are mingled (Fuller, 2010).

The telomere-specific transposition of HeT-A and TART appears to depend on the intranuclear targeting of the Gag proteins encoded by each element. These Gags share amino acid sequence motifs with retroviral Gags, proteins known to be important in intracellular transport of viral RNA. The sequence similarities with retroviral Gags suggest that telomeric Gags are important in intracellular transport of the retrotransposon RNA, a suggestion supported by studies of the intracellular localization of HeT-A and TART Gag proteins. Transient expression of tagged Gag proteins in D. melanogaster cells showed that Gags of both HeT-A and TART localize to nuclei very efficiently. Gags of nontelomeric retrotransposons were also tested in these experiments and found predominantly, if not entirely, in the cytoplasm. Preventing Gags of nontelomeric retrotransposons from entering the nucleus may be one of the mechanisms cells use to protect their genomes from parasitic invaders. In contrast, the telomeric retrotransposons have an essential role in the nucleus and the cell benefits from facilitating nuclear localization of these Gags (Fuller, 2010).

After moving from the cytoplasm into the nucleus, HeT-A Gags form aggregates (Het dots) associated with telomeres in interphase nuclei. HeT-A and TART are intermingled in D. melanogaster telomere arrays so it was surprising that TART Gags formed loose intranuclear clusters with no obvious telomere associations. However, cotransfection experiments showed that when the two Gags are expressed in the same cells, HeT-A Gag dominates the localization and moves TART Gag into telomere-associated Het dots (Rashkova, 2002). Presumably this localization is necessary for transposition to telomeres (Fuller, 2010).

The collaborative localization of the two Gags suggests an explanation for two puzzling observations. The first observation is that all D. melanogaster stocks and cell lines have both HeT-A and TART in their telomeres, suggesting that both elements are needed by the cell. However, the two elements seem to be distributed randomly in telomere arrays, giving no indication that either one has a special role. The second observation is that HeT-A elements do not encode reverse transcriptase, while TART does. Most, if not all, other retrotransposons encode this enzyme. Having the enzyme sequence encoded by the element's RNA would be expected to allow more efficient transposition, as has been shown for human Lines-1 elements. Nevertheless HeT-A transposes efficiently and is significantly more abundant than TART in telomeres of all D. melanogaster stocks and cell lines studied. The finding that HeT-A Gag positions TART for transposition to telomeres suggested that TART might provide the reverse transcriptase for both elements, thereby explaining the need for both elements in the genome. It is suggested that HeT-A is more abundant than TART because HeT-A has stronger telomere targeting (Fuller, 2010).

After these localization studies were finished, a third D. melanogaster telomeric retrotransposon, TAHRE, was reported (Abad, 2004b). TAHRE has both a HeT-A-related Gag protein and a reverse transcriptase closely related to that of TART and thus presumably with the same activity. TAHRE's sequence predicted that it should combine the localization activity of HeT-A with the enzyme activity of TART and transpose more efficiently than either of the other elements, yet TAHRE is actually much less abundant than either HeT-A or TART. Only one full-length copy of this element has been reported and only one full-length copy of its Gag gene is found in the D. melanogaster database. This study examined the intracellular localization of TAHRE Gag to see whether the sequence similarity to HeT-A Gag yields a protein with the remarkable telomere targeting of HeT-A Gag and to shed light on TAHRE's relative rarity in telomeric arrays (Fuller, 2010).

Although the three retrotransposons appear to have similar roles in forming telomere arrays, each Gag protein has a different pattern of localization when expressed by itself. HeT-A Gag localizes to Het dots associated with telomeres in interphase nuclei. TART Gag moves into nuclei but does not show preferential association with telomeres. TAHRE Gag remains predominantly in the cytoplasm with a tendency to concentrate around the nucleus and to colocalize with nuclear lamin. Neither TAHRE nor TART Gags localize to telomeres independently. Both require interaction with HeT-A Gag to reach this localization (Fuller, 2010).

Studies of deletion derivatives of Gag proteins show that association between HeT-A and TART Gags depends on a highly conserved region of each protein that contains the MHR and the zinc knuckle (CCHC box) motifs. This same region directs associations of these two telomeric Gags with TAHRE (Fuller, 2010).

The MHR and zinc knuckle amino acid motifs are hallmarks of retroviral Gag proteins. The MHR (QGX2EX7R) is so named because it is the only region of significant homology among different groups of retroviruses. The zinc knuckle motif has the general formula CX2CX4HX7C, although the spacing of the conserved C and H residues may differ in different elements. Retroviral Gags usually have one or two zinc knuckles; the D. melanogaster retrotransposons described in this study each have three. In both retroviral and retrotransposon Gags, the MHR is slightly N terminal of the zinc knuckle region. These two regions and the sequence between them are strongly conserved, in contrast to the marked sequence variability seen in much of the amino acid sequence of Gag proteins. The MHR-zinc knuckle region appears to have several roles in the retroviral life cycle, including involvement in multimerization of Gags . In these three insect retrotransposons this region also contains a domain, pre_C2HC, of unknown function. This domain occupies most of the sequence between the MHR and the zinc knuckles (Fuller, 2010). HeT-A Gags in the same D. melanogaster genome can differ significantly in amino acid sequence, yet the 151 amino acids of their MHR-zinc knuckle regions align with no gaps in spacing and only 15 residues where one or more of the amino acids differ from the consensus. The only available TAHRE Gag sequence is very similar, having only 20 residues that are not identical to all of the HeT-A Gags in the alignment. Interestingly, 15 of these TAHRE residues are at sites where HeT-A Gags are not all identical and for most sites TAHRE has the amino acid found in the majority of the HeT-A Gags. Therefore most of the differences in the TAHRE sequence are ones that are tolerated in HeT-A Gag as well (Fuller, 2010).

Sequence variation in TART elements is concentrated in the untranslated regions, which define three subfamilies, TART A, TART B, and TART C. The MHR-zinc knuckle regions in Gags of the TART subfamilies also have 151 amino acids, all identical except for two residues in TART C. The TART sequence in this region aligns with the sequences from HeT-A and TAHRE with no gaps and no misalignment of CCHC residues; however, there are more amino acid differences between TART and HeT-A than between TAHRE and HeT-A. TAHRE and the canonical HeT-A have 95% identity in this region but only 50% and 52% identity, respectively with TART. Because HeT-A Gag interacts efficiently with TART Gag, it appears that these amino acid differences are tolerated (Fuller, 2010).

The D. melanogaster genome has many non-LTR retrotransposons that do not transpose into telomeric arrays. Gags of these nontelomeric elements also have a MHR-zinc knuckle region with three zinc knuckles. However, the MHR-zinc knuckle regions of Gag in the nontelomeric elements differ more from the regions in HeT-A, TART, and TAHRE than the regions in the telomeric Gags differ from each other. These differences are easily seen in the spacing of the CCHC residues and in their spacing relative to the MHR. All of the sequences from telomeric Gags have identical spacing while the other three sequences differ in spacing from the telomeric Gags and from each other. Jockey and Doc have only 27%-31% amino acid identity with each other or any of the telomeric Gags, while I factor has ~17% amino acid identity with any of the other sequences. HeT-A Gag does not form functional associations with Gag proteins from Doc, jockey, or I Factor. This specificity is similar to that of the MHR-zinc knuckle region of retroviruses that forms heteromultimers only between genetically related retroviruses. The sequence differences between the nontelomeric Gags and telomeric Gags support the hypothesis that the MHR-zinc knuckle region is involved in the association between telomeric elements (Fuller, 2010).

These sequence comparisons suggest that the MHR-zinc knuckle provides an amino acid code for formation of heteromultimers. They also raise questions about how degenerate the code is. Does the higher similarity of the HeT-A and TAHRE Gags indicate a stronger affinity than either one has for TART Gag or is the code degenerate enough to accommodate the differences seen in this region? The strong interactions between any two of these proteins seen in these experiments indicate that a rigorous answer to this question will require careful quantitative studies with purified proteins. However, as discussed below, the in vivo studies presented in this study suggest that TART Gag's interaction with HeT-A Gag may be favored by its presence in the nucleus in contrast to the more distant position of TAHRE Gag in the cytoplasm (Fuller, 2010).

These studies provide new evidence that Gag protein localization is important in the transposition of the three telomere-specific retrotransposons of D. melanogaster. Of the three telomere-specific retrotransposons only HeT-A encodes a Gag protein that specifically localizes to telomeres. Nevertheless both TAHRE and TART Gags can be directed to telomeres by association with HeT-A Gag. Interactions between any of the three Gag proteins depend on the segment containing the MHR, pre_ C2HC, and zinc knuckle motifs. The amino acid sequence in this region has a highly conserved pattern that is specific for the telomere retrotransposons. The conservation of this segment in these unusually variable proteins suggests the importance of Gag interactions between these retrotransposons (Fuller, 2010).

Gags of the three telomere elements differ in their ability to localize to telomere Het dots. HeT-A Gag can localize to telomeres independently. TART Gag localizes to the nucleus independently but must have the help of HeT-A Gag to associate with telomeres (Rashkova, 2002). Moving into the nucleus puts TART Gag into an optimal position to encounter HeT-A Gag for localization to Het dots. TAHRE Gag requires assistance to move from the cytoplasm so is less efficient than TART Gag in encountering HeT-A Gag for localization to Het dots. Thus TAHRE is less likely to have carried in its RNA for reverse transcription onto telomeres. This could explain the rarity of TAHRE in telomeres, one complete and three truncated copies in the D. melanogaster genome sequenced by the genome project (Abad, 2004b). Similarly, the observation that HeT-A is consistently more abundant than TART in different stocks of D. melanogaster may reflect the fact that TART Gag needs HeT-A Gag for telomere localization. This correlation between the abundance of each element and the efficiency of its Gag in localizing to Het dots provides additional support for the hypothesis that Gag localization is important for targeting telomere-specific transposition (Fuller, 2010).

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, the degree of conservation along HeT-A copies was studied. A small sequence within the 3′ UTR of the element was identified 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 has been 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 (Petit, 2012).

Telomeric retrotransposon HeT-A contains a bidirectional promoter that initiates divergent transcription of piRNA precursors in Drosophila germline

PIWI-interacting RNAs (piRNAs) provide the silencing of transposable elements in the germline. Drosophila telomeres are maintained by transpositions of specialized telomeric retroelements. piRNAs generated from sense and antisense transcripts of telomeric elements provide telomere length control in the germline. This study showed that common regulatory elements are shared by sense and antisense promoters of HeT-A. Therefore, the HeT-A promoter is a bidirectional promoter capable of processive sense and antisense transcription. Ovarian small RNA data show that a solo HeT-A promoter within an euchromatic transgene initiates the divergent transcription of transgenic reporter genes and subsequent processing of these transcripts into piRNAs. These events lead to the formation of a divergent unistrand piRNA cluster at solo HeT-A promoters, in contrast to endogenous telomeres that represent strong dual-strand piRNA clusters. Solo HeT-A promoters are not immunoprecipitated with heterochromatin protein 1 (HP1) homolog Rhino, a marker of the dual-strand piRNA clusters, but are associated with HP1 itself, which provides piRNA-mediated transcriptional repression of the reporter genes. Unlike endogenous dual-strand piRNA clusters, the solo HeT-A promoter does not produce overlapping transcripts. In a telomeric context, however, bidirectional promoters of tandem HeT-A repeats provide a read-through transcription of both genomic strands, followed by Rhi binding. These data indicate that Drosophila telomeres share properties of unistrand and dual-strand piRNA clusters (Radion, 2016).

Specific localization of the Drosophila telomere transposon proteins and RNAs, give insight in their behavior, control and telomere biology in this organism

Drosophila telomeres constitute a remarkable exception to the telomerase mechanism. Although maintaining the same cytological and functional properties as telomerase maintain telomeres, Drosophila telomeres embed the telomere retrotransposons whose specific and highly regulated terminal transposition maintains the appropriate telomere length in this organism. This study reports a detailed study of the localization of the main components that constitute the telomeres in Drosophila, HeT-A and TART RNAs and proteins. The results in wild type and mutant strains reveal localizations of HeT-A Gag and TART Pol that give insight in the behavior of the telomere retrotransposons and their control. TART Pol and HeT-A Gag only co-localize at the telomeres during the interphase of cells undergoing mitotic cycles. In addition, unexpected protein and RNA localizations with a well-defined pattern in cells such as the ovarian border cells and nurse cells, suggest possible strategies for the telomere transposons to reach the oocyte, and/or additional functions that might be important for the correct development of the organism. Finally, it has been possible to visualize the telomere RNAs at different ovarian stages of development in wild type and mutant lines, demonstrating their presence in spite of being tightly regulated by the piRNA mechanism (Lopez-Panades, 2015).

Multiple pathways suppress telomere addition to DNA breaks in the Drosophila germline

Telomeres protect chromosome ends from being repaired as double-strand breaks (DSBs). Just as DSB repair is suppressed at telomeres, de novo telomere addition is suppressed at the site of DSBs. To identify factors responsible for this suppression, an assay an assay was developed to monitor de novo telomere formation in Drosophila, an organism in which telomeres can be established on chromosome ends with essentially any sequence. Germline expression of the I-SceI endonuclease resulted in precise telomere formation at its cut site with high efficiency. Using this assay, the frequency of telomere formation was quantified in different genetic backgrounds with known or possible defects in DNA damage repair. It was shown that disruption of DSB repair factors (Rad51 or DNA ligase IV) or DSB sensing factors (ATRIP or MDC1) resulted in more efficient telomere formation. Interestingly, partial disruption of factors that normally regulate telomere protection (ATM or NBS) also led to higher frequencies of telomere formation, suggesting that these proteins have opposing roles in telomere maintenance vs. establishment. In the ku70 mutant background, telomere establishment was preceded by excessive degradation of DSB ends, which were stabilized upon telomere formation. Most strikingly, the removal of ATRIP caused a dramatic increase in telomeric retrotransposon attachment to broken ends. This study identifies several pathways that suppress telomere addition at DSBs, paving the way for future mechanistic studies (Beaucher, 2012).

In this study de novo telomere formation was induced upon an endonuclease induced DSB. Remarkably, as high as 63% of the progeny on average had acquired a new telomere at the DSB site under continuous I-SceI production through development starting from the earliest stages of embryonic divisions. This high rate of terminal deficiency (TD) recovery in the germline is in startling contrast to Muller's inability to recover terminally deleted chromosomes in Drosophila that had led to the very concept of “telomere” (Muller 1940, Muller and Herkowitz 1954) (Beaucher, 2012).

Four lines of explanation are offered for reconciliation. First, Muller used Xray irradiation as the DSB inducing agent whereas this study used a site-specific endonuclease. Breaks generated by irradiation might need to be processed differently or more extensively than ends from a nuclease digestion to become suitable substrates for telomere formation. Secondly, the DSB at telomeric marker D4A is relatively close to an existing telomere. It is possible that there is a higher concentration of capping proteins surrounding the telomeres in the nucleus making it more likely for a DSB end to be capped as a telomere. Thirdly, I-SceI induces one DSB per diploid genome in this system whereas the number of breaks induced by X-ray was difficult to control and some cells might have more than one. Cells respond differently to DSB dosage. Yeast cells in the G1 phase respond differently to one versus four DSBs induced by a nuclease. This different response might lead to inefficient telomere formation when a cell encounters more than one break. Although the above three factors might contribute to the decreased likelihood of recovering TDs in the Drosophila germline, they remain a priori assumptions to explain Muller's results since TDs can be nevertheless recovered in the female germline by X-ray irradiation (Beaucher, 2012).

A fourth proposition is offered as a key difference between the current experiments and those of Muller's: Muller induced DSBs to male germ cells in advanced stages of spermatogenesis yet the DSB induced in the current assays are limited to the mitotic compartment of the male germline. It was recently discovered that paternal telomeres that have lost the protection of the K81 capping protein engage in highly efficient telomere fusion before the first zygotic division (Gao, 2011). This result implies that first, de novo telomere establishment is highly inefficient on decondensed sperm DNA even in the presence of abundant maternal deposition of capping components; second, DNA repair, particularly end joining, is highly active during the early embryonic cycles. In Muller's experiments, the DSB generated in the male germline likely persist until after fertilization making it unlikely to be capped during the first zygotic division. On the contrary, the DSB in the current assay can be generated throughout development as I-SceI is continuously and ubiquitously expressed. In addition, simple rejoining of an I-SceI-induced DSB or inter-sister GC repair would recreate a functional cut site allowing a second round of cutting. Therefore, the DSB at D4A had multiple opportunities to acquire a telomere during all stages of development. Evidence supporting the last proposition already exists. In light of the increase in TD recovery from irradiated mu2 females, it has been postulated that DNA lesions induced in mu2 oocytes persist through oogenesis followed by telomere establishment on DSBs in the early embryo. A similar increase was not observed when sperm instead of oocytes were irradiated, suggesting DNA lesions on sperm chromatin are poor substrates for telomere formation. In addition, neo-telomere formation on ends of broken dicentric chromosomes can be readily recovered when induced in the mitotic male germline (Beaucher, 2012).

Evidence is also available suggesting that de novo telomere formation can occur very early in the somatic lineages. This was derived from scoring flies that inherited both D4A and a maternal I-SceI gene. In this assay, TD formation leads to the loss of the dominant KrIF mutation restoring the eye to its normal size. In addition, TD formation leads to a variegated eye pigmentation pattern. Flies were often recovered that had variegated eyes with sizes that are fully normal. These eyes likely developed from a cell with D4ATD formed at early stages of development (Beaucher, 2012).

Defective checkpoints allow telomere formation on persisting DSBs The first set of mutants that increase TD recovery in the germline have defects in DNA checkpoint functions: mu2 and mus304. From germlines in both backgrounds, increases were recorded of TD formation that were among the highest in all mutants tested. It is possible that cells able to establish telomeres on DSBs had a survival advantage over cells with persisting DSBs so that cells with TD are selected for in the assay. If this were true and if many cells in mu2 or mus304 were unable to establish telomere at D4A and later died, preferential recovery of the uncut homolog and impaired male fertility due to germ cell loss would have been observed. Neither was observed for any of the mutants that were tested suggesting that apoptosis is not a normal response in these cells defective for damage response or repair. The proposition is supported that defective checkpoints lead to persistence of DSBs allowing more time for telomere establishment (Beaucher, 2012).

The hypomorphic tefu and nbs mutations enhanced TD recovery similarly but to a lesser extent than mu2 and mus304 null mutations. It is possible that the underlying mechanism, i.e. persisting DSBs due to defective checkpoints, is common for both groups of mutants. However, null mutations were used and it was shown that the checkpoint functions of ATM and, to some degrees, MRN are less prominent in Drosophila than the ones controlled by ATR and ATRIP. It is speculated that other functions of ATM and MRN might help inhibit de novo telomere formation. In particular, ATM and MRN are essential for end tethering during DSB repair. It is imaginef that the two ends of the DSB induced at D4A are allowed to separate in great distance in tefu or nbs cells, which would impede repair giving more time for telomere formation. In addition, the MRN complex is important for both HR and NHEJ repair of DSBs. The nbs mutation might affect telomere formation via its function in DSB repair as the results suggest that inhibiting DSB repair facilitates telomere formation (Beaucher, 2012).

Two modes of repair of the DSB at D4A will recreate the I-SceI cut site: precise end joining and recombination with the sister chromatid, making the chromosome susceptible to a second round of cutting. Therefore, any events leading to the disruption of the cut site would be favored in the presence of continuous I-SceI expression. de novo telomere formation represents one of these events. Consistently, when HR was impaired by the spnA mutation or end joining by Lig4, TD recovery rate increased. Loss of SpnA has a larger effect than loss of Lig4, which is consistent with a previous observation that inter-sister HR is the predominant pathway for the repair of I-SceI induced DSBs in the male germline. Therefore, channeling of DSBs is likely the cause for elevated TD recovery in repair defective germline (Beaucher, 2012).

In the assay, scoring progeny that inherited a TD but have lost white sequences helps illustrate the extent of nucleolytic degradation of chromosome ends before and after de novo telomere formation. It is surmised that a longer half-life of DSB in checkpoint mutants would result in more extensive degradation. Consistently, mutants with suspected defects in checkpoint functions (mu2, mus304, tefu and nbs) all led to increased white-loss in TD progeny. However, in tefu and, to a lesser extent, nbs mutants, this increase is disproportionally larger than the increase in total TD recovery. For example, close to half of TD progeny from tefu suffered a loss of white expression. These results suggest that ATM and NBS normally inhibit end degradation at telomere ends. Contrary to mutants that prolong the presence of DSBs, it is considered that mutants defective in individual DSB repair pathways are unlikely to cause extensive end-degradation before telomere formation. Different repair pathways compete for the available DSBs so that when one is defective DSBs are efficiently repaired by others. This suggests that defects in a single repair pathway is unlikely to prolong the presence of DSBs. Consistently, the increases in TD recovery in spnA and Lig4 germline were not accompanied by significantly elevated levels of white-loss (Beaucher, 2012).

The Inverted repeat-binding protein (Irbp) mutant is interesting in that it behaved differently from any other mutants in the assay, causing a dramatic increase of white-loss TD events but without a significant increase in the overall TD recovery. Several points concerning Ku70's function are deduced from these results. First, loss of Ku70 does not impact precise end joining to a degree similar to the Lig4 mutation. Second, imprecise NHEJ is infrequently used for DSB repair at D4A in the male germline so that its disruption by the loss of Ku70 does not lead to significant channeling of DSBs for telomere formation. Thirdly, the excessive end-degradation in Irbp germ cells is likely specific to telomeric ends, and occurs after the commitment of the DSB to a telomeric fate but before the establishment of a functional telomere. This last point was based on the observation that events with white-loss followed by successful NHEJ) were not recovered at a higher frequency in the mutant background. It is also shown that once a functional telomere has been established at D4A, loss of Ku70 has no effect on the rate of end attrition. It is speculated that once a DSB is committed to a telomeric fate, the binding of Ku70 prevents excessive nucleotytic attrition before the functional establishment of a protective cap. Intriguingly, Ku70 seems to have no role in either fate determination of DSB ends or cap establishment on ends (Beaucher, 2012).

Remarkably, a close to 20-fold increase was observed in new telomere formation accompanied by a transposon attachment event in mus304 germ cells. This frequency is likely to be an underestimate due to the fact that transposon attachment to D4A end that has lost part of white could not be identified in the assay that was used (Beaucher, 2012).

In yeast S. ceravisiae, the Mec1/ATR kinase, and presumably its partner ATRIP, prevents accumulation of the Cdc13 protein at DSBs. Cdc13 is a member of the Cdc13-Stn1-Ten1 (CST) telomeric complex essential for telomere protection and the recruitment of telomerase activities to telomeres. Interestingly, the Drosophila Verrocchio (Ver) protein was recently identified as an essential capping protein and shares limited homology with Stn1 proteins from other organisms. This suggests that a similar CST complex might exist in Drosophila. It is speculated that Drosophila CST might accumulate at DSBs in the absence of Mus304/ATRIP, leading to more efficient recruitment of the transposon machinery, similar to CST's role in telomerase recruitment in the other systems (Beaucher, 2012).

Ku70 heterozygosity has been shown to lead to elevated rates of transposon attachment in the female germline. This increase happens over a few successive generations. No evidence was observed of rampant transposon attachment to D4ATD from Southern blot analyses on TDs that have been kept in the Irbp background for several generations. However, the crossing scheme only allowed TDs to be present in the mutant germline from males (Beaucher, 2012).

It was not surprising that almost all mutations tested in this study lead to increases in the recovery of events associated with de novo telomere formation. It is consistent with the idea that telomere formation might be a backup mechanism to all modes of damage repair and response in germ cells. Although the candidate approach in identifying factors essential for telomere establishment is far from comprehensive, a picture has emerged in which factors responsible for the recruitment and execution of damage repair and response activities are also responsible for inhibiting telomere formation. In the absence of these activities, the DNA and chromatin structures at the ends might be sufficient for the recruitment of the protective cap. If this were true, only defects in the protective cap itself would have a negative effect on telomere establishment on DSBs. This hypothesis was not tested due to the lack of hypomorphic mutations in capping components (Beaucher, 2012).

The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response

Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. It has been suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. This study used electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. It was also shown that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, Ver-depleted telomeres were found to form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, these findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin (Cicconi, 2016).

Dealing with chromosome ends represents a major problem for the cell, as they can be mistaken for double strand breaks (DSBs) and activate the DNA damage response (DDR), leading to unwanted repair, telomere fusion and genome instability. Different organisms evolved different protein complexes that specifically bind chromosome ends and help assembly of the telomere, a protective structure that shields DNA termini preventing DSB signaling. In most eukaryotes, telomeric DNA consists of short tandem repeats added by telomerase to chromosome ends. Replication of the lagging strand results in the formation of a terminal 3' G-rich overhang; completion of telomere replication through a fine interplay between exonuclease activities and fill-in DNA synthesis results in 3' overhangs of appropriate length at the ends of both sister chromatids (Cicconi, 2016).

In organisms with telomerase, terminal repeats are specifically recognized by specialized telomere capping complexes. In humans, the TTAGGG repeats are selectively bound by the six-protein (TRF1, TRF2, Rap1, TIN2, TPP1, POT1) shelterin complex, which localizes and function almost exclusively at telomeres. TRF1 and TRF2 bind the TTAGGG duplex and POT1 the 3' overhang; TIN2 and TPP1 bridge POT1 to TRF1 and TRF2. hRap1, a distant homologue of Saccharomyces cerevisiae Rap1, interacts with TRF2, but is not directly implicated in telomere protection or length regulation. TRF2 dysfunction triggers the ATM signaling pathway, and leads to the accumulation of telomere dysfunction foci (TIFs) enriched in γ-H2AX. Loss of POT1 causes the accumulation of RPA (Replication protein A) onto the 3' overhang, which activates the ATR signaling pathway and leads to TIFs. RPA is normally recruited at telomere overhangs during DNA replication, at a time when POT1 is partially released from the telomere, but is replaced by POT1 at the end of DNA replication. Interestingly, transient ATM- and ATR-mediated DNA damage signaling occurs even at normal human telomeres that are completing DNA replication (Cicconi, 2016).

Although 3' overhangs are prevalent among telomeres of organisms with telomerase, in Caenorhabditis elegans 5' overhangs are as abundant as 3' overhangs, and blunt-ended telomeres have been found in Arabidopsis thaliana. 5' overhangs have been also found in mouse and human cells, particularly in G1/S arrested and terminally differentiated cells, as well as in cancer cells that exploit the alternative lengthening of telomeres (ALT) pathway for telomere maintenance (Cicconi, 2016).

In fission yeast, telomeric DNA is protected by a complex that is architecturally reminiscent of shelterin and contains the TRF1 and POT1 homologues Taz1 and SpPot1. In budding yeast, there is not a shelterin complex and the shelterin functions are fulfilled by Rap1 and the RPA-like complex Cdc13-Stn1-Ten1 (CST). Cdc13 does not share homology with POT1, but both proteins use oligonucleotide/oligosaccharide-binding (OB)-fold domains to bind ssDNA. The CST complex exists also in mammals, where it coordinates telomerase-mediated DNA elongation and fill-in synthesis during telomere replication; however, its function is not restricted to telomeres, as it also plays a general role in DNA replication (Cicconi, 2016).

In Drosophila, there is not telomerase and telomeres are elongated by the targeted transposition of three specialized non-LTR retrotransposons (HeT-A, TART and TAHRE). In addition, abundant evidence indicates that Drosophila telomeres can assemble independently of the sequence of the DNA termini. Drosophila telomeres are capped and protected by the terminin complex, which includes HOAP, Moi and Ver. All these proteins interact with each other and share the same features as the shelterin subunits: they are specifically enriched at telomeres throughout the cell cycle and do not perform other functions elsewhere in the genome. Most likely, terminin also includes HipHop, another fast evolving protein that interacts with HOAP and shares the shelterin-like properties of HOAP, Moi and Ver (Cicconi, 2016).

This study focuses on the Verrocchio (Ver) protein, which contains an OB-fold domain with structural similarity to Stn1/RPA2 OB fold. Ver interacts with Modigliani (Moi), and Moi and Ver are both HOAP-dependent and mutually dependent for their telomeric localization. Ver has been also implicated in the recruitment of the HeT-A encoded ORF1p protein and HeT-A transcripts at the telomere. Both electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) showed that Ver binds ssDNA in vitro. Moi was shown not to bind DNA, andVer interaction with Moi was shown to be necessary for Ver localization at telomeres but not for its binding to ssDNA. Finally, it was demonstrated that loss of Ver favors RPA accumulation at telomeres and triggers DNA damage signaling. This suggests that Ver is a functional analog of ssDNA binding proteins such as yeast Cdc13 and human POT1 (Cicconi, 2016).

Previous work has shown that the integrity of the Ver OB-fold domain is dispensable for Ver recruitment at telomeres but is crucial for telomere protection from fusion events. These results suggested but did not prove that Ver possesses ssDNA binding activity. This study provides strong evidence that Ver binds ssDNA. EMSA experiments showed that Ver-GST binds ssDNA probes of different sequence, and that this binding is reduced by competition with ssDNA but not dsDNA. In addition, AFM experiments unambiguously showed that Ver binds DNA with a strong preference for the terminal regions of DNA molecules that end with either 3' or 5' ssDNA overhangs. Collectively, both the results of these experiments and previous studies on Drosophila telomeres strongly suggest that Ver binds ssDNA in a sequence-independent manner. However, it cannot be excluded that diverse DNA sequences could bind Ver with different affinities (Cicconi, 2016).

It was also shown that Ver binds ssDNA as a dimer or a multimer. The protein domain required for Ver-Ver interaction was mapped and it was shown that in the absence of this domain Ver is unable to bind ssDNA and to protect telomeres from fusion events, providing additional evidence that the Ver capping function relies on intact ssDNA binding activity. The presence of ssDNA at Drosophila telomeres has never been directly demonstrated, as the variability of fly telomeric DNA prevented successful application of the commonly used DNA sequence-based methods to characterize the structure of chromosome ends. The findings that Ver binds ssDNA and is required for telomere capping strongly suggests that fly telomeres do in fact terminate with a ssDNA like those of yeasts, plants, and mammals. Studies on C. elegans have shown that this species possesses both 3' and 5' overhangs that are bound by 2 different proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric overhangs, respectively. These data would suggest that Ver could bind both 5' and 3' overhangs. However, they do not prove that these overhangs coexist in living flies (Cicconi, 2016).

The results indicate that Ver binds ssDNA with low affinity, as even high protein concentrations were not sufficient to significantly reduce the amount of unbound probe. However, in a very recent study, Zhang (2016) showed that a trimeric complex formed by recombinant Tea, Moi and Ver, purified with the baculovirus system, has robust sequence independent ssDNA binding activity, while a Moi-capping complexes to maintain an interaction with telomeres (Cicconi, 2016).

Results on Ver provide two important additional pieces of information on the evolution of Drosophila telomeres. First, the findings indicat-Ver subcomplex and ssDNA is probably due to the protein tags and purification methods they used. On the other hand, they clearly showed that Moi, Tea and Ver have high ssDNA binding activity when they act as a trimeric complex. Tea has not obvious ssDNA binding motifs, and remains to be determined whether Tea has its own ssDNA binding activity or simply enhances Ver binding activity (Cicconi, 2016).

The low ssDNA binding affinity of the Ver protein is likely to reflect specific functional requirements. For example, it is conceivable that Ver low affinity for ssDNA prevents unwanted binding of Ver to other ssDNA regions such as those formed during normal DNA replication. It should be noted that telomeric proteins that bind ssDNA with relatively low affinity independently of the sequence have been previously described in yeasts and mammals. For example, Pot1 of S. pombe possesses an N-terminal OB fold that binds DNA in a sequence-dependent fashion, and a C-terminal OB fold with sequence-independent binding properties, a feature that is likely to reflect the need to protect the degenerate telomere sequences present in this yeast species. Another ssDNA binding protein that exhibits no preference for telomeric substrates is C. albicans Cdc13. As a consequence, while S. cerevisiae Cdc13 is recruited at telomeres through sequence-specific interaction with telomeric DNA, recruitment of C. albicans Cdc13 relies on protein-protein interactions. Remarkably, also a high-affinity ssDNA binding complex such as TPP1-POT1 is recruited at telomeres by TIN2, which bridges these ssDNA binding proteins to the dsDNA binding proteins TRF1 and TRF2. Most likely, also Ver recruitment at telomeres depends on interactions with other terminin components and not with telomeric DNA. This is suggested by the behavior of VerΔC. Although this truncated Ver moiety fails to bind ssDNA and to prevent end-to-end fusions, it is normally recruited at telomeres (Cicconi, 2016).

Previous work has shown that Ver and Moi are both mutually dependent and HOAP dependent for their localization at telomeres HOAP binds dsDNA and coats up to 10 kb of telomeric DNA. These findings suggested that HOAP could mediate Ver and Moi recruitment at telomeres. However, recent work has shown that Moi and Ver association with telomeres is also dependent on Tea, which requires HOAP for its telomeric localization. Because HOAP localizes normally at telomeres in tea mutants, these findings suggest that Tea, in the presence of HOAP, could mediate Ver and Moi recruitment at telomeres (Cicconi, 2016).

Although the pathways leading to end-to-end fusion in Drosophila have not been fully elucidated, this study has provided evidence that the early steps of telomere dysfunction recognition are conserved between mammals and flies. This study has shown that fly telomeres depleted of Ver-Moi accumulate RPA and γ-H2AV just as mammalian telomeres lacking TPP1-POT1. It is likely that in the absence of Ver-Moi the telomeric ssDNA binds RPA, which is known to bind ssDNA with high affinity; RPA is then likely to recruit the DNA repair machinery that leads to the formation of telomere associated γ-H2AV foci (Cicconi, 2016).

Several studies in mammalian cells have shown that following POT1 or TPP1-POT1 depletion RPA is recruited at telomeres, leading to the model that loss of POT1 unmasks the single-stranded G overhang, which binds RPA and ATR, eliciting the DDR response. However, it has been recently shown that POT1 is also required for proper telomere replication, probably acting in in the same pathway as CST. These latter findings raise the possibility that RPA localization to POT1-depleted mammalian telomeres is at least in part due to a defect in telomeric DNA replication. The data do not that exclusion of Ver depletion affects telomeric DNA replication in Drosophila. Thus, RPA and γ-H2AV recruitment at ver mutant telomeres could be the consequence of an exposure of the telomeric overhang, a defect in subtelomeric/telomeric DNA replication, or both (Cicconi, 2016).

An interesting issue is how can the ssDNA overhangs of Drosophila telomeres bind Ver in a sequence independent manner and avoid binding by RPA, which has a very strong affinity for ssDNA of any sequence. In human cells, POT1 is less abundant than RPA and, although it specifically recognizes the telomeric DNA sequence, it binds ssDNA with lower affinity than RPA. Nevertheless, after each round of replication, POT1 efficiently replaces RPA at the telomere. The precise mechanism governing this protein switch has not been fully elucidated. It has been proposed that TPP1-POT1 can outcompete RPA when bound to TIN2. An alternative model for the RPA-to-POT1 switch involves TERRA and the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which has an RPA displacing activity. It has been suggested that the low TERRA levels during the late S phase favor the hnRNPA1 activity promoting the RPA replacement with POT1. How can Ver replace RPA at the end of DNA replication? This process might be related to dynamic transformations of the Moi-Tea-Ver complex that could modulate its affinity for ssDNA. It is also possible that the physical interaction between RPA and Ver lowers the affinity of RPA for DNA, thus allowing Ver to outcompete RPA for ssDNA binding. However, the precise mechanism governing RPA to Ver switch is currently unknown and will be a goal of future studies. (Cicconi, 2016).

In all organisms studied so far, specialized OB-fold proteins bind telomeric single stranded overhangs ensuring protection of chromosome ends. Past and current findings on Ver broaden the list of these OB fold proteins, and strengthen the concept that the general architecture of telomere complexes is conserved across evolution, despite a remarkable plasticity in the individual components of the complexes. TRF1 and TRF2 shelterin components bind the DNA duplex and are connected to the ssDNA binding protein POT1 by the non-DNA-binding TIN2 and TPP1; the shelterin-like fission yeast capping complex has similar features. It has been suggested that these shelterin complexes are functionally equivalent to the CST and Rap1-Rif1-Rif2 complexes of budding yeast (Cicconi, 2016).

The telomere-capping complexes of yeast, mammals and Drosophila share similar molecular architectures. The human shelterin and the fission yeast shelterin-like complexes have similar architectural features. In both complexes, the proteins that bind the DNA duplex (TRF1-TRF2 and Taz1) are connected to the ssDNA-binding protein POT1 by non-DNA-binding proteins (TIN2-TPP1 and Poz1-Tpz1). Similarly, in Drosophila terminin, HOAP-HipHop, which bind the DNA duplex, are bridged to the ssDNA-binding Ver by Moi, which does not bind DNA. Tea directly binds Ver and Moi but it is currently unknown whether it binds DNA. It has been suggested that the POT1-TIN2-TPP1 and Pot1-Poz1-Tpz1 subcomplexes are functionally equivalent to the CST complex of budding yeast, which binds ssDNA through its Cdc13 subunit, while the Rap1-Rif1-Rif2 complex binds the DNA duplex (see text for detailed explanation and references (Cicconi, 2016).

The finding that Ver but not Moi binds ssDNA suggests that terminin and shelterin have similar molecular architectures. Drosophila HOAP and HipHop interact with each other and are mutually dependent for their stability. In addition, ChIP analysis has shown that the two proteins are enriched over the terminal 10 kb of the chromosomes. Thus, even if HipHop binding to DNA has never been directly demonstrated, it is likely that the HOAP-HipHop subcomplex binds the DNA duplex. Moi binds both HOAP and Ver, and thus is likely to bridge dsDNA-binding HOAP-HipHop with ssDNA-binding Ver. AP/MS experiments have shown that Moi and CG30007 (Tea) are the most abundant Ver-interacting proteins, suggesting a functionally relevant interaction between the three proteins. Tea does not contain any known DNA binding domain and its DNA binding properties have not so far been investigated. Should Tea fail to bind DNA, then the structural similarity between shelterin and terminin would be even greater. In both complexes, there would be a pair of proteins (TRF1-TRF2 and HOAP-HipHop) that bind the DNA duplex, a single ssDNA binding factor (POT1 and Ver) and two non-DNA-binding proteins (TIN2-TPP1 and Moi-Tea) connecting the dsDNA- and ssDNA-binding subcomplexes. Thus, although the shelterin and terminin components do not share any sequence homology, they form multi-protein complexes with similar molecular architectures (Cicconi, 2016).

It has been proposed that concomitant with telomerase loss Drosophila rapidly evolved terminin, a telomere-specific protein complex that binds and protects chromosome ends independently of their DNA sequence. It was also proposed that Drosophila non-terminin telomere-capping proteins correspond to ancestral telomere-associated proteins that could not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. This hypothesis is supported by the fact that the many non-terminin proteins required for telomere capping (HP1a, ATM, Rad50, Mre11 and Nbs) have homologues playing roles at human and yeast telomeres. Additional support for this hypothesis has been provided by recent findings on separase and pendolino/AKTIP. The conserved protease separase has been shown to be required for telomere protection in both Drosophila and humans. Pendolino (peo) prevents telomeric fusions in flies while its human homologue AKTIP is required for telomere replication. Strikingly, Peo and AKTIP directly bind unrelated terminin and shelterin components, indicating that they co-evolved with divergent capping complexes to maintain an interaction with telomeres (Cicconi, 2016).

Results on Ver provide two important additional pieces of information on the evolution of Drosophila telomeres. First, the findings indicate that the terminin proteins (HOAP, HipHop, Moi, Ver and possibly Tea), although fast-evolving and non conserved outside the Drosophilidae family, are likely to form a telomere-capping complex that is architecturally similar to the shelterin complex. Second, this study has shown that Drosophila telomeres are likely to terminate in ssDNA overhangs that recruit RPA just like the yeast and human telomeres. Moreover, like in human telomeres, the levels of telomere-associated RPA and γH2AV (γH2AX) substantially increase when telomeres are depleted of proteins that bind the terminal ssDNA. Collectively, these results reinforce the idea that apart the capping complexes and the mechanisms of telomere length maintenance, Drosophila telomeres are not as different from human telomeres as generally thought. It is thus believed that Drosophila is an excellent model system for studies on telomere organization and function, which can also be exploited for the identification of novel human proteins involved in telomere maintenance (Cicconi, 2016).

The chromosomal proteins JIL-1 and Z4/Putzig regulate the telomeric chromatin in Drosophila melanogaster

Drosophila telomere maintenance depends on the transposition of the specialized retrotransposons HeT-A, TART, and TAHRE. Controlling the activation and silencing of these elements is crucial for a precise telomere function without compromising genomic integrity. This study describes two chromosomal proteins, JIL-1 and Z4 (also known as Putzig), which are necessary for establishing a fine-tuned regulation of the transcription of the major component of Drosophila telomeres, the HeT-A retrotransposon, thus guaranteeing genome stability. Mutant alleles of JIL-1 were found to have decreased HeT-A transcription, putting forward this kinase as the first positive regulator of telomere transcription in Drosophila described to date. The decrease in HeT-A transcription in JIL-1 alleles correlates with an increase in silencing chromatin marks such as H3K9me3 and HP1a at the HeT-A promoter. Moreover, Z4 mutant alleles show moderate telomere instability, suggesting an important role of the JIL-1-Z4 complex in establishing and maintaining an appropriate chromatin environment at Drosophila telomeres. Interestingly, a biochemical interaction was detected between Z4 and the HeT-A Gag protein, which could explain how the Z4-JIL-1 complex is targeted to the telomeres. Accordingly, it is demonstrated that a phenotype of telomere instability similar to that observed for Z4 mutant alleles is found when the gene that encodes the HeT-A Gag protein is knocked down. A model is proposed to explain the observed transcriptional and stability changes in relation to other heterochromatin components characteristic of Drosophila telomeres, such as HP1a (Silva-Sousa, 2012).

Although in Drosophila the role of JIL-1 in activating transcription has remained controversial, at least in the HeT-A, TART, and TAHRE (HTT) array it could act as a positive regulator of transcription for three different reasons: 1) When telomere elongation is needed, a fast activation of HeT-A transcription should be expected. Accordingly, the mammalian JIL-1 orthologous MSK1/2 have been shown to rapidly induce gene expression on the face of stress or steroid response. 2) HeT-A is embedded into the HTT array, a domain that needs to be protected from the influence of the repressive heterochromatin of the neighboring TAS domain. JIL-1 has been suggested to protect the open chromatin state from the spreading of neighboring repressive chromatin at certain genomic positions. 3) The decrease in expression that was observed in the JIL-1 mutants is moderate. Recent data at genomic level revealed that JIL-1 function agrees with a reinforcement of the transcriptional capability of a particular genomic domain rather than net activation (Silva-Sousa, 2012).

Phalke (2009) suggest that JIL-1 has a role in retrotransposon silencing in general and has no effect on telomere transcription. A possible explanation for this discordance with the current results and hypothesis is that the mutant allele of JIL-1 assayed by Phalke, the JIL-1Su(var)3-1 allele, corresponds to a C-terminal deletion of the JIL-1 protein that causes the protein to miss-localize and phosphorylate ectopic sites. The ectopic phosphorylation caused by the JIL-1Su(var)3-1 allele would activate the expression above wild type levels in those genes that normally are not targeted by JIL-1, as it happens to be the case for the Invader4 retrotransposon. The current study has assayed the JIL-1Su(var)3-1 allele obtaining similar result than for the wild type stock, likely for similar reasons. Supporting this, in addition of the JIL-1Su(var)3-1, data from two more JIL-1 alleles, JIL-1z60 and JIL-1z2, is presented that correspond to loss of function alleles and, in both cases, result in a substantial decrease in HeT-A transcription. Moreover, the changes in telomere transcription reported in this study have been assayed directly on the major component of the HTT array, and not through a reporter. The current data demonstrates that JIL-1 is necessary to maintain active transcription of the telomeric retrotransposon HeT-A or, what is the same, transcription from the telomeres in Drosophila (Silva-Sousa, 2012).

Although it was demonstrated that JIL-1 is necessary to maintain transcription from the HTT array, no decrease was detected in telomere length in the JIL-1 mutant alleles. A reasonable explanation for this observation is that the JIL-1 mutant alleles here analyzed (JIL-1z60 and JIL-1z2) have been maintained as heterozygous. It is therefore possible that one copy of JIL-1 is enough to promote enough HeT-A transcription to elongate significantly the telomeres when needed (Silva-Sousa, 2012).

Although in the case of the hypomorph mutation Z47.1 an increase was observed in HeT-A transcription and HeT-A copy number significantly above the control strain (w1118), the null alleles Z42.1 and pzg66 do not show an up-regulation of HeT-A transcription or an increase in its copy number. Although all the stocks were crossed to the w1118 strain to minimize the effects of the genetic background, it could still have a certain influence when comparing the pzg66 allele with the Z47.1. Nevertheless the Z47.1 and Z42.1 alleles come from the same genetic background. A possible explanation could rely on the fact that the Z47.1 mutation is a hypomorph mutation where a small amount of Z4 protein is still present. By ChIP analyses an increase of JIL-1 protein was detected at the HeT-A promoter above control levels, which could explain in part the major transcription of HeT-A in this mutant background, it is possible that although low, the amount of Z4 present in the Z47.1 allele is enough to recruit JIL-1 to the HeT-A promoter. In the pzg66 and the Z42.1 null alleles, JIL-1 cannot be recruited towards the HeT-A promoter and there is no increase in transcription. Nevertheless, with the current data it cannot be concluded that Z4 directly controls the level of HeT-A transcription (Silva-Sousa, 2012).

A phenotype of telomere instability was detected in all three Z4 mutant alleles Z47.1, Z42.1 and pzg66, suggesting a role of this chromosomal protein in guaranteeing telomere stability in Drosophila. Although a number of genes involved in the capping function in Drosophila still remain unidentified, there is no evidence that Z4 directly participates in the protection of the telomeres. Mutant alleles of genes directly involved in the capping function, such as woc or caravaggio (HOAP), show multiple and more numerous TFs in larval neuroblasts than the ones that were observed in the Z4 mutant alleles. Moreover, it has been possible to detect staining for one of the major capping components, the HOAP protein, in the TFs of Z4 mutant neuroblast cells, indicating that the telomere-capping complex is still loaded to a certain degree. Instead of directly participating in the capping, it is hypothesized that the major chromatin changes caused by the lack of Z4 at the HTT array result in a secondary loss of necessary chromatin and capping components like HP1a (Silva-Sousa, 2012).

Results from the ChIP experiments suggest a relationship between JIL-1, Z4 and HP1a in fine-tuning the chromatin structure at the HTT array. HP1a has a dual role at the telomeres explained by its participation in both the capping function and the repression of gene expression that also exerts in other genomic domains. In the HP1a Su(var)2-505 allele, which it is known to have a major transcription of HeT-A and problems of telomere stability, a pronounced decrease was observed in Z4 and JIL-1. In the Z47.1 allele the decrease in Z4 protein is accompanied by a similar decrease in H3K9me3 and HP1a at the HeT-A promoter. Finally in the JIL-1z60 allele the increase in silencing epigenetic marks like H3K9me3 and HP1a is also accompanied by a decrease in Z4. In particular, the pronounced dependence of the presence of HP1a and Z4, points toward the loss of HP1a and H3K9me3 to a possible cause for telomere instability in the Z4 mutant alleles here studied. Interestingly, in the Su(var)2-504/Su(var)2-505 heteroallelic combination (considered a null mutation), 15% of telomeres involved in telomere associations are still able to recruit the HOAP protein. Therefore the data on HOAP localization in the Z4 mutant alleles is still consistent with the TFs being caused by the decreased availability of HP1a in these cells. The above results demonstrate that Z4 in a coordinated manner together with JIL-1 and HP1a is an important component of the telomere chromatin in Drosophila, which upon its reduction causes significant changes in the chromatin of the HTT array, which are the cause of the observed telomere instability in all the Z4 mutant alleles here studied (Silva-Sousa, 2012).

It has been possible to detect a biochemical interaction between JIL-1 and Z4, and the data suggests that these two proteins can be components of the same protein complex. This interaction had been previously suggested because both proteins have been found co-localizing in different genomic locations, but no direct proof existed to date. In each genomic location where the Z4-JIL-1 complex is needed, a special mechanism of recruitment should exist. Importantly, it has been shown how Z4 specifically interacts with HeT-A Gag. HeT-A Gag is the only protein encoded by the HeT-A element and has been shown to specifically localize at the telomeres. HeT-A Gag has been shown to be in charge of the targeting of the transposition intermediates for the HeT-A element and also for its telomeric partner the TART retrotransposon. Interestingly, when the consequences for telomere stability were studied after knocking down the HeT-A gag gene by RNAi, similar TFs were observed than when knocking down the Z4 gene, further relating the action of both genes in telomere stability. Z4 is known to participate in different protein complexes with roles in different genomic locations. Because it has been demonstrated that Z4 is able to associate with a variety of proteins in these complexes, it is thought that the description of a mechanism for its specific targeting to telomeres through one of the telomeric retrotransposon proteins is especially relevant (Silva-Sousa, 2012).

Integrating information from previous literature and the results exposed by this study, a possible model to describe the state of the chromatin at the HTT array in each of these three mutant scenarios; JIL-1, Z4 and Su(var)2-5, as well as in wild type (see Model of the chromatin environment at the HeT-A promoter). The following phenotypes are proposed: (A) Wild type: Z4 defines a boundary at HeT-A promoter that protects from the action of HP1a and other heterochromatin markers. JIL-1 guarantees a certain level of euchromatin inside the HeT-A promoter in order to allow gene expression, (B) JIL-1 mutants: destabilization of the Z4 boundary and the heterochromatin spreads into the HeT-A promoter (enrichment in HP1 and H3K9me3), (C) Z4 mutants: Disappearance of the Z4 boundary, increase in euchromatin marks (H3K4me3) and decrease in heterochromatin marks (HP1a and H3K9me3). Subtle increase in JIL-1 and in euchromatinization of the HeT-A promoter, (D) In Su(var)2-5 mutants: The lack of HP1a allows relaxation of the Z4 boundary causing a JIL-1 and Z4 spread along the HTT array and a relative decrease of these proteins inside the HeT-A promoter. Although the levels of JIL-1 inside the HeT-A promoter are lower than in wild type, the release of silencing caused by loss of HP1a results in increased HeT-A expression (Silva-Sousa, 2012).

It should be taken into account that 1) HP1a has been shown to spread along the HeT-A sequence. 2) The structure and the phenotypes of the different Z4 mutant alleles suggest a possible role of this protein in setting and maintaining the boundaries between heterochromatin and euchromatin in polytene chromosomes. 3) JIL-1 has been extensively shown to be important to counteract heterochromatinization and, when missing, causes a spreading of heterochromatin markers such as H3K9me2, HP1a and Su(var)3-7. 4) JIL-1 has been found to co-localize with Z4 at the band-inter-band transition in polytene chromosomes and also to co-purify with Z4 in different protein complexes. In addition to this, it has been possible to detect a biochemical interaction between JIL-1 and Z4, as well as, a certain dependence on the presence of JIL-1 for the proper localization of Z4, suggesting a possible role of JIL-1 upstream of Z4. Finally, 5) The ChIP analyses in this study suggest a certain dependence of Z4 on HP1a or onto similar chromatin requirements for the loading of both proteins at the HTT array, more specifically at the HeT-A promoter. Summarizing all of the above, it is proposed that the chromatin at the HeT-A promoter could have the following structure: In a wild type situation, the HeT-A promoter contains intermediate levels of HP1a, JIL-1 and Z4. HP1a would be spread along the HTT array, JIL-1 would be concentrated at the promoter region of HeT-A guaranteeing certain level of expression and Z4 would be important to set the boundary between these two opposite modulators (Silva-Sousa, 2012).

In a JIL-1 mutant, the lack of JIL-1 would disturb the Z4 boundary causing a slight decrease in the Z4 presence. This result is in agreement with a Z4-JIL-1 partial interaction. The decrease in JIL-1 presence and the disturbance of the boundary causes a spreading of HP1a into the HeT-A promoter, increasing its presence and repressing transcription from the HTT array (Silva-Sousa, 2012).

In a Z4 mutant, the disappearance of the boundary together with the significant decrease in H3K9me3 causes a decrease in HP1a binding and a substantial modification of the chromatin at the HTT array. The lack of sufficient HP1a at the HTT array causes a destabilization of the chromatin at the cap domain triggering telomere instability as a result. This scenario applies to the three Z4 mutant alleles present in this study, the hypomorph Z47.1, and the nulls pzg66 and Z42.1. On one hand the loss of some Z4 in Z47.1/Z47.1 genotype produces overexpression of HeT-A because in addition to a relaxation of the chromatin, part of JIL-1 is still recruited to the HeT-A promoter and activates transcription in a more effective way than in a wild type situation (Silva-Sousa, 2012).

Finally, in a Su(var)2-5 mutant background, the lack of HP1a along the HeT-A sequence allows a relaxation of the boundary causing a spread of JIL-1 and Z4 from the HeT-A promoter towards the rest of the array and creating as a consequence, permissive chromatin environment releasing HeT-A silencing (Silva-Sousa, 2012).

The model does not completely explain the complex relationships that regulate telomere chromatin, likely because other important components are yet to be described or associated with the ones presented in this study. For example, other chromatin regulatory components that have been associated with Drosophila telomeres included the deacetylase Rpd3, with a regulatory role on chromatin structure, and the histone methyltransferase SetDB1 and the DNA methylase Dnmt2 which by acting in the same epigenetic pathway repress transcription of HeT-A as well as of retroelements in general. Future in depth studies on additional chromatin components will allow completion and detailing even more the description of the chromatin at the HTT array, and allow a better understanding of the mechanism of retrotransposon telomere maintenance and the epigenetic regulation of eukaryote telomeres in general. In the meantime, this study describes a plausible scenario in the view of the transcription and ChIP data (Silva-Sousa, 2012).

The results shown in this study demonstrate the role of JIL-1 as the first described positive regulator of telomere (i.e. HeT-A) expression in Drosophila. Because HeT-A is in charge of telomere maintenance in Drosophila, these results are key to understand how telomere elongation is achieved in retrotransposon telomeres. It was also demonstrated that Z4 is necessary to guarantee telomere stability. The data presented in this study strongly suggest that JIL-1 and Z4 exert these functions by maintaining an appropriate telomere chromatin structure by a coordinated action together with other known telomere components such as HP1a. Moreover, this study shows that JIL-1 and Z4 interact biochemically. Last, and importantly for understanding how the specific role of the Z4-JIL-1 complex at the telomeres is defined and differentiated from its role in other genomic regions, it was shown that Z4 might interact with the HeT-A Gag protein, providing evidence for a targeting mechanism that specifically retrieves this complex to the telomeres (Silva-Sousa, 2012).

The JIL-1 kinase affects telomere expression in the different telomere domains of Drosophila

In Drosophila, the non-LTR retrotransposons HeT-A, TART and TAHRE build a head-to-tail array of repetitions that constitute the telomere domain by targeted transposition at the end of the chromosome whenever needed. As a consequence, Drosophila telomeres have the peculiarity to harbor the genes in charge of telomere elongation. Understanding telomere expression is important in Drosophila since telomere homeostasis depends in part on the expression of this genomic compartment. Recent studies have shown that the essential kinase JIL-1 is the first positive regulator of the telomere retrotransposons. JIL-1 mediates chromatin changes at the promoter of the HeT-A retrotransposon that are necessary to obtain wild type levels of expression of these telomere transposons. The present study shows how JIL-1 is also needed for the expression of a reporter gene embedded in the telomere domain. This analysis, using different reporter lines from the telomere and subtelomere domains of different chromosomes, indicates that JIL-1 likely acts protecting the telomere domain from the spreading of repressive chromatin from the adjacent subtelomere domain. Moreover, the analysis of the 4R telomere suggests a slightly different chromatin structure at this telomere. In summary, these results strongly suggest that the action of JIL-1 depends on which telomere domain, which chromosome and which promoter is embedded in the telomere chromatin (Silva-Sousa, 2013).

Both the experiments of telomere position effect on the mini-white insertions and the real-time PCR quantifications of the expression of the different telomeric elements, indicate that the lower amount of JIL-1 present in the mutant alleles JIL-1z60 and JIL-1h9 assayed in this study results in a decreased expression of the HTT array. These experiments, in accordance with recent published data, confirm that JIL-1 is necessary to obtain wild type levels of gene expression from the HTT array (Silva-Sousa, 2013).

Additionally, these experiments have also revealed that the effect of JIL-1 is stronger over the HeT-A promoter than over the mini-white promoter existent in the reporter lines used in this study. Similarly, when the effect was assayed of mutations of the Su(var)2-5 gene, known to greatly de-repress the expression of the HeT-A retrotransposon, only a faint de-repression was obtained of the mini-white gene of the lines EY08176, EY09966 and EY00453. In agreement with these observations, another study found similar results using two additional Su(var)2-5 mutant alleles, Su(var)2-502 and Su(var)2-504, over the same reporter lines (Silva-Sousa, 2013).

In summary, JIL-1 and HP1a control gene expression from the telomere domain in Drosophila, being the HeT-A promoter especially sensitive to their effect (Silva-Sousa, 2013).

Although JIL-1 has not been found to localize at the subtelomere domain, TAS, the observation that gene expression varies in this domain in a JIL-1 mutant background, suggests a role of this protein related with a putative boundary between the telomere and subtelomere domains. A pronounced increase was observed in mini-white expression at the TAS domains from the 2L and 2R telomeres when placed in a JIL-1 trans-heterozygous (JIL-1z60/JIL-1h9) mutant background. Accordingly, the ChIP data reveals a significant decrease of H3K27me3 upstream of the mini-white insertion when a JIL-1 trans-heterozygous background is present. Integrating these results, a model is proposed in which the JIL-1 kinase acts as a boundary protecting the promoters of the HTT retrotransposons from the highly compacted chromatin of the adjacent TAS domain. This model is in agreement with an increase of repressive chromatin at the HTT array in JIL-1 mutations. Moreover, the model also reflects the previously reported role of JIL-1 in the protection of the excessive spreading of heterochromatin to adjacent domains, and suggests that JIL-1 could exert a barrier function at the HTT-TAS boundary, based on the results obtained from this study (Silva-Sousa, 2013).

A different behavior was not observedin the control of gene expression when studying the HTT array of the 4th and the 2nd chromosomes. Interestingly, a significant difference was found when looking at the subtelomere of the 4R telomere. In the reporter lines 39C-72 and 118E-5 from the subtelomere domain of the 4th chromosome, it was found that JIL-1 is important to allow gene expression and HP1a is necessary to repress it. This finding indicates that the chromatin of the subtelomere domain in the 4th chromosome is not equivalent to the TAS chromatin in the other chromosomes (Silva-Sousa, 2013).

In contrast, the results indicate that the chromatin at the 4R subtelomere of the reporter lines used in this study is less compacted and more permissive to gene expression than the TAS domains from the other chromosomes, and even than the HTT array. This scenario suggests that in the 4R telomere of these lines the compaction of the chromatin is in the opposite orientation than in the rest of the telomeres. It would be interesting to study if this reversed chromatin organization with respect to the other telomeres has a particular role in the general telomere function in Drosophila (Silva-Sousa, 2013).

This study has reported how the presence of the JIL-1 kinase is needed at the telomere in Drosophila in order to allow gene expression of the promoters embedded in this domain. The lack of JIL-1 causes an increase of silencing at the HTT array and a release of silencing at the subtelomeric domain likely by the spreading of heterochromatin towards adjacent domains. Finally, it was discovered that the telomere and subtelomere domain of the 4R arm of some lines might have a different chromatin organization with respect to the other Drosophila telomeres (Silva-Sousa, 2013).

The analysis of pendolino (peo) mutants reveals differences in the fusigenic potential among Drosophila telomeres

Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin. This study shows that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations shows that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. The study thus hypothesizes that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping (Cenci, 2015).

Telomeric repeat silencing in germ cells is essential for early development in Drosophila

The germline-specific role of telomeres consists of chromosome end elongation and proper chromosome segregation during early developmental stages. Despite the crucial role of telomeres in germ cells, little is known about telomere biology in the germline. This study analyzed telomere homeostasis in the Drosophila female germline and early embryos. A novel germline-specific function of deadenylase complex Ccr4-Not in the telomeric transcript surveillance mechanism is reported. Depletion of Ccr4-Not complex components causes strong derepression of the telomeric retroelement HeT-A in the germ cells, accompanied by elongation of the HeT-A poly(A) tail. Dysfunction of transcription factors Woc and Trf2, as well as RNA-binding protein Ars2, also results in the accumulation of excessively polyadenylated HeT-A transcripts in ovaries. Germline knockdowns of Ccr4-Not components, Woc, Trf2 and Ars2, lead to abnormal mitosis in early embryos, characterized by chromosome missegregation, centrosome dysfunction and spindle multipolarity. Moreover, the observed phenotype is accompanied by the accumulation of HeT-A transcripts around the centrosomes in early embryos, suggesting the putative relationship between overexpression of telomeric transcripts and mitotic defects. These data demonstrate that Ccr4-Not, Woc, Trf2 and Ars2, components of different regulatory pathways, are required for telomere protection in the germline in order to guarantee normal development (Morgunova, 2015).

Mod(mdg4) variants repress telomeric retrotransposon HeT-A by blocking subtelomeric enhancers

Telomeres in Drosophila are composed of sequential non-LTR retrotransposons HeT-A, TART and TAHRE. Although they are repressed by the PIWI-piRNA pathway or heterochromatin in the germline, the regulation of these retrotransposons in somatic cells is poorly understood. This study demonstrated that specific splice variants of Mod(mdg4) repress HeT-A by blocking subtelomeric enhancers in ovarian somatic cells. Among the variants, it was found that the Mod(mdg4)-N variant represses HeT-A expression the most efficiently. Subtelomeric sequences bound by Mod(mdg4)-N block enhancer activity within subtelomeric TAS-R repeats. This enhancer-blocking activity is increased by the tandem association of Mod(mdg4)-N to repetitive subtelomeric sequences. In addition, the association of Mod(mdg4)-N couples with the recruitment of RNA polymerase II to the subtelomeres, which reinforces its enhancer-blocking function. These findings provide novel insights into how telomeric retrotransposons are regulated by the specific variants of insulator proteins associated with subtelomeric sequences (Takeuchi, 2022).

Identification of the Telomere elongation Mutation in Drosophila

Length maintenance of Telomeres in Drosophila relies on the transposition of the specialized retrotransposons Het-A, TART, and TAHRE, rather than on the activity of the enzyme telomerase as it occurs in most other eukaryotic organisms. The length of the telomeres in Drosophila thus depends on the number of copies of these transposable elements. Previous work has led to the isolation of a dominant mutation, Tel(1), that caused a several-fold elongation of telomeres. In this study, the Tel(1) mutation was molecularly identified by a combination of transposon-induced, site-specific recombination and next-generation sequencing. Recombination located Tel(1) to a 15 kb region in 92A. Comparison of the DNA sequence in this region with the Drosophila Genetic Reference Panel of wild-type genomic sequences delimited Tel(1) to a 3 bp deletion inside intron 8 of Ino80. Furthermore, CRISPR/Cas9-induced deletions surrounding the same region exhibited the Tel(1) telomere phenotype, confirming a strict requirement of this intron 8 gene sequence for a proper regulation of Drosophila telomere length (Reddy, 2022).

The hnRNP A1 homolog Hrb87F/Hrp36 is important for telomere maintenance in Drosophila melanogaster

Unlike the telomerase-dependent mammalian telomeres, HeT-A, TART, and TAHRE (HTT) retroposon arrays regulate Drosophila telomere length. Cap prevents telomeric associations (TAs) and telomeric fusions (TFs). Results from this study suggest important roles of Hrb87F in telomeric HTT array and cap maintenance in Drosophila. All chromosome arms, except 2L, in Df(3R)Hrb87F homozygotes (Hrb87F-null) display significantly elongated telomeres with amplified HTT arrays and high TAs, all of which resolve without damage. Presence of FLAG-tagged Hrb87F (FLAG-Hrb87F) on cap and subtelomeric regions following hsFLAG-Hrb87F transgene expression in Df(3R)Hrb87F homozygotes suppresses TAs without affecting telomere length. A normal X-chromosome telomere expands within five generations in Hrb87F-null background and displays high TAs, but not when hsFLAG-Hrb87F is co-expressed. Tel 1 /Gaiano line or HP1 loss-of-function mutant-derived expanded telomeres carry Hrb87F on cap and HTT arrays while Hrb87F-null telomeres have HP1 and HOAP on caps and expanded HTT arrays. ISWI, seen only on cap on normal telomeres, is abundant on Hrb87F-null expanded HTT arrays. Together, these suggest complex interactions between members of the proteome of telomeres so that absence of any key member leads to telomere expansion and/or enhanced TAs/TFs. HTT expansion in Hrb87F-null condition is not developmental but a germline event presumably because absence of Hrb87F in germline may deregulate HTT retroposition/replication leading to telomere elongation (Singh, 2015).

Targeting of P element reporters to heterochromatic domains by transposable element 1360 in Drosophila melanogaster

Heterochromatin is a common DNA packaging form employed by eukaryotes to constitutively silence transposable elements. Determining which sequences to package as heterochromatin is vital for an organism. This study used Drosophila to study heterochromatin formation, exploiting position effect variegation, a process whereby a transgene is silenced stochastically if inserted in proximity to heterochromatin, leading to a variegating phenotype. Previous studies identified the transposable element 1360 as a target for heterochromatin formation. This study used transgene reporters with either one or four copies of 1360 to determine if increasing local repeat density can alter the fraction of the genome supporting heterochromatin formation. Including 1360 in the reporter increases the frequency with which variegating phenotypes are observed. This increase is due to a greater recovery of insertions at the telomere-associated sequences (~50% of variegating inserts). In contrast to variegating insertions elsewhere, the phenotype of telomere-associated sequence insertions is largely independent of the presence of 1360 in the reporter. Variegating and fully expressed transgenes were found to be located in different types of chromatin, and variegating reporters in the telomere-associated sequences differ from those in pericentric heterochromatin. Indeed, chromatin marks at the transgene insertion site can be used to predict the eye phenotype. This analysis reveals that increasing the local repeat density (via the transgene reporter) does not enlarge the fraction of the genome supporting heterochromatin formation. Rather, additional copies of 1360 appear to target the reporter to the telomere-associated sequences with greater efficiency, thus leading to an increased recovery of variegating insertions (Huisinga, 2015).

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. It was found that mutations in pendolino (peo) cause telomeric fusions (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. These analyses lead to the hypothesis 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 (Marzullo, 2016).

he Deadbeat paternal effect of uncapped sperm telomeres on cell cycle progression and chromosome behavior in Drosophila melanogaster

Telomere-capping complexes (TCCs) protect the ends of linear chromosomes from illegitimate repair and end-to-end fusions and are required for genome stability. The identity and assembly of TCC components have been extensively studied, but whether TCCs require active maintenance in non-dividing cells remains an open question. This study shows that Drosophila melanogaster requires Deadbeat (Ddbt), a sperm nuclear basic protein (SNBP) that is recruited to the telomere by the TCC and is required for TCC maintenance during genome-wide chromatin remodeling that transforms spermatids to mature sperm. Ddbt-deficient males produce sperm lacking TCCs. Their offspring delay the initiation of anaphase as early as cycle 1 but progress through the first two cycles. Persistence of uncapped paternal chromosomes induces arrest at or around cycle 3. This early arrest can be rescued by selective elimination of paternal chromosomes and production of gynogenetic haploid or haploid mosaics. Progression past cycle 3 can also occur if embryos have reduced levels of the maternally provided checkpoint kinase Chk2. The findings provide insights into how telomere integrity affects the regulation of the earliest embryonic cell cycles. They also suggest that other SNBPs, including those in humans, may have analogous roles and manifest as paternal effects on embryo quality (Yamaki, 2016).

Recurrent innovation at genes required for telomere integrity in Drosophila

Telomeres are nucleoprotein complexes at the ends of linear chromosomes. These specialized structures ensure genome integrity and faithful chromosome inheritance. Recurrent addition of repetitive, telomere-specific DNA elements to chromosome ends combats end-attrition, while specialized telomere-associated proteins protect naked, double-stranded chromosome ends from promiscuous repair into end-to-end fusions. Although telomere length homeostasis and end-protection are ubiquitous across eukaryotes, there is sporadic but building evidence that the molecular machinery supporting these essential processes evolves rapidly. Nevertheless, no global analysis of the evolutionary forces that shape these fast-evolving proteins has been performed on any eukaryote. The abundant population and comparative genomic resources of Drosophila melanogaster and its close relatives offer a unique opportunity to fill this gap. This study leverages population genetics, molecular evolution, and phylogenomics to define the scope and evolutionary mechanisms driving fast evolution of genes required for telomere integrity. Evidence was uncovered of pervasive positive selection across multiple evolutionary timescales. Prolific expansion, turnover, and expression evolution was documented in gene families founded by telomeric proteins. Motivated by the mutant phenotypes and molecular roles of these fast-evolving genes, four alternative, but not mutually exclusive, models were proposed of intra-genomic conflict that may play out at very termini of eukaryotic chromosomes. The findings set the stage for investigating both the genetic causes and functional consequences of telomere protein evolution in Drosophila and beyond (Levine, 2016).

Short and long-term evolutionary dynamics of subtelomeric piRNA clusters in Drosophila

Two Telomeric Associated Sequences, TAS-R and TAS-L, form the principal subtelomeric repeat families identified in Drosophila melanogaster. They are PIWI-interacting RNA (piRNA) clusters involved in repression of Transposable Elements. This study revisited TAS structural and functional dynamics in D. melanogaster and in related species. In silico analysis revealed that TAS-R family members are composed of previously uncharacterized domains. This analysis also showed that TAS-L repeats are composed of arrays of a region termed 'TAS-L like' (TLL) identified specifically in one TAS-R family member, X-TAS. TLL were also present in other species of the melanogaster subgroup. Therefore, it is possible that TLL represents an ancestral subtelomeric piRNA core-cluster. Furthermore, all D. melanogaster genomes tested possessed at least one TAS-R locus, whereas TAS-L can be absent. A screen of 110 D. melanogaster lines showed that X-TAS is always present in flies living in the wild, but often absent in long-term laboratory stocks and that natural populations frequently lost their X-TAS within 2 years upon lab conditioning. Therefore, the unexpected structural and temporal dynamics of subtelomeric piRNA clusters demonstrated in this study suggests that genome organization is subjected to distinct selective pressures in the wild and upon domestication in the laboratory (Asif-Laidin, 2017).

Chromosome healing is promoted by the telomere cap component Hiphop in Drosophila

The addition of a new telomere onto a chromosome break, a process termed healing, has been studied extensively in organisms that utilize telomerase to maintain their telomeres. In comparison, relatively little is known about how new telomeres are constructed on broken chromosomes in organisms that do not use telomerase. Chromosome healing was studied in somatic and germline cells of Drosophila melanogaster, a non-telomerase species. It was observed, for the first time, that broken chromosomes can be healed in somatic cells. In addition, overexpression of the telomere cap component Hiphop increased the survival of somatic cells with broken chromosomes, while the cap component HP1 did not, and overexpression of the cap protein HOAP decreased their survival. In the male germline, Hiphop overexpression greatly increased the transmission of healed chromosomes. These results indicate that Hiphop can stimulate healing of a chromosome break. It is suggested that this reflects a unique function of Hiphop: it is capable of seeding formation of a new telomeric cap on a chromosome end that lacks a telomere (Kurzhals, 2017).

Chronic low-dose pro-oxidant treatment stimulates transcriptional activity of telomeric retroelements and increases telomere length in Drosophila

It has been proposed that oxidative stress, elicited by high levels of reactive oxygen species, accelerates telomere shortening by erosion of telomeric DNA repeats. While most eukaryotes counteract telomere shortening by telomerase-driven addition of these repeats, telomeric loss in Drosophila is compensated by retrotransposition of the telomeric retroelements HeT-A, TART and TAHRE to chromosome ends. This study tested the effect of chronic exposure of flies to non-/sub-lethal doses of paraquat, which is a redox cycling compound widely used to induce oxidative stress in various experimental paradigms including telomere length analyses. Indeed, chronic paraquat exposure for five generations resulted in elevated transcriptional activity of both telomeric and non-telomeric transposable elements, and extended telomeric length in the tested fly lines. It is proposed that low oxidative stress leads to increased telomere length within Drosophila populations. For a mechanistic understanding of the observed phenomenon two scenarios are discusser: adaption, acting through a direct stimulation of telomere extension, or positive selection favoring individuals with longer telomeres within the population (Korandova, 2018).

Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis

The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. The biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development was studied with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, this study showed that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, it was demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, it is suggested that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants (Kordyukova, 2018).

Diversification and collapse of a telomere elongation mechanism

In most eukaryotes, telomerase counteracts chromosome erosion by adding repetitive sequence to terminal ends. Drosophila melanogaster instead relies on specialized retrotransposons that insert exclusively at telomeres. This exchange of goods between host and mobile element-wherein the mobile element provides an essential genome service and the host provides a hospitable niche for mobile element propagation-has been called a "genomic symbiosis." However, these telomere-specialized, jockey family retrotransposons may actually evolve to "selfishly" overreplicate in the genomes that they ostensibly serve. Under this model, rapid diversification is expected of telomere-specialized retrotransposon lineages and, possibly, the breakdown of this ostensibly symbiotic relationship. This study reports data consistent with both predictions. Searching the raw reads of the 15-Myr-old melanogaster species group, de novo jockey retrotransposon consensus sequences were generated, and phylogenetic tree-building was used to delineate four distinct telomere-associated lineages. Recurrent gains, losses, and replacements account for this retrotransposon lineage diversity. In Drosophila biarmipes, telomere-specialized elements have disappeared completely. De novo assembly of long reads and cytogenetics confirmed this species-specific collapse of retrotransposon-dependent telomere elongation. Instead, telomere-restricted satellite DNA and DNA transposon fragments occupy its terminal ends. It is infered that D. biarmipes relies instead on a recombination-based mechanism conserved from yeast to flies to humans. Telomeric retrotransposon diversification and disappearance suggest that persistently "selfish" machinery shapes telomere elongation across Drosophila rather than completely domesticated, symbiotic mobile elements (Saint-Leandre, 2019).

Rapid evolution at the Drosophila telomere: transposable element dynamics at an intrinsically unstable locus

Drosophila telomeres have been maintained by three families of active transposable elements (TEs), HeT-A, TAHRE, and TART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. This study leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85  Drosophila melanogaster genomes and compared their variation to other more typical TE families. Occasional large-scale copy number expansions of both HTTs and other TE families occured, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, it is instead suggested that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. This study has extended previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than one-third do not appear to have been healed with an HTT insertion. It is also reported that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. It is further suggested that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. It is proposed that HTT evolution is driven by multiple processes, with niche specialization and telomere instability being previously underappreciated and likely predominant (McGurk, 2021).

Evolutionary mode for the functional preservation of fast-evolving Drosophila telomere capping proteins

DNA end protection is fundamental for the long-term preservation of the genome. In vertebrates the Shelterin protein complex protects telomeric DNA ends, thereby contributing to the maintenance of genome integrity. In the Drosophila genus, this function is thought to be performed by the Terminin complex, an assembly of fast-evolving subunits. Considering that DNA end protection is fundamental for successful genome replication, the accelerated evolution of Terminin subunits is counterintuitive, as conservation is supposed to maintain the assembly and concerted function of the interacting partners. This problem extends over Drosophila telomere biology and provides insight into the evolution of protein assemblies. In order to learn more about the mechanistic details of this phenomenon this study investigated the intra- and interspecies assemblies of Verrocchio and Modigliani, two Terminin subunits using in vitro assays. Based on the results and on homology-based three-dimensional models for Ver and Moi, it is concluded that both proteins contain Ob-fold and contribute to the ssDNA binding of the Terminin complex. It is proposed that the preservation of Ver function is achieved by conservation of specific amino acids responsible for folding or localized in interacting surfaces. This study also provides the first evidence on Moi DNA binding (Vedelek, 2021).

Loss of telomere silencing is accompanied by dysfunction of Polo kinase and centrosomes during Drosophila oogenesis and early development

Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. The mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila was studied. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. This study found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction (Morgunona, 2021).

Paramutation-like Epigenetic Conversion by piRNA at the Telomere of Drosophila virilis

First discovered in maize, paramutation is a phenomenon in which one allele can trigger an epigenetic conversion of an alternate allele. This conversion causes a genetically heterozygous individual to transmit alleles that are functionally the same, in apparent violation of Mendelian segregation. Studies over the past several decades have revealed a strong connection between mechanisms of genome defense against transposable elements by small RNA and the phenomenon of paramutation. For example, a system of paramutation in Drosophila melanogaster has been shown to be mediated by piRNAs, whose primary function is to silence transposable elements in the germline. This paper characterizes a second system of piRNA-mediated paramutation-like behavior at the telomere of Drosophila virilis. In Drosophila, telomeres are maintained by arrays of retrotransposons that are regulated by piRNAs. As a result, the telomere and sub-telomeric regions of the chromosome have unique regulatory and chromatin properties. Previous studies have shown that maternally deposited piRNAs derived from a sub-telomeric piRNA cluster can silence the sub-telomeric center divider gene of Drosophila virilis in trans. This paper shows that this silencing can also be maintained in the absence of the original silencing allele in a subsequent generation. The precise mechanism of this paramutation-like behavior may be explained by either the production of retrotransposon piRNAs that differ across strains or structural differences in the telomere. Altogether, these results show that the capacity for piRNAs to mediate paramutation in trans may depend on the local chromatin environment and proximity to the uniquely structured telomere regulated by piRNAs. This system promises to provide significant insights into the mechanisms of paramutation (Dorador, 2022).

Telomeric retrotransposons show propensity to form G-quadruplexes in various eukaryotic species

Canonical telomeres (telomerase-synthetised) are readily forming G-quadruplexes (G4) on the G-rich strand. However, there are examples of non-canonical telomeres among eukaryotes where telomeric tandem repeats are invaded by specific retrotransposons. Drosophila melanogaster represents an extreme example with telomeres composed solely by three retrotransposons-Het-A, TAHRE and TART (HTT). Even though non-canonical telomeres often show strand biased G-distribution, the evidence for the G4-forming potential is limited. Using circular dichroism spectroscopy and UV absorption melting assay this study has verified in vitro G4-formation in the HTT elements of D. melanogaster. Namely 3 in Het-A, 8 in TART and 2 in TAHRE. All the G4s are asymmetrically distributed as in canonical telomeres. Bioinformatic analysis showed that asymmetric distribution of potential quadruplex sequences (PQS) is common in telomeric retrotransposons in other Drosophila species. Most of the PQS are located in the gag gene where PQS density correlates with higher DNA sequence conservation and codon selection favoring G4-forming potential. The importance of G4s in non-canonical telomeres is further supported by analysis of telomere-associated retrotransposons from various eukaryotic species including green algae, Diplomonadida, fungi, insects and vertebrates. Virtually all analyzed telomere-associated retrotransposons contained PQS, frequently with asymmetric strand distribution. Comparison with non-telomeric elements showed independent selection of PQS-rich elements from four distinct LINE clades. These findings of strand-biased G4-forming motifs in telomere-associated retrotransposons from various eukaryotic species support the G4-formation as one of the prerequisites for the recruitment of specific retrotransposons to chromosome ends and call for further experimental studies (Jedlicka, 2023).


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Zygotically transcribed genes

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