The Interactive Fly
Zygotically transcribed genes
Two distinct domains in Drosophila melanogaster telomeres
Genomic organization of the Drosophila telomere retrotransposable elements
HP1 is distributed within distinct chromatin domains at Drosophila telomeres
HipHop interacts with HOAP and HP1 to protect Drosophila telomeres in a sequence-independent manner
Gag proteins of Drosophila telomeric retrotransposons: collaborative targeting to chromosome ends
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).
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).
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).
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
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).
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).
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).
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).
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