caravaggio: Biological Overview | References
Gene name - caravaggio
Synonyms - HOAP, HP1/ORC-associated protein
Cytological map position - 95F2-95F2
Function - chromatin component
Keywords - capping of telomeres
Symbol - cav
FlyBase ID: FBgn0026257
Genetic map position - 3R:20,046,864..20,049,254 [+]
Classification - rapidly evolving proteins
Cellular location - nuclear
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 (reviewed in Rong, 2008a). 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 (Bi, 2005; Ciapponi, 2006; Oikemus, 2006). Several other proteins serving capping function in Drosophila have homologs in other organisms: HP1, UbcD1, Woc (Raffa, 2005) and the H2A.Z histone variant (Rong, 2008b). 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 (Shareef, 2001; Cenci, 2003). 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 (Gao, 2008), 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 (Komonyi, 2009; Raffa, 2009). 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 (Cenci, 2003), 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 (Diede, 2001; Goudsouzian. 2006; Negrini, 2007). 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).
Eukaryotic nuclei contain regions of differentially staining chromatin (heterochromatin), which remain condensed throughout the cell cycle and are largely transcriptionally silent. RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability. This study reports a similar phenotype for the telomeric partner of HP1, HOAP (Caravaggio), and roles for both proteins in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the masterswitch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, SxlPe, is essential to females, as it provides SXL protein to initiate the productive female-specific splicing of later Sxl transcripts, which are transcribed from the maintenance promoter (SxlPm) in both sexes. HOAP mutants show inappropriate SxlPe firing in males and the concomitant inappropriate splicing of SxlPm-derived transcripts, while females show premature firing of SxlPe. HP1 mutants, by contrast, display SxlPm splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with SxlPe sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to SxlPe are severely compromised. These genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at SxlPe (Li, 2011).
The canonical heterochromatin protein HP1 is most commonly associated with constitutive heterochromatin and gene repression. This study reports a critical role for it in regulating one of the earliest decisions in metazoan development, whether to embark on a female or male path of sexual differentiation and dosage compensation. The role of heterochromatin in mammalian dosage compensation has been recognized from early work on the mouse. Although Drosophila utilizes a different mechanism to equalize X-linked gene dose, through hyper-activation of the single male X chromosome via chromatin modification, this study provides the first evidence of a role for heterochromatin proteins in the early events of Drosophila sex determination. HP1, together with its telomere partner HOAP, influence the critical decision in sex determination - activation of SxlPe, the Sxl establishment promoter (Li, 2011).
Reductions in HOAP preferentially compromise male viability. This was observed for two different cav mutant alleles and by reducing HOAP through RNAi. The presence of SxlPm-derived transcripts that have been spliced in the female mode in cav mutant males suggested inappropriate Sxl activation to be responsible for this reduced viability. In situ data indicating inappropriate firing of SxlPe in male embryos from cav2248 heterozygous parents support this view, as does the rescue of the cav2248 male viability defect by Sxl loss of function mutations. The more pronounced male lethality observed from reducing HOAP by RNAi expression driven by maternal, versus paternal, contribution of Actin5C GAL4 is consistent with such an early requirement for HOAP for male viability (Li, 2011).
Previous reports have shown that reducing HP1 by RNAi similarly reduces male viability preferentially. RT-PCR assays of SxlPm transcripts in HP1 mutants, however, suggested a more complex scenario as incorrect sex specific transcripts were observed in both sexes. This pointed to an activation, as well as repressor, role for HP1. Consistent with an activation role, reduction of maternal HP1 severely compromised female viability when the dose of Sxl was also reduced in the progeny, and ChIP assays of embryos from this cross showed recruitment of RNAP II to SxlPe to be impaired. This effect of reducing HP1 on female viability was strictly maternal, as was the antagonizing effect of simultaneously reducing maternal HOAP. Moreover, the partial rescue of the Su(var)205 maternal effect by the C-terminally truncated cav1 allele, which produces a protein that is compromised for HP1-binding, points to an involvement of HP1 in the antagonizing activity of HOAP. Finally, ChIP assays show a dependence of HP1 on HOAP for its association with SxlPe. Combined, these data indicate both antagonistic and cooperative roles for these heterochromatin proteins in regulating SxlPe, whereby HOAP acts as a repressor and HP1 acts as both an activator and repressor. The reliance of HP1 on HOAP for recruitment to the promoter would suggest HOAP may also have a role in the activation function of HP1 at the promoter, although this was not readily apparent in the assays used in this study (Li, 2011).
Although the data clearly show maternal roles for HOAP and HP1 in regulating the activity of SxlPe, for both HOAP and HP1, RNAi knockdown data indicate a substantial zygotic component in their effects on male viability. These zygotic effects, observed only in progeny carrying both an interference RNA transgene and a GAL4 driver transgene, suggest additional later sex-specific roles for both proteins. Such roles could be related to those observed for HP1 and SU(VAR)3-7 in male dosage compensation. Because the effect of reducing these proteins on the chromosomal distribution of DCC proteins is the opposite of those observed for males that are deficient for DCC proteins, as predicted to occur with inappropriate SxlPe expression, the activities of heterochromatin proteins in dosage compensation appear to be distinct from the early roles of HP1 and HOAP at SxlPe. In addition, there may be zygotic roles for heterochromatin proteins in sex-specific gene expression, as proposed for HP1 (Li, 2011).
Previous analysis of SxlPe indicated that 400 bp immediately upstream of the promoter are sufficient for sex-specific regulation, but distal sequences, extending to -1700 bp, are required for wild type levels of expression, E-box binding sites for antagonistically acting bHLH proteins, which are encoded by zygotically expressed X-linked and autosomal signal elements (XSE and ASE) and direct an X counting mechanism, are distributed throughout both regions (Li, 2011).
Both HP1 and HOAP are enriched in the region proximal to SxlPe which contains binding sites for both positive and negative E-box proteins. Within the SxlPe promoter distal region, HOAP alone is enriched in two peaks where there is a striking relationship with E-box binding sites for positive factors, but those for negative factors appear essentially devoid of HOAP. HOAP may antagonize positive factors but permit negative factors to bind in the SxlPe distal region, in an HP1-independent repressing role. Whereas loss of HOAP de-represses SxlPe in males, the strength and uniformity of expression does not approach that in wild type females. This indicates continued influence from the X counting mechanism in cav mutant males. SxlPe is also expressed prematurely in female embryos. This de-repression by reduced levels of maternal HOAP in both sexes indicates that HOAP is present at SxlPe in both sexes of wild type embryos. However, whether the proximal and distal SxlPe regions have the same or different compositions of HOAP and HP1 in the two sexes cannot be determined from ChIP assays, as the embryos are of mixed sexual identity (Li, 2011).
The interdependency of HOAP and HP1 for their binding to the SxlPe proximal region, most notably the dependence of HP1 on HOAP, also indicates both proteins are in this region in, at least, wild type female embryos. In spite of this interdependency, the genetic data show HOAP repression antagonizes HP1 activation. HOAP repression appears to also be partly HP1-dependent; the mutant HOAP protein from the cav1 allele which lacks HP1-binding also antagonizes HP1 activation. This combination of antagonistic and cooperative interactions suggests a model in which maternal HOAP and HP1 first cooperate to repress SxlPe prior to its activation. The repressive structure formed by maternal HOAP and HP1 likely serves to reduce the sensitivity of SxlPe to spurious fluctuations in zygotic XSE levels, ensuring it is only activated in females where an effective ratio of activating to repressing transcription factors exists. HP1 is retained at SxlPe during its activation in females, where it presumably switches into an activation role. In early embryos constitutive heterochromatin proteins may be more appropriate for such regulation than the Polycomb Group of facultative heterochromatin proteins, as they would not be subject to cross regulatory signals from body plan specification pathways (Li, 2011).
How HP1 switches over to transcriptional activation mode in the SxlPe proximal region is unclear. Changes in HP1 phosphorylation and/or association with other factors could alter its activity. Several XSE (X-linked element) binding sites are nearby, making them strong candidates. Presumably, this would only occur in females where the XSE dose surpasses a threshold and SxlPe is activated (Li, 2011).
This report provides the most clearly defined role for HP1 in developmental control of a euchromatic gene in a metazoan species, and the first evidence of a bifunctional regulatory role for it in such a context. Prior reports describing HP1 in transcriptional activation have focused on it in the context of transcription elongation. ChIP data at SxlPe, however, show a requirement of it for association of RNAP II with the promoter, more consistent with a role in transcription initiation. A role in initiation is also in keeping with the position of HP1 on the gene; very little HP1 is found elsewhere on the Sxl gene, even during the time of SxlPe activity. This dependence of RNAP II association on HP1 is similar to what is observed in the accumulation of noncoding RNAs at S. pombe centromeric repeats and mating type locus. Nonetheless, it is possible that the loss of RNAP II at SxlPe reflects reduced stability of all RNAP II isoforms as a consequence of an early defect in transcription elongation, rather than a defect in RNAP II recruitment to the promoter (Li, 2011).
Pausing of RNAP II in promoter proximal regions prior to activation has been observed in a high proportion of genes under developmental control in Drosophila embryos, and such pauses have also been implicated in regulation of alternative splicing. While SxlPm appears to have the features of a promoter with paused RNAP II in a genome wide RNAP II ChIP study of 0-4 hr embryos, RNAP II was absent from SxlPe. It is likely that the collection window for this study did not precisely coincide with the time of SxlPe activity. A more narrowly timed collection indicates paused RNAP II at SxlPe, suggesting that, like SxlPm, it is a pre-loaded promoter. A preloaded SxlPe also readily explains how generalized up-regulation of phosphorylation of the RNAP II CTD by the loss of Nanos, causes SxlPe activation in males with an unchanged X:A ratio (Li, 2011).
Finally, the dominant negative activity of the cav2248 allele suggests a role for the partially deleted SRY-like HMG box in HOAP association with SxlPe. ChIP data show HOAP association with the SxlPe proximal region is required for HP1 association. This proposed role for the HMG box of HOAP in SxlPe regulation is of particular interest with regards to a recent report linking HP1 and KAP-1 (TIF1β) to SRY-dependent repression of testis-specific genes in the ovary. Because mammalian sex determination is inextricably linked to gonad sex determination, SRY and HOAP each appear to constitute early decision points in their respective sex determination pathways. There are, perhaps, unexpected parallels between these divergent pathways (Li, 2011).
In both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions (Cenci, 2003). This study shows that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. These results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition (Musarò, 2008).
In most organisms, telomeres contain arrays of tandem G-rich repeats added to the chromosome ends by telomerase. Drosophila telomeres are not maintained by the activity of telomerase, but instead by the transposition of three specialized retrotransposons to the chromosome ends. In addition, whereas yeast and mammalian telomeres contain proteins that recognize telomere-specific sequences, Drosophila telomeres are epigenetically determined, sequence-independent structures. Nonetheless, Drosophila telomeres are protected from fusion events, just as their yeast and mammalian counterparts are. Genetic and molecular analyses have thus far identified eight loci that are required to prevent end-to-end fusion in Drosophila: effete (eff, also known as UbcD1), which encodes a highly conserved E2 enzyme that mediates protein ubiquitination; Su(var)205 and caravaggio (cav), encoding HP1 and HOAP, respectively; the Drosophila homologs of the ATM, RAD50, MRE11A
To determine whether mutations in genes required for telomere capping also affect cell cycle progression, DAPI-stained preparations of larval brains from seven of these eight telomere-fusion mutants were examined. Mutant brains were examined for the mitotic index (MI) and the frequency of anaphases (AF). The mitotic indices observed for the eff, Su(var)205, mre11, rad50, woc and tefu mutants ranged from 0.46 to 0.75, values that were slightly lower than the mitotic index observed for the wild type (0.86). However, brains from cav mutants showed a fourfold reduction of the mitotic index (0.19) with respect to the wild type. cav mutants also had a very low frequency of anaphases (1.7%-1.9%) compared to the wild type (13.2%), whereas in the other mutants, frequency of anaphases ranged from 8.6% to 12.5%. Reductions in both the mitotic index and the frequency of anaphases were rescued by a cav+ transgene, indicating that these phenotypes were indeed due to a mutation in cav (Musarò, 2008).
These results prompted a focus on cav mutations in order to determine how unprotected telomeres might influence cell cycle progression. The cav allele used in this study is genetically null for the telomere-fusion phenotype (Cenci, 2003). cav homozygotes and cav1/Df(3R)crb-F89-4 hemizygotes show very similar mitotic indices and frequencies of anaphases, indicating that cav is also null for these cell cycle parameters. The cav-encoded HOAP protein localizes exclusively to telomeres; cav produces a truncated form of HOAP that fails to accumulate at chromosome ends (Musarò, 2008).
The low frequencies of anaphases observed in cav mutant cells suggest that they may be arrested in metaphase. To confirm a metaphase-to-anaphase block, mitoses were filmed of cav and wild-type neuroblasts expressing the GFP-tagged H2Av histone. Control cells entered anaphase within a few minutes after chromosome alignment in metaphase, whereas cav cells remained arrested in metaphase for the duration of imaging (Musarò, 2008).
It was hypothesized that the cav-induced metaphase arrest was the result of SAC activation. As in all higher eukaryotes, unattached Drosophila kinetochores recruit three SAC protein complexes (Mad1-Mad2, Bub1-BubR1-Cenp-meta and Rod-Zw10-Zwilch) that prevent precocious sister chromatid separation by negatively regulating the ability of Cdc20 to activate the anaphase-promoting complex or cyclosome (APC/C). Mutations in genes encoding components of these complexes lead to SAC inactivation and allow cells to enter anaphase even if the checkpoint is not satisfied. To ask whether the low frequency of anaphases in cav mutant brains was due to SAC activation, zw10 cav and bubR1 cav double mutants were analyzed. In both cases, the frequency of anaphases was significantly higher than in the cav single mutant, whereas the frequency of telomere fusions remained unchanged. These results imply that the low frequency of anaphases in cav mutants is indeed due to SAC activation (Musarò, 2008).
SAC activation would be expected to increase the mitotic index through the accumulation of metaphase cells; however, in cav single mutants, the mitotic index is abnormally low. One explanation for this apparent paradox is that the cell cycle in cav cells is also delayed before M-phase, as a result of the DNA damage response (DDR). To ask whether HOAP-depleted telomeres activate any DNA damage checkpoints, double mutants were generated for cav and genes known to be involved in these checkpoints: mei-41 and telomere fusion (tefu), encoding the fly homologs of ATR and ATM, respectively; mus304, which encodes the ATR-interacting protein ATRIP grapes (grp), which specifies a CHK1 homolog and rad50, whose product is part of the Mre11-Rad50-Nbs complex. DAPI-stained preparations of larval brain cells from these double mutants showed that mei-41, mus304, grp and rad50 mutations alleviate the cell cycle block induced by cav, causing a ~2.5-fold increase of the mitotic index relative to that observed in the cav single mutant. In contrast, the tefu mutation did not affect the cav- induced interphase block. These effects are unrelated to variations in the frequency of telomere fusions, as the telomere fusion frequencies in double mutants were very similar to those in the cav single mutant. It is thus concluded that the interphase arrest in cav mutants occurs independently of ATM and is mediated by a signaling pathway involving ATR, ATRIP, Chk1 and Rad50. This signaling pathway is known to activate DNA damage checkpoints during the G1/S transition, the S phase and the G2/M transition. However, the current results do not allow identification of the particular checkpoint(s) activated by HOAP-depleted telomeres (Musarò, 2008).
Notably, in all double mutants for cav and any one of the genes associated with the DDR, including tefu (ATM), a significant increase was also observed in the frequencies of anaphases relative to that of the cav single mutant, suggesting that these genes are involved in the cav-induced metaphase arrest. This finding reflects a role of these DDR-associated genes in the peculiar mechanism by which uncapped Drosophila telomeres activate SAC (Musarò, 2008).
To obtain further insight about the cav-induced metaphase arrest, the localization of Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) was determined by immunofluorescence. In wild-type Drosophila cells, these proteins begin to accumulate at kinetochores during late prophase and remain associated with kinetochores until the chromosomes are stably aligned at the metaphase plate. Treatments with spindle poisons (for example, colchicine) disrupt microtubule attachment to the kinetochores, leading to metaphase arrest with SAC proteins accumulated at the centromeres. Immunostaining for Zwilch, Zw10, Cenp-meta or BubR1 showed that in all cases, the frequencies of cav metaphases with strong centromeric signals were comparable to those observed in colchicine-treated wild-type cells, and they were significantly higher than those seen in untreated wild-type metaphases. These findings support the view that HOAP-depleted telomeres activate the canonical SAC pathway (Musarò, 2008).
Through a detailed examination of cav metaphases immunostained for SAC proteins, an unexpected connection was found between uncapped telomeres and the localization of at least one SAC component. Although Zwilch, Zw10 and Cenp-meta accumulated exclusively at kinetochores, BubR1 was concentrated at both kinetochores and telomeres. BubR1 localized at both unfused (free) and fused telomeres; most (94.4%) cav metaphases showed at least one telomeric BubR1 signal. To better resolve the chromosome tangles seen in cav metaphases, cells were treated with hypotonic solution, allowing a focus on free telomeres, which can be reliably scored. It was found that 25% of the free telomeres in cav metaphases show an unambiguous BubR1 signal. BubR1 accumulations were not observed at wild-type telomeres or at the breakpoints of X-ray-induced chromosome breaks. BubR1 localization at telomeres was not caused by the formation of ectopic kinetochores at the chromosome ends, since cav telomeres did not recruit the centromere and kinetochore marker Cenp-C. Low frequencies of BubR1-labeled telomeres were also observed in other mutant strains with telomere fusions including eff, Su(var)205 and woc. These results indicate that BubR1 specifically localizes at uncapped telomeres (Musarò, 2008).
It was next asked whether mutations in mei-41, grp, mus304, tefu, rad50 and zw10 affect BubR1 localization at cav mutant telomeres. Whereas mutations in zw10 did not affect BubR1 localization at cav chromosome ends, double mutants for cav and any of the other genes all showed significant reductions in the frequency of BubR1-labeled free telomeres with respect to cav single mutants. Considered together, these results indicate that when the canonical SAC machinery is intact (in all cases except in zw10 cav double mutants), there is a strong negative correlation between the frequency of BubR1-labeled telomeres and the frequency of anaphases. These findings suggest that BubR1 accumulation at telomeres can activate the SAC (Musarò, 2008).
Finally it was asked whether mutations in DDR-associated genes can allow cells to bypass the SAC when it is activated by spindle abnormalities rather than by uncapped telomeres. The spindle was disrupted in two ways: with the microtubule poison colchicine and with mutations in abnormal spindle (asp). Both situations activated the SAC and caused metaphase arrest; neither mei-41 nor grp or tefu mutations allowed cells to bypass this arrest, whereas mutations in zw10 led such cells to exit mitosis. These findings indicate that the DDR-associated genes regulate BubR1 accumulation at cav telomeres but are not directly involved in the SAC machinery (Musarò, 2008).
Collectively, these results suggest a model for the activation of cell cycle checkpoints by unprotected Drosophila telomeres. It is proposed that uncapped telomeres activate DDR checkpoints, leading to interphase arrest through a signaling pathway involving mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50, but not tefu (ATM). This pathway is independent of telomeric BubR1, because mutations in tefu, which strongly reduce BubR1 accumulation at chromosome ends, do not rescue cav-induced interphase arrest. Uncapped telomeres can also activate the SAC by recruiting BubR1 through a pathway requiring mei-41, mus304, grp, rad50 and tefu functions. Once accumulated at the telomeres, BubR1 may negatively regulate either Fizzy (Cdc20) or another APC/C subunit so as to cause metaphase arrest. This model posits that certain DDR-associated genes, such as rad50, function both in the DDR pathway and in the pathway that mediates BubR1 recruitment at telomeres. This explains why rad50 and mre11 mutants show only mild reductions of the mitotic index and the frequency of anaphases even though HOAP is substantially depleted from their telomeres (Musarò, 2008).
It is proposed that uncapped telomeres can induce an interphase arrest independently of BubR1 through a signaling pathway that involves ATR, ATRIP, CHK1 and Rad50 but not ATM. The same proteins, including ATM, are required for the recruitment of BubR1 at unprotected telomeres. Telomeric BubR1 may negatively regulate the activity of the Cdc20-APC complex, leading to a metaphase-to-anaphase transition block. The metaphase arrest caused by Cdc20-APC inhibition is likely to cause an accumulation of SAC proteins on the kinetochores, reinforcing SAC activity. Consistent with this view, mutations in ida, which encodes an APC/C subunit, lead to a metaphase arrest phenotype with BubR1 accumulated at the kinetochores (Musarò, 2008).
Several recent reports have suggested possible relationships between DNA damage, SAC and telomeres. In both Drosophila and mammalian cells, DNA breaks can activate the SAC, presumably by disrupting kinetochore function. In Schizosaccharomyces pombe, Taz1-depleted telomeres result in Mph1p- and Bub1p-mediated SAC activation, and mutations in yKu70 affecting Saccharomyces cerevisiae telomere structure also activate the SAC. However, these previous studies did not explain how telomere perturbations might be perceived by the SAC. This study has found that unprotected Drosophila telomeres recruit the BubR1 kinase as do the kinetochores that are unconnected to spindle microtubules. Thus, it is possible that telomere-associated BubR1 inhibits anaphase through molecular mechanisms similar to those that govern SAC function at the kinetochore. Consistent with this possibility, a single BubR1 accumulation at either a centromere or a telomere seems competent to block anaphase onset. It will be of interest in the future to establish whether deprotected mammalian telomeres can also activate the SAC and, if so, whether BubR1 recruitment to the damaged telomeres mediates this response (Musarò, 2008).
Several proteins have been identified that protect Drosophila telomeres from fusion events. They include UbcD1, HP1, HOAP, the components of the Mre11-Rad50-Nbs (MRN) complex, the ATM kinase, and the putative transcription factor Woc. Of these proteins, only HOAP has been shown to localize specifically at telomeres. This study shows that the modigliani gene encodes a protein (Moi) that is enriched only at telomeres, colocalizes and physically interacts with HOAP, and is required to prevent telomeric fusions. Moi is encoded by the bicistronic CG31241 locus. This locus produces a single transcript that contains 2 ORFs that specify different essential functions. One of these ORFs encodes the 20-kDa Moi protein. The other encodes a 60-kDa protein homologous to RNA methyltransferases that is not required for telomere protection (Drosophila Tat-like). Moi and HOAP share several properties with the components of shelterin, the protein complex that protects human telomeres. HOAP and Moi are not evolutionarily conserved unlike the other proteins implicated in Drosophila telomere protection. Similarly, none of the shelterin subunits is conserved in Drosophila, while most human nonshelterin proteins have Drosophila homologues. This suggests that the HOAP-Moi complex, named in this study 'terminin,' plays a specific role in the DNA sequence-independent assembly of Drosophila telomeres. It is speculated that this complex is functionally analogous to shelterin, which binds chromosome ends in a sequence-dependent manner (Raffa, 2009).
This study has shown that the Moi protein is enriched exclusively at telomeres, where it colocalizes and physically interacts with both HOAP and HP1. Moi is not required for HOAP accumulation at telomeres, whereas Moi localization requires the wild-type functions of cav and mre11. These results suggest a mechanism for Moi localization at telomeres. It is proposed that the Drosophila chromosome ends, which contain variable DNA sequences, are processed and shaped by the MRN complex so as to allow binding of HOAP, which would in turn recruit Moi. The Moi-HOAP complex shares several analogies with shelterin, a 6-protein complex that protects human chromosome ends, allowing cells to distinguish telomeres from sites of DNA damage (Palm, 2008). Shelterin is comprised of 3 polypeptides that directly bind the TTAGGG telomeric repeats (TRF1, TRF2, and POT1) interconnected by 3 additional proteins (Tin2, TPP1, and Rap1). The shelterin subunits share 3 properties that distinguish them from the nonshelterin telomere-associated proteins. They are specifically enriched at telomeres; they are present at telomeres throughout the cell cycle; and their functions are limited to telomere maintenance. With the exception of Tin2 and TPP1, shelterin-related proteins have been found in most eukaryotes. However, none of the shelterin subunits are conserved in Drosophila. This is not surprising as Drosophila telomeres are DNA sequence-independent structures, while the core subunits of shelterin are sequence-specific DNA binding proteins (Raffa, 2009 and references therein).
The Moi and HOAP proteins have the same properties of the shelterin subunits: they accumulate only at telomeres; they are likely to be associated with telomeres throughout the cell cycle, as they colocalize in discrete aggregates present in all interphase nuclei and are enriched at polytene chromosome telomeres; and they appear to function only at telomeres. HP1 interacts with both Moi and HOAP but does not share their properties; it localizes to multiple chromosomal sites and its function is not limited to telomere maintenance. Notably, Moi and HOAP are not conserved in either yeasts or mammals, consistent with the fact that both proteins associate with telomeres in a sequence-independent fashion. Thus, it is proposed that Moi and HOAP are the founding components of a Drosophila telomere complex, named here 'terminin,' which acts like human shelterin. It is suggested that terminin accumulation at chromosome ends prevents both checkpoint activation and telomere fusion and helps in recruiting nonterminin components of Drosophila telomeres. This hypothesis posits that the nonterminin proteins of Drosophila telomeres should be conserved in humans and play roles in telomere maintenance. Similarly, nonshelterin components of human telomeres should have conserved Drosophila homologues. Indeed, all of the nonterminin proteins specified by the Drosophila telomere-fusion mutants so far identified have human counterparts. UbcD1 and Woc have highly conserved human homologues but it is currently unknown whether any of them is involved in telomere maintenance. HP1 too is conserved in humans, and HP1 homologues have been found at mouse telomeres where they appear to control telomere length. The ATM kinase and the components of the MRN complex (Mre11, Rad50, and Nbs) have highly conserved human orthologues (Zhu, 2000; Karlseder, 2004), which bind shelterin and help to regulate human telomere organization (Raffa, 2009).
In addition to ATM, Mre11, Rad50, and Nbs1, the human nonshelterin factors include Ku70 and Ku80 and their associated DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the ATR kinase, PARP1 and PARP2, Rad51, the ERCC1/XPF endonuclease, the Apollo nuclease, and the RecQ family members WRN and BLM, which are mutated in the Werner and Bloom syndrome, respectively (Palm, 2008). With the exception of DNA-PKcs, all these nonshelterin proteins have Drosophila homologues. There is also evidence that Drosophila ATM and ATR cooperate to prevent telomere fusion, and that Ku70 and Ku80 act as negative regulators of Drosophila telomere elongation by transposition. However, it is not currently known whether the fly homologues of Rad51, ERCC1, Apollo, WRN, and BLM play roles at Drosophila telomeres (Raffa, 2009).
In summary, it is clear that the terminin and shelterin components are not evolutionarily conserved. In contrast, the nonterminin and nonshelterin proteins are largely conserved from flies to mammals, and many of them play telomere-related functions in both Drosophila and humans. This suggests that the main difference between Drosophila and human telomeres is in the protective complexes that specifically associate with the DNA termini. Thus, apart from the different mechanisms of elongation, Drosophila and human telomeres might not be as different as it is generally thought (Raffa, 2009).
A critical function of telomeres is to prevent fusion of chromosome ends by the DNA repair machinery. In Drosophila somatic cells, assembly of the protecting capping complex at telomeres notably involves the recruitment of HOAP, HP1, and their recently identified partner, The hiphop gene was duplicated before the radiation of the melanogaster subgroup of species, giving birth to K81, a unique paternal effect gene specifically expressed in the male germline. This study shows that K81 specifically associates with telomeres during spermiogenesis, along with HOAP and HP1, and is retained on paternal chromosomes until zygote formation. In K81 mutant testes, capping proteins are not maintained at telomeres in differentiating spermatids, resulting in the transmission of uncapped paternal chromosomes that fail to properly divide during the first zygotic mitosis. Despite the apparent similar capping roles of K81 and HipHop in their respective domain of expression, it was demonstrated by in vivo reciprocal complementation analyses that they are not interchangeable. Strikingly, HipHop appeared to be unable to maintain capping proteins at telomeres during the global chromatin remodeling of spermatid nuclei. These data demonstrate that K81 is essential for the maintenance of capping proteins at telomeres in postmeiotic male germ cells. In species of the melanogaster subgroup, HipHop and K81 have not only acquired complementary expression domains, they have also functionally diverged following the gene duplication event. It is proposed that K81 specialized in the maintenance of telomere protection in the highly peculiar chromatin environment of differentiating male gametes (Dubruille, 2010).
K81 encodes a new telomere capping protein required for the transmission of functional paternal chromosomes to the diploid zygote. This finding elucidates the origin of the unique paternal effect lethal phenotype associated with K81. K81 is the first identified Drosophila telomere protein specifically expressed in the male germline. In fact, the structure and organization of telomeres in Drosophila male germ cells have remained largely unexplored. This study shows that during spermiogenesis, K81 accumulates in a small number of foci, where it is systematically associated with the HOAP and HP1 capping proteins. In contrast to HOAP, which is essentially a telomere-specific protein, HP1 is mainly enriched in pericentric heterochromatin in somatic nuclei. In addition, HP1 is also detected at telomeres and at numerous euchromatic sites on polytene chromosomes. In this regard, it is remarkable that HP1 is only retained at telomeric regions in spermatid nuclei, suggesting that its sole function in differentiating male germ cells is in capping telomeres. The lethality associated with cav (encoding HOAP) and Su(var)205 (encoding HP1) loss-of-function mutant alleles prevents direct testing of their respective roles during spermiogenesis. This study shows, however, that both HOAP and HP1 are lost from spermatid telomeres in K81 mutant testes. This loss of telomere capping proteins does not interfere with male gamete differentiation and maturation. Instead, the K81 mutant phenotype manifests itself only after fertilization and results in the incapacity of paternal chromosomes to segregate during the first zygotic mitosis. This initial defect leads to the formation of aneuploid embryos, which arrest development after a few abnormal nuclear divisions, or to the occasional escaping of haploid gynogenetic embryos that die shortly before hatching. The systematic and specific bridging of paternal chromatin during the first anaphase most likely results from the presence of chromosome end-to-end fusions. Although telomere fusions can be easily observed in cultured cells or in squashed preparations of larval brains, where they form chains of connected chromosomes, these defects appeared to be very difficult to observe in detail in Drosophila zygotes. Nonetheless, chromatin bridges associated with telomere dysfunction have been reported in syncytial embryos from mothers bearing hypomorphic alleles of mre11 or nbs (Gao, 2009), thus indicating that the DNA repair machinery presumably responsible for the fusion of uncapped telomeres is already active during early cleavage divisions (Dubruille, 2010).
The distribution of telomere capping protein foci in spermatid nuclei indicates that telomeres tend to associate within clusters during spermiogenesis. Interestingly, telomere clustering seems to be a conserved feature of animal spermiogenesis, such as in mammals, in which telomeres from the same chromosome are frequently associated in pairs. In Drosophila, telomere clustering is apparently the rule in late spermatids, as well as in the decondensing male pronucleus, because a single major focus of capping proteins is frequently observed in these nuclei. It is likely that this spectacular gathering of telomeres in a limited nuclear volume could favor the occurrence of paternal chromosome end-to-end fusions in K81 mutants (Dubruille, 2010).
Despite their critical role in chromosome protection, telomere proteins are rapidly evolving from yeasts to mammals. This tendency is observed in Drosophila, where important capping proteins such as HOAP, Verrocchio, Modigliani, and HipHop are encoded by fast-evolving genes. Previous work has shown that K81 is a relatively young gene that is restricted to the nine species comprising the melanogaster subgroup (Loppin, 2005). K81 originated after the duplication of its paralog, hiphop (originally known as CG6874/l(3)neo26), presumably through a retroposition mechanism. The predicted K81 transcription start site is only about 100 bp from the 5' end of the Rb97D gene, which is expressed in primary spermatocytes and is required for male fertility. The selection of both hiphop and K81 genes was thus likely favored by the immediate acquisition of male germline-specific expression of the duplicated copy, after its landing close to Rb97D, followed by loss of hiphop expression in this lineage. In a less parsimonious, alternative scenario, an ancestral male germline-specific hiphop gene could have evolved a somatic and female germline expression following the duplication. However, this possibility does not fit with the expected requirement of HipHop for telomere protection in somatic cells. Interestingly, with a single exception, all Drosophila sequenced species outside the melanogaster subgroup have a single member of the hiphop/K81 gene family. For instance, D. ananassae, D. pseudoobscura, and D. persimilis have hiphop with the same conserved synteny as in melanogaster species but lack K81. In these three species, hiphop is thus expected to protect telomeres in all cells, including male germ cells. Most interestingly, phylogenetic analysis reveals the existence of a second, independent duplication of hiphop in the lineage leading to D. willistoni. Moreover, this D. willistoni hiphop duplicate presents a male-biased expression, allowing the possibility that it could be required in the male germline, like K81 in D. melanogaster. Although functional studies are not currently feasible in non-melanogaster species, developmental in situ expression analysis of members of this gene family may support these predictions (Dubruille, 2010).
In their respective cellular environments, HipHop and K81 are both specifically localized at telomeres, and they are required for the maintenance of the HOAP and HP1 capping proteins at chromosome ends. However, and despite the apparently identical molecular functions of K81 and HipHop, these experiments demonstrate that they cannot replace one another in vivo. When ectopically expressed in the male germline, GFP::HipHop is able to transiently restore the localization of HOAP and HP1 at spermatid telomeres in a K81 mutant background. In this genetic context, telomeres remained capped until the global replacement of histones with sperm-specific nuclear proteins. What actually triggers the loss of HipHop, HP1, and HOAP in these spermatids is not known. The fact that these proteins disappear concomitantly with the onset of global spermatid chromatin remodeling suggests a causal link, although this remains to be established. In mammals, although telomere integrity in male gametes is essential for zygote formation, little is known about the organization of telomeres in germ cells. However, a few studies point to the peculiar composition of telomere complexes in human sperm, suggesting that the unique organization of sperm chromatin imposes constraints on the structure and function of telomeres. Similarly, this study suggests that K81 specialized in the epigenetic maintenance of telomere identity in the highly peculiar chromatin environment of male gametes. This scenario also implies that HipHop lost its ability to protect sperm telomeres after the emergence of K81 function. Phylogenetic analysis of the hiphop and K81 coding sequences actually supports this subfunctionalization scenario. First, hiphop and K81 genes show a symmetrical acceleration of evolution in the melanogaster subgroup of species. Second, synonymous and nonsynonymous nucleotide substitution analysis of the coding sequences indicates that hiphop and K81 evolved under purifying selection. Finally, K81 expression in somatic cells does not rescue the zygotic lethality of hiphop mutants, thus confirming the functional divergence of both proteins (Dubruille, 2010).
The maternal expression of hiphop is apparently sufficient to protect telomeres during embryo development, as observed with mutations in other telomere capping genes. Accordingly, this study has shown that maternally expressed GFP::HipHop decorates both paternal and maternal telomeres as soon as the diploid zygote is formed. However, the early larval zygotic lethality of hiphop mutants prevented a more detailed in vivo phenotypic analysis using third instar larvae polytene chromosomes or neuroblast mitotic chromosomes. Although both mRFP1::K81 and GFP::K81 are fully able to associate with somatic telomeres, these experiments could only be carried out in a wild-type hiphop genetic background. It is thus not know whether K81 associates with somatic telomeres autonomously or through its association with other capping proteins, such as HOAP and/or HP1, in a HipHop-dependent manner (Dubruille, 2010).
The functional divergence of HipHop and K81 could reflect their adaptation to different chromatin environments. However, as new Drosophila telomere proteins are regularly discovered, it is also reasonable to consider the possibility that K81 and HipHop require one or more yet-unknown protein partners to function properly. For instance, K81 could not protect telomeres in somatic cells if its capping activity requires another factor only expressed in spermatids. Interestingly, the HP1-related protein Umbrea/HP6, which has been recently proposed to function in telomere protection (Joppich, 2009), is mainly expressed in the adult testis. Future studies should thus aim at determining whether other capping proteins are specialized in the protection of telomeres in germ cells, like K81 (Dubruille, 2010).
In conclusion, this study demonstrates that HipHop and K81 diverged not only in their domain of expression, but also in their ability to protect telomeres in their respective cellular environments. A challenge will be to understand the nature of the evolutionary pressure that ultimately shaped the diversification of the hiphop/K81 gene family in the genus Drosophila (Dubruille, 2010).
Drosophila telomeres are elongated by transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the terminal DNA sequence. Drosophila telomeres are protected by terminin, a complex that includes the HOAP (Heterochromatin Protein 1/origin recognition complex-associated protein) and Moi (Modigliani) proteins and shares the properties of human shelterin. This study shows that Verrocchio (Ver), an oligonucleotide/oligosaccharide-binding (OB) fold-containing protein related to Rpa2/Stn1, interacts physically with HOAP and Moi, is enriched only at telomeres, and prevents telomere fusion. These results indicate that Ver is a new terminin component; it is speculated that, concomitant with telomerase loss, Drosophila evolved terminin to bind chromosome ends independently of the DNA sequence (Raffa, 2010).
It has been suggested that fly telomeres are capped by the HOAP-Moi complex, which was called terminin, and which has the same properties of shelterin: a specific telomeric localization throughout the cell cycle, and a telomere-limited function (Raffa, 2009). This study has shown that ver mutants exhibit a very high frequency of telomeric fusions (about five per cell), comparable with those observed previously in cav (HOAP) and moi mutants (Cenci, 2003; Musarò, 2008; Raffa, 2009). Consistent with these findings, Ver is enriched exclusively at telomeres like HOAP and Moi, and colocalizes precisely and interacts physically with both these proteins. In addition, the current analyses indicate that Ver functions only at telomeres. These findings strongly suggest that Ver is a component of the terminin complex (Raffa, 2010).
The results indicate that Ver contains an OB fold domain that shares structural similarity with the Rpa2/Stn1 OB fold. Interestingly, the Drosophila genome does not appear to contain homologs of the shelterin subunits and the other CST subunits. However, all of the nonshelterin and non-CST components of human telomeres are conserved in flies. Conversely, with the exception of HOAP and Moi, all of the Drosophila telomere-related proteins identified so far have clear human counterparts (Cenci, 2005; Raffa, 2009). Thus, it is hypothesized that, concomitant with telomerase loss, Drosophila lost the shelterin and the CST homologs that bind DNA in a sequence-specific fashion, and evolved terminin to bind chromosome ends independently of the DNA sequence (Raffa, 2010).
The hypothesis on terminin evolution generates several expectations. It is logical to assume that telomerase loss resulted in a divergence of terminal DNA sequences, accompanied by a strong selective pressure toward the evolution of sequence-independent telomere-binding factors. It is also conceivable that the evolutionary pressure on these factors was higher than that exerted on telomere proteins not specifically involved in capping. Therefore, one would predict that proteins involved directly and exclusively in telomere capping evolved more rapidly than the other telomere-associated proteins. This prediction is verified by the finding that HOAP, Moi, and Ver are fast-evolving proteins, while the other Drosophila telomere proteins, including HP1, are not (Raffa, 2010).
Although the frequencies of telomeric fusions elicited by loss of each terminin component are fully comparable, Ver, Moi, and HOAP do not play identical roles at Drosophila telomeres. HOAP localizes at telomeres independently of Ver and Moi, which are both HOAP-dependent and mutually dependent for telomeric localization. In addition, while loss of HOAP triggers both the DNA damage and the spindle assembly (SAC) response (Musarò, 2008), depletion of either Ver or Moi (Raffa, 2009) does not appear to elicit these checkpoint responses. These results suggest that HOAP is crucial for masking chromosome ends to avoid their recognition as double-strand breaks. Ver and Moi are not required for terminal DNA protection so as to prevent checkpoint responses. However, Ver and Moi are essential to hide chromosome ends from the DNA repair machineries that mediate telomere fusion. A Ver protein with mutations in the OB fold domain is still recruited at telomeres, but is unable to prevent telomere fusion. This suggests that the integrity of the Ver OB fold domain is crucial to prevent inappropriate repair of terminal DNA, and implies that Drosophila telomeres terminate with a single-strand overhang like their yeast, plant, and mammalian counterparts (Raffa, 2010).
Drosophila HP1-interacting protein (Hip) is a partner of heterochromatin protein 1 (HP1) and is involved in transcriptional epigenetic gene silencing and the formation of heterochromatin. Recently, it has been shown that HP1 interacts with the telomere capping factor HP1/ORC (origin recognition complex)-associated protein (HOAP). Telomeres, complexes of DNA and proteins at the end of linear chromosomes, have been recognized to protect chromosome ends from degradation and fusion events. Both proteins are located at telomeres and prevent telomere fusions. This study reports the identification and characterization of the Hip-interacting protein Umbrea (identical to the recently described HP6 encoding gene) (Greil. 2007). Umbrea interacts directly with Hip, HP1 and HOAP in vitro. Umbrea, Hip and HP1 are partners in a protein complex in vivo and completely co-localize in the pericentric heterochromatin and at telomeres. Using a Gal4-induced RNA interference system, it was found that after depletion of Umbrea in salivary gland polytene chromosomes, they exhibit multiple telomeric fusions. Taken together, these results suggest that Umbrea cooperates with Hip, HP1 and HOAP and plays a functional role in mediating normal telomere behaviour in Drosophila (Joppich, 2009).
This study identified and characterized the heterochromatin protein Umbrea by searching a yeast two-hybrid database for predicted interacting partners of the previously characterized HP1-interacting protein Hip (Schwendemann. 2008). This study not only confirmed the predicted interaction of Umbrea and Hip but it was also found that Umbrea is able to interact with HP1. This direct interaction is not reported from the Drosophila interaction database (Joppich, 2009).
In contrast to the current results results, Greil (2007) performed no additional protein-protein interaction studies to verify the predicted interactions. For localization studies, Greil used epitope-tagged HP6 and HP1 in transfected Drosophila Kc cells. Whereas HP1 is enriched at the heterochromatic chromocentre, for HP6 localization they found a uniform nuclear staining. However, they did not detect a clear co-localization of HP6 and HP1 in the chromocentre. In a different experiment, Greil used the DamID large-scale mapping technique in transfected cell culture Drosophila Kc cells for co-localization studies with HP1 (Greil, 2007). In contrast, in this experiment they found binding of HP6 in pericentric regions of the major chromosomes and on the small chromosome 4. HP6 localization was only subtly affected after HP1 depletion. On the basis of this result, Greil speculate that an additional interaction might play a key role in targeting HP6 to heterochromatin. To functionally characterize HP6, Greil tested whether mutation of HP6 is a suppressor of PEV. However, the assay they used did not reveal such a function for HP6, suggesting that HP6 is not needed for heterochromatic transgene silencing (Joppich, 2009).
Both HP1 and Umbrea contain a chromo shadow domain. This domain mediates homodimerization of HP1 and this domain mediates heterodimerization between HP1 and Umbrea in vitro. This finding is supported by immunoprecipitation assays. Hip and HP1 are co-precipitated with Umbrea, suggesting that all three proteins are associated in a protein complex in vivo. It should be noted that three HP1-binding interfaces have been identfied in the Hip protein (Schwendemann, 2008). The presence of three binding interfaces in Hip implies a mode of cooperative binding suited to cross-linking of multiple chromo shadow domain-containing molecules like HP1 and Umbrea. It therefore cannot be ruled out that the in vivo interaction between Umbrea and HP1 is only indirect, mediated by the bridging protein Hip (Joppich, 2009).
In agreement with this model, it was found that Umbrea and HP1 use the same three binding modules within the Hip sequence. Both the chromo shadow domains of Umbrea and HP1 interact independently with the three binding interfaces of Hip. The interaction of the two different proteins with the same interaction modules in Hip supports the idea of a novel chromo shadow domain binding interface in Hip (Schwendemann, 2008). The Umbrea protein appears unique among other heterochromatin proteins since it is almost reduced to its chromo shadow domain. What might be the functional mechanism of a protein that consists of a single domain that is similar to the HP1 chromo shadow domain? In HP1 this domain provides the surface for the interaction with various other chromosomal proteins and displays the HP1 protein partner promiscuity. In agreement with this, Umbrea was shown to interact in the same way with at least three proteins. Binding of Umbrea could block the binding surface of an interacting partner to prevent the interaction with other proteins. It is known that HP1 is essential for heterochromatin localization of many proteins. It has recently been shown that Hip binding to heterochromatin depends on HP1 (Schwendemann, 2008). In this study it was found that HP1 also serves as a binding platform for Umbrea. For this experiment HP1-deficient third-instar larvae were used and the result is not consistent with experiments of Greil (2007). Greil used RNAi to reduce HP1 levels. They found that chromosomal localization of HP6 (identical to Umbrea) was only subtly affected by HP1 depletion. It is speculated that residual low amounts of HP1 after RNAi might be sufficient for Umbrea binding to chromatin (Joppich, 2009).
Umbrea binding along the arms of polytene chromosomes seems to be unaffected by Hip depletion. Given the interaction of Umbrea with both Hip and HP1, it is likely that the Umbrea/HP1 interaction is sufficient to target Umbrea to chromatin in the absence of Hip. In turn, this seems to be the case for HP1 and Hip. Their binding appeared to be unaffected after RNAi-induced Umbrea depletion. In contrast, Umbrea association with chromocentre heterochromatin depends on Hip. Different requirements of Hip for Umbrea association with chromocentre and chromosomal arms suggest occurrence of heterochromatin protein complexes of different composition that differentially regulate the assembly of Umbrea-containing complexes. Taking these findings together, it is speculated that HP1 is a key player for heterochromatin targeting and serves as an essential binding platform for chromatin localization of Hip and Umbrea and many other proteins (Joppich, 2009).
The Drosophila HOAP and HP1 proteins are stable components of telomeres and both proteins specifically interact with each other (Shareef, 2001; Badugu, 2003). Cytogenetic studies revealed that Umbrea also localizes to telomeres. However, 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 retrotransposon array of He-T-A/TAHRE/TART elements; and the subterminal TAS repeats. Protein attachment to telomeric structures is not sufficient to establish that a protein is a component of the cap. Thus, from cytogenetic analyses it is not possible to assign Umbrea localization to one of the three domains in telomere ends of polytene chromosomes. But given the association of HP1 and HOAP with the cap region and the direct protein interaction of Umbrea with both HP1 and HOAP, it is speculated that Umbrea also localizes to the cap region (Joppich, 2009 and references therein).
Mutations in Su(var)2-5 and cav cause extensive telomere-telomere fusions, indicating that the encoded proteins are essential for telomere stability and required for telomere capping and telomere fusion protection. It was also shown that Umbrea physically interacts not only with HP1 but also with HOAP. Cytogenetic studies revealed that Umbrea is a component of all telomeres. On the basis of these results, a similar telomeric function for Umbrea was expected. However, cytological analysis of larval brain cells displayed neither end-to-end attachments of metaphase chromosomes nor abnormal metaphase configurations. For analysis of mutant brain cells different approaches were used. The lethality of the umbrea P-element mutant line did not allow cytological analysis since homozygous animals die early during embryogenesis. In another approach, progeny of an umbrea specific RNAi line under the control of UAS were examined in combination with different neuronal and ubiquitous Gal4 driver lines. Again, lethality precludes mutant characterization of metaphase chromosomes. Interestingly, the RNAi-induced depletion of Umbrea in salivary glands reveals a mutant phenotype. Frequent telomere-telomere attachments were found in polytene nuclei. Given the localization of Umbrea at telomeres and the interaction of Umbrea with the telomere-associated proteins HP1 and HOAP, this result is not really surprising at first glance. However, the mechanisms by which telomeres attach to each other in polytene nuclei are not currently understood. It is speculated that mitotic and polytene chromosomes have different mechanisms of telomere protection. In polytene chromosomes, telomere associations depend largely on the length of the retrotransposon arrays. On the other hand, in contrast to mitotic chromosomes, defects in the cap protein structure have not been shown to modify the frequencies of polytene telomere fusions. Important differences observed between the polytene and mitotically dividing cells are speculated to be due to the fact that salivary gland differentiation and transition from mitotic divisions to endocycles takes place in early embryogenesis. In this respect, maternally contributed HP1 from heterozygous Su(var)2-5 mutants is still sufficient to suppress telomeric fusions. However, a different RNAi-mediated approach was used to deplete Umbrea using the early embryonic driver line G61. Given the observed telomere-telomere fusion of polytene chromosomes, it is speculated that the fusion potential depends critically on the onset of Umbrea protein reduction (Joppich, 2009).
It is known that mutations in Su(var)2-5 cause both telomere fusion and telomere retrotransposon elongation. Ultimately, on the basis of umbrea-specific RNAi analyses, the telomere fusion cannot be attributed to defects in the protein cap structure or to the presence of excessive retrotransposon arrays. It might even be possible that Umbrea, like HP1, exhibits functions in both mechanisms. However, the results clearly indicate that umbrea elicits a phenotype similar to that observed in mutants in the HP1- and HOAP-encoding genes cav and Su(var)2-5. It is assumed that Umbrea, together with HP1 and HOAP (and perhaps numerous additional proteins), forms a telomere-capping complex and is required for telomere function (Joppich, 2009).
HP1 associates with heterochromatin, telomeres and multiple euchromatic sites. It is speculated that the different locations of HP1 are related to multiple different functions. Umbrea is located not only at telomeres but also in the pericentric heterochromatin, at regions along the euchromatic arms and, interestingly, in the nucleolus. Given these different positions, it is assumed that the function of Umbrea is not limited to telomeres. The gene umbrea is essential for normal development since both the umbrea P-element mutant and RNAi depletion of Umbrea are lethal. Further studies are required for understanding the function of the chromo shadow domain protein Umbrea and its relationship with other heterochromatin binding proteins (Joppich, 2009).
HOAP (HP1/ORC-associated protein) has recently been isolated (Shareef, 2001) from Drosophila melanogaster embryos as part of a cytoplasmic complex that contains heterochromatin protein 1 (HP1) and the origin recognition complex subunit 2 (ORC2). This study shows that caravaggio, a mutation in the HOAP-encoding gene, causes extensive telomere-telomere fusions in larval brain cells, indicating that HOAP is required for telomere capping. These analyses indicate that HOAP is specifically enriched at mitotic chromosome telomeres, and strongly suggest that HP1 and HOAP form a telomere-capping complex that does not contain ORC2 (Cenci, 2003).
caravaggio (cav) was identified in the course of an extensive screen for mutations that affect Drosophila chromosome behaviour. Flies homozygous for the cav mutation die at the larval/pupal boundary, allowing cytological analysis of larval brain cells. DAPI-stained metaphases from squashes of colchicine-treated cav brains displayed frequent end-to-end attachments (TAs) that involve all Drosophila telomeres. Approximately 99% of cav mutant metaphases had at least one TA, with a mean number of four TAs per cell. Brain preparations from larvae bearing cav over Df(3R)crb-F89-4, a deficiency that removes cav+, display a frequency of telomeric fusions comparable with that in cav homozygotes (4.17 fusions per cell), suggesting that cav is a functionally null mutant. cav mutant brains also exhibit both polyploid metaphases (21/310) and metaphases with chromosome rearrangements (15/310), which are probably the consequence of unresolved chromatin bridges. These results indicate that cav elicits a cytological phenotype similar to that observed in mutants in the HP1-encoding gene Su(var)2-5 (Cenci, 2003).
Deficiency mapping showed that cav is uncovered by Df(3R)crb-F89-4. This deficiency removes the anonymous fast evolving 1G5 (anon fe 1G5) gene, which encodes HOAP (Shareef, 2001). The amino-terminal part of HOAP contains a region that is similar to sequence-specific HMG proteins, whereas its carboxyl terminus contains three copies of a novel repeated sequence. Given that HP1 and HOAP form a complex (Shareef, 2001), it was asked whether cav was the HOAP-encoding gene. Thus, flies were constructed that were homozygous for cav and carried an anon fe 1G5 transgene. This transgene rescued both lethality and the telomere fusion phenotype of cav homozygotes. Moreover, sequencing of the cav mutant gene revealed a 5 base pair deletion that generates a truncated HOAP protein. Western blotting with an anti-HOAP antibody demonstrated that this truncated protein is stable and can be detected in cav mutants (Cenci, 2003).
Previous analysis with an anti-HOAP antibody showed that the protein is enriched at polytene chromosome telomeres but undetectable at mitotic telomeres (Shareef, 2001). Given that HOAP seems to be required for telomere capping, immunostaining of mitotic chromosomes was repeated with the same antibody, buta different fixation technique was used. This analysis identified a specific enrichment of HOAP at all mitotic telomeres. The interphase nuclei of brain cells exhibit several discrete HOAP positive foci, which probably correspond to interphase telomeres. Examination of cav brains immunostained for HOAP demonstrated that HOAP does not accumulate at either telomeres or interphase nuclei. These results indicate that the truncated form of HOAP present in cav mutants is unable to accumulate at telomeres, supporting the conclusion that cav is functionally null (Cenci, 2003).
Next, it was asked whether binding of HOAP at telomeres requires a specific telomeric sequence. Drosophila telomeres contain several copies of HeT-A and TART retrotransposons, which specifically transpose to chromosome ends. These sequences are not essential for Drosophila telomere stability, as terminally deleted X chromosomes can be transmitted over many generations without re-acquiring telomeric transposons. To assess whether HOAP binding at telomeres requires HeT-A and TART, four different terminally deleted X chromosomes (y RT85, y RT184, y RT814 and y RT852), were used that lack HeT-A and TART sequences at their XL termini). Immunostaining for HOAP showed that the XL ends of all four terminally deleted chromosomes do not differ from their wild-type Oregon-R counterpart in relation to the intensity of HOAP staining. Thus, HOAP, as shown previously for HP1 (Fanti, 1998), binds to the ends of Drosophila chromosomes in a sequence-independent manner (Cenci, 2003).
To determine the functional relationships between HOAP and HP1, Su(var)2-504/Su(var)2-505 mutant brains were immunostained for HOAP. Previous studies have shown that this hetero-allelic combination is equivalent to a null mutation. In HOAP-stained wild-type metaphases, the percentage of labelled telomeres is 98%. In Su(var)2-504/Su(var)2-505 mutant brains, 70% of telomeres not involved in TAs have a clear HOAP signal, whereas only 15% of telomeres involved in TAs have a HOAP signal. This is consistent with a role for HOAP in mitotic telomere capping and indicates that HP1 is not absolutely required for HOAP localization at mitotic chromosome ends. HP1 immunostaining of cav mitotic chromosomes could not be performed, because HP1 binds at many sites along mitotic euchromatin, preventing unambiguous recognition of telomeric signals. Thus, it is currently unknown whether HOAP is required for localization of HP1 at mitotic telomeres (Cenci, 2003).
Although HOAP, HP1 and ORC2 form a cytoplasmic complex in early Drosophila embryos, several data indicate that ORC2 is not involved in telomere capping. First, immunolocalization studies have shown that ORC2 is not enriched at telomeres. Second, immunostaining of brain preparations from two different orc2 mutants, l(3)k43 and l(3)b51, has shown that HOAP normally localizes at mitotic telomeres. Finally, previous studies have not reported telomeric fusions in orc2 mutant brains; these results were confirmed by re-examining brain preparations of l(3)k43 and l(3)b51 mutants. These observations indicate that ORC2 is not required for telomere capping and suggest that HOAP and HP1 form a telomere-capping complex that does not contain ORC2 (Cenci, 2003).
The specific enrichment of HOAP at all Drosophila telomeres prompted an exploration of the mechanisms underlying HOAP accumulation at chromosome ends. Studies in mammalian cells have led to the view that telomere proteins are recruited to chromosome ends through interactions with TRF1 and TRF2 proteins, which specifically bind telomeric TTAGGG repeats. Gel mobility shift assays have shown that HOAP can bind different types of double-stranded DNA in vitro (Shareef, 2001), suggesting that HOAP can bind telomeric DNA in vivo. However, both the current results and previous findings strongly suggest that the nucleoprotein complexes comprising Drosophila telomeres can assemble independently of telomeric DNA sequence. Thus, it is suggested that HOAP is tethered to chromosome ends through interactions with an unknown telomere-binding factor. An intriguing possibility is that HOAP is recruited to chromosome ends through interactions with DNA end-binding proteins, such as the Ku or the Mre11 complex. The protein components of these DNA repair complexes are conserved in flies (Hopfner, 2002) and have been shown to be essential for telomere maintenance both in yeast and mammals (Cenci, 2003).
Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. This study shows direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain). This study found heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin (Badugu, 2003; full text of article).
Search PubMed for articles about Drosophila HOAP
Badugu, R., Shareef, M. M. and Kellum, R. (2003). Novel Drosophila heterochromatin protein 1 (HP1)/origin recognition complex-associated protein (HOAP) repeat motif in HP1/HOAP interactions and chromocenter associations. J. Biol. Chem. 278(36): 34491-8. PubMed ID: 12826664
Bi, X., Wei, S. C. and Rong, Y. S. (2004), Telomere protection without a telomerase; the role of ATM and Mre11 in Drosophila telomere maintenance. Curr. Biol. 14: 1348-1353. PubMed ID: 15296751
Cenci, G., Siriaco, G., Raffa, G. D., Kellum, R. and Gatti, M. (2003). The Drosophila HOAP protein is required for telomere capping. Nat. Cell Biol. 5: 82-84. PubMed ID: 12510197
Cenci, G., Ciapponi, L. and Gatti, M. (2005). The mechanism of telomere protection: A comparison between Drosophila and humans. Chromosoma 114: 135-145. PubMed ID: 16012858
Ciapponi, L., Cenci, G. and Gatti, M. (2006). The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability. Genetics 173: 1447-1454. PubMed ID: 16648644
Diede, S. J. and Gottschling, D. E. (2001). Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere. Curr. Biol. 11: 1336-1340. PubMed ID: 11553326
Dubruille, R., et al. (2010). Specialization of a Drosophila capping protein essential for the protection of sperm telomeres. Curr. Biol. 20(23): 2090-9. PubMed ID: 21093267
Fanti, L., Giovinazzo, G., Berloco, M. and Pimpinelli, S. (1998). The heterochromatin protein 1 prevents telomere fusions in Drosophila. Mol. Cell 2: 527-538. PubMed ID: 9844626
Gao, G., McMahon, C., Chen, J. and Rong, Y. S. (2008). A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila. Proc. Natl. Acad. Sci. 105: 13999-14004. PubMed ID: 18772376
Gao, G., Bi, X., Chen, J., Srikanta, D. and Rong, Y. S. (2009). Mre11-Rad50-Nbs complex is required to cap telomeres during Drosophila embryogenesis. Proc Natl Acad Sci 106(26): 10728-33. PubMed ID: 19520832
Gao, G., Walser, J. C., Beaucher, M. L., Morciano, P., Wesolowska, N., Chen, J. and Rong, Y. S. (2010). HipHop interacts with HOAP and HP1 to protect Drosophila telomeres in a sequence-independent manner. EMBO J. 29(4): 819-29. PubMed ID: 20057353
Goudsouzian, L. K., Tuzon, C. T., Zakian, V. A. (2006). S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association. Mol. Cell 24: 603-610. PubMed ID: 17188035
Greil, F., de Wit, E., Bussemaker, H. J., van Steensel, B. (2007). HP1 controls genomic targeting of four novel heterochromatin proteins in Drosophila. Embo J. 26: 741-751. PubMed ID: 17255947
Hopfner, K.-P., Putnam, C. D. and Tainer, J. A. (2002). Curr. Opin. Struct. Biol. DNA double-strand break repair from head to tail. 12: 115-122. PubMed ID: 1183949
Joppich, C., Scholz, S., Korge, G. and Schwendemann, A. (2009). Umbrea, a chromo shadow domain protein in Drosophila melanogaster heterochromatin, interacts with Hip, HP1 and HOAP. Chromosome Res. 17(1): 19-36. PubMed ID: 19190990
Karlseder, J., et al. (2004). The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response. PLoS Biol. 2: E240. PubMed ID: 15314656
Khurana, J. S., Xu, J., Weng, Z. and Theurkauf. W. E. (2010). Distinct functions for the Drosophila piRNA pathway in genome maintenance and telomere protection. PLoS Genet. 6(12): e1001246. PubMed ID: 21179579
Komonyi, O., Schauer, T., Papai, G., Deak, P., Boros, I. M. (2009). A product of the bicistronic Drosophila melanogaster gene CG31241, which also encodes a trimethylguanosine synthase, plays a role in telomere protection. J. Cell. Sci. 122: 769-74. PubMed ID: 19240120
Li, H., et al. (2011). Cooperative and antagonistic contributions of two heterochromatin proteins to transcriptional regulation of the Drosophila sex determination decision. PLoS Genet. 7(6): e1002122. PubMed ID: 21695246
Loppin, B. et al. (2005). Origin and neofunctionalization of a Drosophila paternal effect gene essential for zygote viability. Curr. Biol. 15: 87-93. PubMed ID: 15668163
Lydall, D. (2009). Taming the tiger by the tail: modulation of DNA damage responses by telomeres. EMBO J. 28: 2174-2187. PubMed ID: 19629039
Musarò, M., Ciapponi, L., Fasulo, B., Gatti, M. and Cenci, G. (2008). Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint. Nat. Genet. 40(3): 362-6. PubMed ID: 18246067
Negrini, S., Ribaud, V., Bianchi, A. and Shore, D. (2007). DNA breaks are masked by multiple Rap1 binding in yeast: implications for telomere capping and telomerase regulation. Genes Dev. 21: 292-302. PubMed ID: 17289918
Oikemus, S. R., et al. (2006). Epigenetic telomere protection by Drosophila DNA damage response pathways. PLoS Genet. 2: e71. PubMed ID: 16710445
Palm, W, and de Lange, T. (2008), How shelterin protects mammalian telomeres. Annu. Rev. Genet. 42: 301-334. PubMed ID: 18680434
Raffa, G. D., et al. (2005). The putative Drosophila transcription factor woc is required to prevent telomeric fusions. Mol. Cell 20(6): 821-31. PubMed ID: 16364909
Raffa, G. D., et al. (2009). The Drosophila modigliani (moi) gene encodes a HOAP-interacting protein required for telomere protection. Proc. Natl. Acad. Sci. 106: 2271-2276. PubMed ID: 19181850
Rong, Y. S. (2008a). Telomere capping in Drosophila: dealing with chromosome ends that most resemble DNA breaks. Chromosoma 117: 235-242. PubMed ID: 18193446
Rong, Y. S. (2008b). Loss of the histone variant H2A.Z restores capping to checkpoint-defective telomeres in Drosophila. Genetics 180: 1869-1875. PubMed ID: 18845840
Schwendemann, A., et al. (2008). Hip, an HP1-interacting protein, is a haplo- and triplo-suppressor of position effect variegation. Proc. Natl. Acad. Sci. 105: 204-209. PubMed ID: 18162556
Shareef, M. M., et al. (2001). Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing. Mol. Biol. Cell 12: 1671-1685. PubMed ID: 11408576
Villasante, A., de Pablos, B., Méndez-Lago, M. and Abad, J. P. (2008). Telomere maintenance in Drosophila: rapid transposon evolution at chromosome ends. Cell Cycle 7: 2134-2138. PubMed ID: 18635962
Zhu, X. D., Kuster, B., Mann, M., Petrini, J. H. and de Lange, T. (2000). Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat. Genet. 25: 347-352. PubMed ID: 10888888
date revised: 17 August 2012
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