InteractiveFly: GeneBrief

HP1-HOAP-interacting protein: Biological Overview | References |

Drosophila chromosomes are elongated by retrotransposon attachment, a process poorly understood. This study characterized a mutation affecting the HipHop telomere-capping protein. In mutant ovaries and the embryos that they produce, telomere retrotransposons are activated and transposon RNP accumulates. Genetic results are consistent hiphop mutation weakening the efficacy of HP1-mediated silencing while leaving piRNA-based mechanisms largely intact. Remarkably, mutant females display normal fecundity suggesting that telomere de-silencing is compatible with germline development. Moreover, unlike prior mutants with overactive telomeres, the hiphop stock does not over-accumulate transposons for hundreds of generations. This is likely due to the loss of HipHop's abilities both to silence transcription and to recruit transposons to telomeres in the mutant. Furthermore, embryos produced by mutant mothers experience a checkpoint activation, and a further loss of maternal HipHop leads to end-to-end fusion and embryonic arrest. Telomeric retroelements fulfill an essential function yet maintain a potentially conflicting relationship with their Drosophila host. This study thus showcases a possible intermediate in this arms race in which the host is adapting to over-activated transposons while maintaining genome stability. These results suggest that the collapse of such a relationship might only occur when the selfish element acquires the ability to target non-telomeric regions of the genome. HipHop is likely part of this machinery restricting the elements to the gene-poor region of telomeres. Lastly, the hiphop mutation behaves as a recessive suppressor of PEV that is mediated by centric heterochromatin, suggesting its broader effect on chromatin not limited to telomeres (Cui, 2021).

Transposable elements (TEs) are omnipresent in eukaryotic genomes. They are primarily viewed as a threat to the host organism as TE insertions can disrupt gene expression and function, or induce secondary genome instability as a result of illegitimate recombination. The presence of certain TEs is nevertheless beneficial to the host, and one of the best examples concerns telomeres in the model of Drosophila, where chromosome ends are populated by telomere specific retrotransposons. These elements serve to elongate chromosome ends thus counteracting sequence loss due to incomplete end replication. In Drosophila melanogaster, three classes of non-LTR type retrotransposons make up telomeric DNA: HeT-A, TART and the HeT-A-related TAHRE, with HeT-A being the most abundant class. HeT-A has a single open-reading-frame (orf) encoding a Gag-like protein (Orf1p) but lacks the accompanying orf2, encoding the reverse transcriptase found in typical non-LTR elements. This suggests that HeT-A lacks the ability to transpose on its own. TART and TAHRE each carry both orfs. Whether they control HeT-A transposition in addition to their own is not known. Full-length elements are not found elsewhere in the genome suggesting that their transposition is limited to chromosome ends. Remarkably, these elements have the ability to attach to a chromosome end that does not terminate on one of the normal telomeric transposons. How this remarkable target specificity is achieved remains poorly understood. In an earlier study, it was showed that the Orf1p protein from HeT-A is present in the nucleus of somatic cells in S phase, and forms large spherical structures that are attached to one and sometimes multiple chromosome ends. These 'HeT-A Spheres' consist of a proteinaceous shell made of Orf1p that encapsulates sense transcripts from HeT-A. It is speculated that HeT-A Sphere represents an intermediate in the molecular events leading to HeT-A transpositions (Cui, 2021).

In addition to serving a function of timely elongation of chromosome ends, telomere performs another essential function: to prevent the recognition and illicit repair of telomeres as broken DNA ends. This capping function relies on protein complexes that are specifically enriched on telomeric chromatin: the capping complex. In Drosophila, two potentially separate complexes essential for the prevention of telomere fusion have been identified. First, occupying primarily the double stranded portion of telomeric chromatin is the HipHop-HOAP complex, which includes the previously identified HOAP protein. HipHop-HOAP may also recruit Heterochromatin Protein 1 (HP1) to telomeres for transcriptional regulation. In addition, telomeres end in a 3' overhang in most systems studied. In Drosophila, a second Moi-Tea-Ver (MTV) complex is proposed to occupy this overhang based on in vitro results. MTV includes the Tea protein, and the Moi and Ver proteins which were previously identified. A previous study showed that the Ver subunit of MTV, and quite possibly the other two, is required for HeT-A Sphere formation on telomeres (Zhang, 2014), and it was proposed that this single-strand binding complex participates in the recruitment of transposon RNP to Drosophila telomeres (Zhang, 2016). This would be similar to the recruitment of telomerase by the CST complexes in other organisms. Therefore, Drosophila capping machinery likely limits TE attachment only to telomeres, setting up a potential conflict between the two entities. The group of proteins enriched at Drosophila telomeres have been collectively called 'Terminin', similar to the concept of 'Shelterin' proposed for telomerase-maintained systems (Cui, 2021).

Transcriptional regulation is another layer of control placed on telomeric TEs by the Drosophila host. Although transposon transcripts and proteins were readily detectable in proliferating tissues in the soma, their presence is very low in the germline owing at least partially to the transcriptional regulation mediated by piRNAs. In piRNA mutant germlines, transcripts from telomeric TEs are overproduced just as the other TEs genome wide. These mutations commonly result in the loss of female fertility. In cases where limited fertility was allowed so that mutant stocks were maintained for many fly generations, an increase of transposon copy numbers at telomeres has been reported. These results suggest that retrotransposon overexpression might be sufficient for 'excess' telomere accumulation of retro-elements. However, there are also cases in which telomere over-elongation was not associated with apparent de-silencing at telomeres. A recent survey of copy number variations in laboratory stocks identified a large range of telomeric array sizes under seemingly wildtype backgrounds, confounding the notion that extra copies of telomeric elements might be detrimental to the host. Moreover, converting fitness loss in the laboratory setting to that in nature has always been difficult (Cui, 2021).

Drosophila telomeres display signs of accelerated evolution. First, telomere arrays not only display copy number variations but also a high degree of sequence diversity. In the most extreme cases, telomeres can lose all the telomeric elements, either naturally or artificially, and still retain a normal capping function. Moreover, most if not all components of the capping complexes that are specifically enriched at chromosome ends are poorly conserved at the primary sequence level and show signs of fast protein evolution among Drosophilids. It is plausible that the fast evolution of telomeric proteins is driven by the highly diverse DNA sequences underlying fly telomeres. However, since the function of the capping complex requires no DNA sequence component, it is difficult to formulate a model in which highly variable DNA sequences drive the evolution of proteins that bind to them. In addition, the difficulty in extracting any fitness consequence in stocks with extra-long telomeres seems to suggest that the host plays a passive role in the evolutionary arms race with the TEs. How the host capping complexes limit telomere TEs propagation is largely unclear since strong loss of their functions invariably impair viability. Recently, Saint-Leandre et al. (Saint-Leandre, 2020) conducted the first study in which a replacement of HOAP protein of D. melanogaster with one from its close relative D. yakuba resulted in transposon de-repression in the germline (Cui, 2021).

This study showed that a novel hiphop mutation in D. melanogaster leads to a specific de-repression of telomeric elements in the germline via a mechanism that is largely independent of the piRNA-mediated pathway but possibly dependent on HP1 functions. Remarkably, this de-repression did not result in 'extra-long' telomeres, most likely because the mutation simultaneously makes HipHop less effective in recruiting the elements to chromosome ends. Although this study was primarily designed to investigate the mechanisms by which the capping machinery regulates telomeric transposons, the results nevertheless shed new insights into the evolutionary relationship between the host and the TEs. It is speculated that the ability of the capping machinery to target all transpositions to chromosome ends represents the most important weapon in the host's response to the presence of these active transposons (Cui, 2021).

Telomere in Drosophila serves as an excellent model for the study of evolutionary forces that balance the fitness cost of having active TEs with the essential function that these elements fulfill for the host organism. At the interface of this interaction lies the capping complex, the host machinery in direct contact with the TEs, whose subunits are prime candidates through which the host exerts its control. However, it has been difficult to assess the role of capping proteins in transposon regulation, particularly in the germline, as a strong loss of the capping function results in lethality due to end-to-end fusion in somatic cells. An ideal situation for such a study is the availability of separation-of-function mutations that support one but not the other function of the complex. Whether the hiphop^HA mutation represents a true separation-of-function mutation is debatable. It nevertheless supports viability and fertility allowing a study of the effects of transposon hyperactivation on germline and embryonic development, and inference of host mechanisms that mitigate the effect of having active transposons in the genome (Cui, 2021).

Similar to TEs in other parts of the Drosophila genome, telomeric TEs are under the piRNA-mediated surveillance in the germline in that TE transcripts serve as precursors for the biogenesis of small RNAs targeting the elements themselves. These small RNAs and their associated protein factors control the level of transposon RNAs via two major mechanisms: by directing RNA degradation in the cytoplasm or by maintaining a specialized chromatin structure in cis for transcriptional silencing. This second mode of regulation involves the HP1 proteins at telomeres. Given the fact that HipHop is predominantly a telomeric protein, it is likely that the defect(s) leading to transposon de-repression in hiphop^HA germline is in cis such as an altered chromatin state at the telomeres. The genetic results support such a proposition in that a reduction of HP1 dosage further aggravates the silencing defects (Cui, 2021).

The results however beg the question of what role the piRNA surveillance plays in transposon silencing under the mutant background. The fact that non-telomeric elements are not de-repressed in hiphop mutant ovaries implies that the piRNA mechanisms are fully functional. How could the excess telomeric transcripts escape the surveillance of a functional piRNA machinery? It is possible that the extent of telomeric overexpression exceeds the capacity of piRNA-mediated transcript degradation. This is considered unlikely on the grounds that that telomeric de-repression under a piRNA-mutant background is much greater than that in the hiphop mutant germline. Therefore, the capacity of piRNA mediated RNA degradation is not likely to have reached its capacity in the mutant. Alternatively, piRNA production might be defective in the mutant as suggested by a genetic interaction study with the rhino mutation. Since this proposed defect is unlikely due to the lack of telomeric transcription per se, it is suggested that the aberrant telomeric transcripts might not be conducive to piRNA production, or that these aberrant transcripts are themselves resistant to piRNA mediated degradation. This hypothesis is not favored, however, since some of the HeT-A transcripts are functional in producing the Orf1p protein (Cui, 2021).

A common organismal phenotype associated with transposon de-repression is the loss of female fertility as demonstrated in many studies of piRNA and related pathways. As transposon RNPs are deposited into the egg, lethality of the resulting embryos has also been reported. Since de-repression happens genome-wide and to different classes of elements in these earlier studies, it has been difficult to determine whether any particular class of elements exerts a disproportionally large effect on organismal fitness, or whether the mere presence of excessive amounts of RNA and protein molecules is sufficient to impose the detrimental effect (Cui, 2021).

This study showed that hiphop-mutant ovaries specifically accumulate a large amount of telomeric transcripts and the Orf1p protein from HeT-A. Therefore, the specific effects of highly active telomeres on development could be studied. Despite telomere hyperactivation in the germline, mutant females laid normal number of fertilized eggs. These results are consistent with ones reported earlier suggesting that telomere specific de-repression does not have a catastrophic effect on germline development (Cui, 2021).

Contrary to the lack of effect on female fecundity, the presence of excess telomeric RNPs is associated with defects in embryonic development, and those defects can be rescued partially with a chk2 mutation suggesting that they were due to checkpoint activation. However, checkpoint activation by excessive transposon RNPs does not necessarily result in embryonic lethality since over 80% of the embryos laid by hiphop^HA homozygous mothers hatch (normalized over the hatching rate from the heterozygotes) even though they too have inherited abundance of transposon RNPs. Therefore, telomere specific de-repression does not necessarily have a catastrophic effect on embryonic development either, suggesting that telomeric over-activation is well tolerated and more sensitive methods are needed to uncover its effects on organismal fitness (Cui, 2021).

Contrary to hiphop^HA homozygous females that have good fertility, majority of embryos laid by hiphop^HA hemizygous mothers arrest very early during development with defects in chromosome segregation. Telomere fusion was identified as likely the major cause for this embryonic lethality, which was based on two independent assays of 'fusion PCR' and 'mitotic chromosome squash'. This contrasts with the lack of capping defects in somatic tissues of the same mutant, which suggests that the HipHop^HA protein retains sufficient capping function in post-embryonic somatic cells but not during the rapid embryonic cycles. Earlier work showed that hypomorphic mutations in the Mre11-Rad50-Nbs complex or the ATM checkpoint kinase supports normal somatic development but causes severe uncapping in early embryos. These results are consistent with the propositions that the syncytial cycles place an exquisite requirement on telomere capping, and that hiphop^HA is a hypomorphic mutation for telomere capping (Cui, 2021).

It is considered unlikely that telomere uncapping in hiphop-embryos is secondary to the primary defect of having hyperactive telomeres. Based on limited cytological evidence, it has been previously claimed that a high level of telomeric transcripts is accompanied by telomere fusions in embryos functionally defective for piRNA, or for the degradation of telomeric transcripts. It is difficult to envision how an overabundance of telomeric RNA induces uncapping since the source of these RNAs is primarily maternal. If there were wide-spread telomere uncapping in those transcriptionally inert embryos, it might be more likely caused by the reduction of maternal factors essential for the loading of capping complexes. (Cui, 2021).

The capping complex could be intimately involved in transcriptional regulation by maintaining a chromatin structure at telomeres, and a common chromatin feature may be essential for both transcription regulation and end capping. In support of this model are the findings that mutations in HP1 impair both processes, and that the HipHop-HOAP complex at telomeres contains HP1. However, the two chromatin domains, one for end protection and the other for transcriptional silencing are not identical. First of all, it has been shown that different telomeric domains support different transcriptional activities from the same transgene. Moreover, it has been reported that the size of the telomeric array can vary greatly, up to a 288-fold range in laboratory stocks. Therefore, the machinery essential for transcription regulation likely has a wider and more variable distribution on telomeres than the capping complex, which is limited to the more immediate vicinity of the actual chromosome ends. For example, HP1 and other transcriptional regulators might be initially recruited to chromosome ends by their interactions with the capping complex, and subsequently spread to neighboring regions for transcriptional silencing. In the case of a partial loss of the capping proteins, fewer silencing proteins might be recruited to the ends resulting in regions of the transposon arrays lacking silence chromatin. On the other hand, the level of capping proteins might be sufficient to cover a much smaller region still ensuring normal end protection (Cui, 2021).

Therefore, it is conceivable that both transposon de-repression and embryonic uncapping are caused by the same partial loss of HipHop function in the mutant. Based on this proposition a model was raised that is consistent with the empirical data presented in this study. The model assumes that the silencing function is more sensitive to the perturbation of HipHop, and it predicts that no individual mutation would be defective in capping but proficient in transposon silencing. Such a prediction might be tested with a systematic mutagenesis of hiphop or cav. Since end capping might be considered as a more conserved function of HipHop or HOAP, and since neither the viable cav allele reported by Saint-Leandre (2020) nor the hiphop_HA allele entails replacement of highly conserved residues in the protein sequences, it is predicted that separation-of-function alleles likely arise from changes of these more conserved residues, a direction that is being actively pursuing (Cui, 2021).

There does not appear to be an optimal length of telomeric arrays in Drosophila as mounting evidence suggest that Drosophila melanogaster can sustain a large variation in copy numbers of these elements. Therefore, 'extra-long' telomeric arrays do not necessarily pose a grave fitness cost in flies. On the other hand, a telomeric element would be a significant detriment to host fitness had it gained the ability to insert elsewhere in the genome. Restricting the insertional site, but not the expression level, of these elements might represent the most important tool that the host evolves to balance the potential cost of having active transposons. This proposition is prompted by the remarkable finding that homozygous stocks of hiphop^HA does not accumulate longer telomeric arrays over generations even in the continuing presence of excess amount of transposon RNPs. This contrasts with the outcome in previous examples with a similar condition of telomere hyperactivation. The results suggest that the same hiphop mutation that leads to telomeric de-repression also suffers a partial loss in its ability to recruit the machinery needed for transposition (Cui, 2021).

The mechanism for transposon targeting to chromosome ends remains poorly understood for the Drosophila elements. Prior study suggests a multi-faceted mechanism that directs the formation of an elaborate structure of transposon RNP, which is both cell-cycle regulated and coincides with on-going telomeric transcription. More importantly, this study has provided the first line of evidence that the host capping complex is intimately involved in transposon recruitment to the ends (Cui, 2021).

How is it then that the elements unable to land at the telomere in hiphop^HA mutants also do not insert somewhere else in the genome? It has been proposed that Drosophila telomeric elements are actively targeted to a gene-poor region of the genome. In other similar cases of transposon targeting, host factors are known to be essential for the process as expected. For examples, the Ty elements in budding yeast target RNA pol III transcribed tRNA genes by interacting with subunits of the polymerase complex. The fission yeast Tbf1 elements are targeted to genomic regions specifically bound by the host Sap1 protein. Remarkably, loss of Sap1 drastically reduces Tbf1 transposition efficiency suggesting that Sap1 not only targets but is itself necessary for Tbf1 transpositions. It is speculated that HipHop plays a similar role for the transposition of telomeric elements in Drosophila so that most of the excess transcripts in a hiphop^HA mutant germline are unable to insert anywhere in the genome due to the same partial loss of HipHop function. The model predicts that the same hiphop^HA mutation should also prevent over-elongation of telomeres under another genetic background that is permissive to over-elongation, e.g., the Tel background. Unfortunately, a combination of hiphop^HA and Tel causes female sterility complicating the test of the hypothesis (Cui, 2021).

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

Telomeres prevent chromosome ends from being repaired as double-strand breaks (DSBs). Telomere identity in Drosophila is determined epigenetically with no sequence either necessary or sufficient. To better understand this sequence-independent capping mechanism, proteins were isolated that interact with the HP1/ORC-associated protein (HOAP) capping protein, and HipHop was identified as a subunit of the complex. Loss of one protein destabilizes the other and renders telomeres susceptible to fusion. Both HipHop and HOAP are enriched at telomeres, where they also interact with the conserved HP1 protein. A model telomere lacking repetitive sequences was developed to study the distribution of HipHop, HOAP and HP1 using chromatin immunoprecipitation (ChIP). It was discovered that they occupy a broad region >10 kb from the chromosome end and their binding is independent of the underlying DNA sequence. HipHop and HOAP are both rapidly evolving proteins yet their telomeric deposition is under the control of the conserved ATM and Mre11-Rad50-Nbs (MRN) proteins that modulate DNA structures at telomeres and at DSBs. 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 D4A^TD 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 D4A^TD. 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 D4A^TD versus its homologous telomere is observed in immunostaining experiments. Similar observations were documented for HP1 on polytene and HOAP on mitotic telomeres using TDs (Gao. 2010).

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

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

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

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

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

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

Previous studies reported that MRN is important for the telomere localization of HOAP and telomere stability. Therefore, MRN is thought to be involved in HOAP telomere recruitment. However, whether MRN interacted with HOAP was not clear (On, 2021).

This study reports that the MRN components, Mre11 and Nbs, interact with HOAP. HOAP is known for binding to terminal double-strand DNA (dsDNA) regions of the telomere (Gao, 2010) while MRN binds to ends of dsDNA (Myler, 2017); therefore, both HOAP and MRN tend to associate to ends of dsDNA. Thus, there is a possibility that ends of dsDNA could act as a scaffold to promote HOAP-MRN interaction. It was also shown that the nuclear localization of Mre11-Rad50 is dependent on Nbs, but HOAP does not affect the cytoplasmic localization of Mre11 in spite of their detected interaction by IP. Therefore, it is predicted that HOAP and Mre11 interact in nucleus after Nbs-Mre11 transport from cytoplasm to nucleus. In addition, co-localized foci of Nbs-HOAP could not be detected after detergent treatment. It is suspected to be due to the weak chromatin binding of Nbs. In mammals, the accumulation of NBS1 to a DSB site is dependent on the DNA-binding activity of MRE11-RAD50 (Myler, 2017). Hence, Nbs could also bind to chromatin tightly via the interaction with the Mre11-Rad50 complex in Drosophila. Therefore, it is predicted that the telomeric foci of Nbs after detergent treatment would be detected in Mre11-Rad50-Nbs overexpressing cells. In mammals, NBS1 interacts with TRF2, a mammalian telomere duplex region binding protein, and maintains telomere-specific loop formation. It is therefore possible that, as for the interactions of HOAP-Mre11 and HOAP-Nbs, MRN also interacts with HOAP to promote loop formation in Drosophila, and the interaction of MRN-HOAP is essential for maintenance of telomere structure. In addition to HOAP, the association of HP1a at telomere regions is also reduced in the MRN mutants. HP1a is known as a heterochromatin protein and interacts with HOAP. Moreover, a recent study reports that Nbs also interacts with HP1a. The interaction that was detected between Nbs and HOAP suggests that both Nbs-HP1a and Nbs-HOAP interactions may contribute to telomere stability (On, 2021).

Nbs was shown to be essential for the nuclear transport of Mre11-Rad50. In addition, several previous studies showed that Rad50 formed nuclear foci in WT flies, but not in mre11 and nbs mutants. This strongly supports the IF results that both Mre11 and Nbs are essential for nuclear localization of Rad50. Overexpressed Rad50 was detected as huge foci in the cytoplasm. Rad50 is known to have long coiled-coil domains and form a homodimer or a heterotetramer via Zn+. In addition in Drosophila, it was reported that endogenous Rad50 formed large nuclear foci in embryos. Therefore it seems that Rad50 tends to aggregate, suggesting that overexpressed Rad50 also forms dimers and/or tetramers, which associate as huge blobs in the cytoplasm. Although it was not verified the DSB repair function of overexpressed MRN components, the results suggested that nuclear transport system and protein-protein interactions are conserved even in the overexpression experiments. Taken together, it is concluded that Nbs is essential for the nuclear localization of MRN in Drosophila. In addition, mammalian MRN uses a similar transport mechanism as Drosophila, suggesting that nuclear localization system of MRN is conserved between mammals and Drosophila (On, 2021).

The N-terminal region of HOAP is reported to be essential for its co-localization with HipHop. Although HOAP has no functional domains, several studies reported that the N-terminus of HOAP has sequence similarity with the high mobility group (HMG) box domains. The HMG-box domain is found in both sequence-dependent and sequence-independent DNA-binding domains. Therefore, the N-terminal region of HOAP is expected to be important in telomeric DNA binding. This study showed the first evidence that the N-terminal region of HOAP is necessary for its localization at putative telomeres. Moreover, the Nbs interacting site was mapped to the N-terminus of HOAP, suggesting that Nbs might regulate the DNA-binding activity of HOAP (On, 2021).

The C-terminal truncated HOAP1-286 showed clear telomere dot-signals with HipHop. However, the C-terminal region of HOAP was essential for its telomere binding. This contradiction could be explained by differences in the deletion sites used in the two studies. In an earlier study the C-terminal truncated HOAP altered the aa sequence from residue 261 and caused a stop codon at aa 281. This truncated HOAP is shorter than HOAP1-286 that was used in this study. Therefore, the C-terminal 261-281 aa region is likely to be essential for the telomere-binding function of HOAP. It was also shown that the conserved STQ (279-281) in the C-terminus of HOAP was not necessary for its telomere localization. As TRF2 is phosphorylated by ATM and ERK1/2 in mammals, the conserved STQ may be a target for serine/threonine kinases in the DDR pathway or MAPK signaling pathway (On, 2021).

In mammals, TRF2 binds telomeric DNA in a sequence-dependent manner. However, several studies recently reported that TRF2 also possesses DNA sequence-independent binding for its accumulation at DSB sites. In addition, it has been reported that the accumulation of TRF2 at DSB sites can be stabilized by the interaction of MRE11 and NBS1. Several studies have presented evidence indicating that HOAP binds to telomeres in a DNA sequence-independent manner, but no evidence has shown that HOAP is involved in the DSB repair pathway. The present finding of the interactions of HOAP-Mre11 and HOAP-Nbs suggests functional similarities between TRF2 and HOAP. They also provide new approaches to study the DNA sequence-independent binding mechanism of telomere capping proteins and suggest that Drosophila is a useful model for such studies (On, 2021).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Search PubMed for articles about Drosophila HipHop

Cicconi, A., Micheli, E., Verni, F., Jackson, A., Gradilla, A. C., Cipressa, F., Raimondo, D., Bosso, G., Wakefield, J. G., Ciapponi, L., Cenci, G., Gatti, M., Cacchione, S. and Raffa, G. D. (2016). The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response. Nucleic Acids Res 45(6):3068-3085. PubMed ID: 27940556

Cui, M., Bai, Y., Li, K. and Rong, Y. S. (2021). Taming active transposons at Drosophila telomeres: The interconnection between HipHop's roles in capping and transcriptional silencing. PLoS Genet 17(11): e1009925. PubMed ID: 34813587

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 Citation: 20057353

Kurzhals, R. L., Fanti, L., Gonzalez Ebsen, A. C., Rong, Y. S., Pimpinelli, S. and Golic, K. G. (2017). Chromosome healing is promoted by the telomere cap component Hiphop in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 28942425

Myler, L. R., Gallardo, I. F., Zhou, Y., Gong, F., Yang, S. H., Wold, M. S., Miller, K. M., Paull, T. T. and Finkelstein, I. J. (2016). Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins. Proc Natl Acad Sci U S A 113(9): E1170-1179. PubMed ID: 26884156

On, K., Crevel, G., Cotterill, S., Itoh, M. and Kato, Y. (2021). Drosophila telomere capping protein HOAP interacts with DSB sensor proteins Mre11 and Nbs. Genes Cells PubMed ID: 33556205

Saint-Leandre, B., Christopher, C. and Levine, M. T. (2020) Adaptive evolution of an essential telomere protein restricts telomeric retrotransposons. Elife 9:e60987. PubMed ID: 33350936

Zhang, L., Beaucher, M., Cheng, Y. and Rong, Y. S. (2014). Coordination of transposon expression with DNA replication in the targeting of telomeric retrotransposons in Drosophila. EMBO J. 33(10):1148-58. PubMed ID: 24733842

Zhang, Y., Zhang, L., Tang, X., Bhardwaj, S. R., Ji, J. and Rong, Y. S. (2016). MTV, an ssDNA Protecting Complex Essential for Transposon-Based Telomere Maintenance in Drosophila. PLoS Genet 12(11): e1006435. PubMed ID: 27835648

date revised: 10 October 2022

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