InteractiveFly: GeneBrief

caravaggio: Biological Overview | References

BIOLOGICAL OVERVIEW

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 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 (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 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 (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).

Cooperative and antagonistic contributions of two heterochromatin proteins to transcriptional regulation of the Drosophila sex determination decision

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, Sxl_Pe, 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 (Sxl_Pm) in both sexes. HOAP mutants show inappropriate Sxl_Pe firing in males and the concomitant inappropriate splicing of Sxl_Pm-derived transcripts, while females show premature firing of Sxl_Pe. HP1 mutants, by contrast, display Sxl_Pm splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with Sxl_Pe sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to Sxl_Pe are severely compromised. These genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at Sxl_Pe (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 Sxl_Pe, 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 Sxl_Pm-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 Sxl_Pe 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 Sxl_Pm 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 Sxl_Pe 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 Sxl_Pe. Combined, these data indicate both antagonistic and cooperative roles for these heterochromatin proteins in regulating Sxl_Pe, 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 Sxl_Pe, 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 Sxl_Pe expression, the activities of heterochromatin proteins in dosage compensation appear to be distinct from the early roles of HP1 and HOAP at Sxl_Pe. In addition, there may be zygotic roles for heterochromatin proteins in sex-specific gene expression, as proposed for HP1 (Li, 2011).

Previous analysis of Sxl_Pe 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 Sxl_Pe which contains binding sites for both positive and negative E-box proteins. Within the Sxl_Pe 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 Sxl_Pe distal region, in an HP1-independent repressing role. Whereas loss of HOAP de-represses Sxl_Pe 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. Sxl_Pe 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 Sxl_Pe in both sexes of wild type embryos. However, whether the proximal and distal Sxl_Pe 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 Sxl_Pe 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 Sxl_Pe prior to its activation. The repressive structure formed by maternal HOAP and HP1 likely serves to reduce the sensitivity of Sxl_Pe 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 Sxl_Pe 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 Sxl_Pe 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 Sxl_Pe 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 Sxl_Pe, 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 Sxl_Pe 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 Sxl_Pe 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 Sxl_Pm 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 Sxl_Pe. It is likely that the collection window for this study did not precisely coincide with the time of Sxl_Pe activity. A more narrowly timed collection indicates paused RNAP II at Sxl_Pe, suggesting that, like Sxl_Pm, it is a pre-loaded promoter. A preloaded Sxl_Pe also readily explains how generalized up-regulation of phosphorylation of the RNAP II CTD by the loss of Nanos, causes Sxl_Pe 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 Sxl_Pe. ChIP data show HOAP association with the Sxl_Pe proximal region is required for HP1 association. This proposed role for the HMG box of HOAP in Sxl_Pe 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).

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).

Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint

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 and NBN (also known as NBS1) genes; and without children (woc), whose product is a putative transcription factor (Musarò, 2008).

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).

The Drosophila modigliani (moi) gene encodes a HOAP-interacting protein required for telomere protection

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).

Specialization of a Drosophila capping protein essential for the protection of sperm telomeres

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).

Verrocchio, a Drosophila OB fold-containing protein, is a component of the terminin telomere-capping complex

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).