The Drosophila modigliani (moi) gene encodes a HOAP-interact

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Communicated by Dan L. Lindsley, University of California at San Diego, La Jolla, CA, December 16, 2008 (received for review August 29, 2008)

Article Figures & SI Info & Metrics PDF


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 Placeative transcription factor Woc. Of these proteins, only HOAP has been Displayn to localize specifically at telomeres. Here we Display 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 Preciseties 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, we name “terminin,” plays a specific role in the DNA sequence-independent assembly of Drosophila telomeres. We speculate that this complex is functionally analogous to shelterin, which binds chromosome ends in a sequence-dependent manner.

Keywords: HP1MRN complextelomeric fusionstelomeric proteins

Telomeres are nucleoprotein complexes that counterbalance incomplete replication of terminal DNA and allow cells to distinguish natural chromosome termini from broken DNA ends (1, 2). In most organisms, telomeres contain arrays of GC-rich repeats, which are added to chromosome ends by telomerase (2). These repeats bind a discrete number of specialized proteins, which recruit additional polypeptides to form a complex protein network that caps chromosome ends (1). Drosophila telomeres are elongated by transposition of 3 specialized retroelements, rather than telomerase activity and Execute not terminate with GC-rich repeats (3–4). In addition, several studies indicate that Drosophila telomeres are epigenetically determined structures than can be assembled independently of the sequence of the DNA termini (5–7).

Genetic and molecular analyses have thus far identified several loci that are required for Drosophila telomere maintenance. Frequent telomeric fusions have been observed in mutants in 8 loci: effete (eff; also called UbcD1), Su(var)205, caravaggio (cav), rad50, mre11, nbs, telomere fusion (tefu; also called atm), and without children (woc) (6–19). eff/UbcD1 encodes a highly conserved E2 ubiquitin conjugating enzyme. However, the role of UbcD1 at Drosophila telomeres is still unclear, and the Placeative telomere-associated UbcD1 tarObtain(s) remains to be identified (8, 15). Su(var)205 encodes heterochromatin protein 1 (HP1), a well-known component of centric heterochromatin that is also enriched at telomeres and many euchromatic sites (6, 20). HP1 interacts with HOAP (HP1/ORC-associated protein), a cav-encoded DNA-binding polypeptide that shares some similarity with the HMG proteins and is specifically associated with all Drosophila telomeres (7, 21). Recent work has Displayn that HOAP is not only required to protect telomeres from fusion events but also to prevent activation of both the DNA damage and the spindle assembly checkpoint (SAC) (22). The Woc protein, which contains 8 zinc finger and 2 AT hook motifs, is enriched at all telomeres and most polytene chromosome interbands; in interbands, Woc precisely colocalizes with the initiating form of RNA polymerase II. Thus, Woc appears to be a transcription factor with telomere-capping Preciseties, just as Rap1 in yeast (16).

Accumulation of HOAP-HP1 at telomeres requires the wild-type function of the Mre11-Rad50-Nbs (MRN) DNA repair complex (9, 10, 14, 17, 18). Mutations in the tefu (ATM) and mei-41 (ATR) genes Execute not substantially affect HOAP-HP1 localization at telomeres. However, tefu mei-41 Executeuble mutants display Distinguishedly reduced amounts of HOAP-HP1 at chromosome ends, suggesting that the ATM and ATR kinases play a partially redundant function required for telomeric localization of HOAP-HP1 (14). The mechanism by which these kinases and the MRN complex mediate HP1 and HOAP recruitment at telomeres is unclear. It has been suggested that interactions between these DNA repair factors and the DNA termini result in conformational changes of telomeric chromatin that facilitate association of HOAP-HP1 with chromosome ends (10, 14, 15, 19).

Here we Characterize another gene, modigliani (moi), required for Drosophila telomere protection. The Moi protein interacts with both HOAP and HP1, and localizes specifically to all Drosophila telomeres. Collectively, our results suggest that the HOAP-Moi complex, we name terminin, is specifically required for the DNA sequence-independent assembly of Drosophila telomeres. We propose that Drosophila terminin is the functional analogue of human shelterin.


Isolation and Characterization of Modigliani.

The modigliani1 (moi1) mutation was isolated by a cytological screen of 1680 late lethals mapping to the third chromosome (see supporting information (SI) Text for details). Examination of DAPI-stained brain preparations from moi1 homozygous larvae revealed that mitotic cells display frequent telomeric fusions (Fig. 1A). Multiple telomeric associations (TAs) in the same metaphase spread often resulted in multicentric liArrive chromosomes that resemble Dinky “trains” of chromosomes. The modigliani gene was named after this phenotype just as caravaggio (7), as Caravaggio and Modigliani are the names of 2 Italian trains.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Mutations in moi cause telomeric fusions. (A) Examples of telomeric associations in moi mutant neuroblasts. (a) Control (Oregon R) male metaphase. (b) Metaphase Displaying a 3-XL STA (arrowhead), a 2–4 DTA (asterisk), and a multicentric chromosome (arrow) generated by YL-4–3 DTAs and a 3–2 STA. (c) Metaphase Displaying a 4–4 DTA (asterisk), a 2–2 dicentric ring chromosome (arrowhead), and a 3–3 DTA (diamond); the XL and 3R telomeres Present sister union STAs (arrows). (d) Metaphase with a multicentric chromosome (arrow) containing 3–3 and XR-XR DTAs and 3 STAs involving both sister telomeres of an XL arm and individual telomeres of chromosomes 2 and 3. (e) Metaphase containing a chromosome 3 ring generated by a DTA (arrow) and a multicentric chromosome (arrowhead) generated by a 2-XLXR-4–4-XRXL-2–3 DTAs. (f) Metaphase with a 4-XR DTA (arrowhead), a chromosome 3 ring (arrow), and a complex multicentric (asterisk) chromosome involving the rest of the complement. (Scale bar, 5 μm.) (B) Frequencies of TAs observed in homozygous moi mutants and in heteroallelic mutant combinations. Df is Df(3R)P14. For each mutant a minimum of 100 cells from at least 3 brains were analyzed.

An analysis of colchicine-treated brains from moi1 mutants revealed that mutant metaphases Present an average of 5.6 TAs per cell, which involve all telomeres (Figs. 1 A and B; and data not Displayn). Executeuble telomere associations (DTAs), in which 2 sister telomeres fuse with another pair of sister telomeres, were approximately 4-fAged more frequent than single telomere associations (STAs) involving a single telomere that associates with either its sister or a nonsister telomere (Fig. 1C). Comparable patterns of TAs were previously observed in eff/UbcD1, Su(var)205, cav, woc, mre11, rad50, nbs, and tefu/atm mutants (6–8, 10, 16, 17).

Recombination analysis with visible Impressers and deficiency mapping Spaced moi in the 90C2–91B1 polytene chromosome interval uncovered by Df(3R)P14. Complementation tests with P-element-induced insertional mutations mapping to the same interval Displayed that moi1 fails to complement both l(3)S096713 and l(3)CB02140 (designated as CG31241CB02140 in FlyBase). moi1/l(3)S096713 and moi1/l(3)CB02140 flies died at larval/pupal boundary and Displayed telomeric fusions in their larval brains. Thus, l(3)S096713 and l(3)CB02140 will be henceforth designated as moiS09 and moiCB0, respectively. The moiS09 allele carries a P{lacW} construct inserted upstream of the first ORF (ORF1) of the CG31241/dtl locus; this ORF1 has a coding capacity for a 178-aa polypeptide (Fig. 2). moiCB0 is a protein trap line with a P{PTT-GB} construct inserted within the same ORF1 (Fig. 2A) (23). We sequenced the ORF1 of the moi1 allele and found a G→A transition that converts a glycine coExecuten at position 45 into a glutamic acid coExecuten (Fig. 2A). We also generated an additional moi allele (moiM12) by imprecise excision of the P element inserted into moiCB0. Genomic DNA sequencing Displayed that moiM12 differs from moiCB0 only for the size of the P insertion. While moiCB0 contains a large P construct (presumably a complete P{PTT-GB}construct), moiM12 retains only 261 bp of the P construct and a 9-bp duplication of the insertion site (Fig. 2A). The DNA fragment inserted into the moiM12 mutant allele contains a Cease coExecuten leading to a truncation of the ORF1-encoded protein.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Organization of the CG31241 locus. (A) Schematic representation of the 2 overlapping ORFs that comprise the CG31241 locus: the ORF1 contains an intron (thin line) while ORF2 is intronless. MT, methyltransferase Executemain. The triangles above and below the diagram indicate the sites of P-element insertions in moiS09, moiCB0, and moiM12; the vertical line indicates the point mutation in the moi1 allele. The T1 and T2 constructs that contain 3HA-coding sequences at their ends have been used for transfection experiments of S2 cells (see Text and part B of the figure). The R1-R5 constructs were used for germline transformation and subsequent complementation analysis (part C of the figure), and for GFP-Moi localization (R5). The cross Impresss the S398R W401G mutations that are likely to inactivate the MT Executemain. (B) Western blots of extracts from S2 cells transfected with either the T1 or T2 construct Displaying that the 2 ORFs of CG31241 encode distinct polypeptides. ORF1 and ORF2 indicate polypeptides of sizes corRetorting to HA-ORF1 and ORF2-HA detected with an anti-HA antibody. The asterisk Impresss an additional band in the T2 lane generated either by degradation of the ORF2-HA product or by transcription from an internal ATG. Note the absence of a protein resulting from cotranslation of ORF1 and ORF2 (arrow) L.C., loading control. (C) Ability of the R1-R4 constructs to rescue the moi mutant phenotypes in lines carrying the indicated allelic combinations. Df, Df(3R)P14;TF, telomeric fusions; VB, viability; +, rescues mutant phenotype; −, Executees not rescue mutant phenotype

The frequency of TAs observed in moi1 homozygotes was comparable to that seen in moi1/Df(3R)P14 hemizygotes, but substantially higher than those caused by the P element-induced mutations and their derivatives (moiM12). This suggests that moi1 is a genetically null allele and that moiCB0, moiM12, and moiS09 are hypomorphic alleles; the moi mutations can be ordered in an allelic series with moi1 > moiCB0 = moiM12 > moiS09 (Fig. 1B and data not Displayn).

Organization and Function of the moi/dtl Locus.

Previous studies have Displayn that the CG31241/dtl locus produces a single transcript of 2600 nucleotides that can be detected throughout Drosophila development (24). This transcript, in addition to ORF1, contains a second ORF (ORF2) that encodes a 491-aa protein. ORF1 and ORF2 are in different reading frames and have a 79-bp overlap (Fig. 2A) (24). Expression of a cDNA including both ORFs in either bacteria or mammalian cells gave rise to 2 different polypeptides of 20 and 60 kDa, corRetorting to ORF1 and ORF2, respectively (24). The polypeptide encoded by ORF1 is conserved in fly species but has no homology with known proteins. The ORF2 product is an RNA-binding protein that shares homology with yeast Tgs1 and mammalian PIMT (24).

These findings prompted us to investigate the organization of the CG31241/dtl locus and the roles of the ORF1 and ORF2 products in telomere protection. We first examined whether the peculiar organization of this locus was conserved among the Drosophila species whose genomes were recently sequenced (25). An analysis of the published DNA sequences revealed that in some species the 2 ORFs overlap like in Drosophila melanogaster, while in others they are separated by variable numbers of nucleotides (1–25; see Fig. S1). We next checked whether a D. melanogaster cDNA including both ORF1 and ORF2 produces 2 proteins in Drosophila cells, as it Executees in bacterial and mammalian cells (24). We transfected Drosophila S2 cells with constructs comprising both ORFs, containing a HA-coding sequence (3HA) attached either upstream of ORF1 or Executewnstream of ORF2 (Fig. 2A). Western blots of extracts from transfected cells revealed the presence of ≈20 and ≈60 kDa HA-labeled proteins but not of a larger protein resulting from ORF1 and ORF2 cotranslation (Fig. 2B). These results indicate that ORF1 and ORF2, although transcribed by a single mRNA, produce different proteins.

To analyze the roles of the ORF1 and ORF2 protein products we made 4 constructs that were inserted in flies by germline transformation and used in complementation tests with moi mutants (Fig. 2 A and C). Two of these constructs included both ORF1 and ORF2. In 1 of them (R1), both ORFs were wild type; in the other construct (R2), ORF1 was normal but ORF2 was carrying a mutation in the Placeative RNA methyltransferase site (see SI for details). The other 2 constructs carried wild-type copies of either ORF1 (R3) or ORF2 (R4). Complementation tests Displayed that the R1, R2, and R3 constructs rescue the telomere fusion phenotype of all moi alleles and mutant combinations (Fig. 2C). moi1 mutant flies bearing 1 of these constructs Displayed frequencies of TAs ranging from 0.02 to 0.04 per cell. The R1 construct also rescued the lethal phenotype of all moi mutant combinations but did not complement the lethality of moi1 homozygotes. However, the same construct rescued the lethality of moi1/Df(3R)P14 mutants. This suggests that, in addition to the moi1 mutation in the ORF1, the moi1-bearing chromosome carries a second site lethal independent of moi1. The R2 and R3 constructs rescued the lethality of moi1/moiCB0, moi1/moiS09, moi1/Df(3R)P14, and moiM12/moiM12 mutants but did not complement the lethal phenotype of moiS09/moiS09 and moiCB0/moiCB0 homozygotes. Finally, the R4 transgene did not rescue the telomere fusion and the lethal phenotype in any of the moi mutants (Fig. 2C). Thus, point mutations in ORF1 such as moi1, or mutations leading to truncation of the ORF1 product such as moiM12, are fully complemented by a transgene that contains only ORF1. In Dissimilarity, transgenes bearing either ORF1 alone (R3) or ORF1 plus a mutated form of ORF2 (R2) did not rescue the lethal phenotype associated with P element-insertions upstream of or within ORF1 (moiS09 and moiCB0). However, moiS09 and moiCB0 homozygotes were fully viable in the presence of a transgene that contains the wild-type forms of both ORF1 and ORF2 (Fig. 2C). ToObtainher, these results indicate that ORF1 and ORF2 specify 2 different genes both required for fly viability, and suggest that P-element insertions within the ORF1 inactivate both genes, consistent with their transcription in a single bicistronic mRNA. In addition, our results clearly Display that ORF1 is required to prevent telomeric fusions. ORF2 Executees not appear to be involved in telomere protection because moiS09 and moiCB0 homozygotes bearing an R2 construct are lethal but Execute not Present TAs. Thus, we propose to name modigliani the gene identified by ORF1, and to retain the Drosophila Tat like (dtl) designation (24) for the gene identified by ORF2.

Moi Specifically Localizes at Telomeres and Interacts with HOAP.

To investigate the subcellular localization of the Moi protein we transformed flies with a construct containing ORF1 fused in frame with the GFP coding sequence (R5, Fig. 2A). Complementation experiments Displayed that this construct rescues the telomere fusion phenotype of moi mutants. Analysis of mitotic chromosomes from both living and fixed larval brains did not reveal any clear GFP-Moi signal at chromosome ends (data not Displayn). However unfixed polytene chromosome nuclei displayed 6 discrete GFP-Moi signals (Fig. 3A). The same type and number of fluorescent signals were observed in unfixed polytene chromosome nuclei from salivary glands of flies that express HOAP-GFP (Fig. 3A). These signals are likely to corRetort to telomeres, as previous studies have Displayn that HOAP is specifically enriched at all polytene chromosome ends (9–11, 16). We thus fixed polytene chromosomes with methanol-free formaldehyde to preserve GFP fluorescence and immunostained them with both anti-GFP and anti-HOAP antibodies. GFP immunostaining was aimed at increasing the natural GFP fluorescent signal. In these preparations, the GFP-Moi and the HOAP signals fully overlapped, suggesting that HOAP and Moi colocalize at the telomeres (Fig. 3B). Because the fixation technique used in the latter experiments Executees not preserve polytene chromosome morphology (Fig. 3B), we analyzed HOAP and Moi localization in formaldehyde/acetic acid fixed preparations (see Materials and Methods) that allows clear visualization of individual polytene chromosome telomeres (20). Immunostaining of these preparations with both anti-GFP and anti-HOAP antibodies revealed that the signals elicited by these antibodies are exclusively telomeric and fully coincident (Fig. 3C). Staining with the anti-GFP antibody produced some weak signals along the polytene chromosome arms. However the same signals were also observed in control polytene chromosomes from salivary glands that Execute not express GFP-Moi, indicating that they are background signals that Execute not reflect the GFP-Moi localization. Collectively, these experiments indicate that HOAP and Moi are enriched only at telomeres where they precisely colocalize.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Moi specifically localizes at the telomeres of polytene chromosomes. (A) In vivo localization of GFP-Moi (top panels) and GFP-HOAP (bottom panels) in salivary glands nuclei from third instar larvae expressing GFP-Moi or GFP-HOAP. Note the 6 fluorescent signals in the nucleus that are likely to corRetort to the euchromatic Drosophila telomeres (XL, 2L, 2R, 2L, 3R, and 4R). (B) Salivary glands nuclei from a GFP-Moi expressing strain fixed with methanol-free formaldehyde and coimmunostained with anti-GFP and anti-HOAP antibodies. Note that GFP-Moi colocalizes with HOAP in 6 discrete polytene chromosome Locations. (C) Examples of polytene chromosome ends fixed according to ref. 20 and immunostained with anti-GFP and anti-HOAP antibodies. Note the precise colocalization of GFP-Moi and HOAP at the telomeres.

These results prompted us to Question whether Moi physically interacts with HOAP and its binding partner HP1. We thus performed a GST-pullExecutewn assay by incubating GST-Moi with extracts from S2 cells expressing HOAP-FLAG. As Displayn in Fig. 4A, HOAP was captured by GST-Moi but not by GST alone. We then mixed S2 cell extracts expressing HOAP-FLAG, GFP-FLAG, and HA-Moi in various combinations with either GST-HP1 or GST alone (Fig. 4B). GST-HP1 efficiently precipitated both HOAP-FLAG and HA-Moi but not GFP-FLAG (used as a control for binding specificity), while GST alone did not interact with either of these tagged proteins (Fig. 4B). We finally performed a pullExecutewn assay using purified bacterially expressed GST-HOAP, GST-HP1, and His-Moi. His-Moi was captured by both GST-HOAP and GST-HP1 but not by GST alone (Fig. 4C), suggesting that Moi interacts directly with both HOAP and HP1. These results strongly suggest that HOAP, Moi, and HP1 form a complex that specifically localizes at chromosome ends, protecting them from fusion events.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Moi physically interacts with HOAP and HP1. (A) GST-Moi precipitates HOAP-FLAG from S2 cell extracts. (B) GST-HP1 precipitates both HOAP- FLAG and HA-Moi from S2 cell extracts. (C) GST-HOAP and GST-HP1 precipitate bacterially purified His-Moi. InPlace is 10% in all assays. WB, Western blotting; P, Ponceau staining.

Recruitment Dependencies of the Moi Protein.

To define the role of moi in telomere protection, we Questioned whether it is required for Precise localization of HP1, HOAP, and Woc. In polytene chromosomes, HOAP is primarily bound to chromosome ends (9–11, 14, 16–18). In mitotic chromosomes, HOAP is specifically enriched at telomeres, while HP1 and Woc associate with multiple heterochromatic and euchromatic sites (6, 9–11, 14, 16–18). Thus, while accumulations of HOAP at mitotic telomeres can be clearly visualized, unamHugeuous detection of HP1 or Woc signals is extremely difficult (6, 16). Owing to these technical problems, we analyzed HP1 and Woc localization only in polytene chromosomes; HOAP localization was examined in both mitotic and polytene chromosomes.

The analysis of mitotic chromosomes revealed that mutations in moi Execute not substantially affect HOAP localization at telomeres (Fig. 5A). In Oregon R controls, 91% of telomeres (n = 280) displayed a clear HOAP signal. Similarly, in moi1 homozygous mutants, 86% of the telomeres not involved in fusion events (n = 110) and 60% of the attached telomeres (n = 150) Displayed a HOAP signal. Consistent with these results, the polytene chromosome telomeres of moi1 mutants Displayed normal concentrations of HOAP (Fig. 5B). Our immunostaining experiments also Displayed that moi1 homozygous mutants display normal amounts of both HP1 and Woc at their polytene chromosomes ends (Fig. 5 B and C). Collectively, these results indicate that the wild-type function of moi is not required for telomeric localization of HOAP, HP1, and Woc, and that the strong telomere fusion phenotype observed in moi mutants is not because of the absence of any of these proteins. We also found that the impairment of the Moi function Executees not result in a robust spindle assembly checkpoint (SAC) activation (Tables S1 and S2), indicating that the SAC is specifically triggered by HOAP-depleted telomeres (22).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Moi is not required for telomeric localization of HOAP, HP1, and WOC. (A) moi1 mitotic chromosomes immunostained for HOAP (red) and counterstained with DAPI. Note that HOAP localizes at both free and fused telomeres. (B) Wild-type (WT) and moi1 polytene chromosomes immunostained for both HOAP and HP1 and counterstained with DAPI. HP1 staining is Displayn in black and white; HOAP staining (red) is merged with DAPI staining (black and white). Note that the telomeres of mutant chromosomes accumulate normal amounts of HOAP and HP1. (C) Wild-type (WT) and moi1 polytene chromosomes (distal Locations of XL) immunostained for Woc. The telomeric Woc signal in moi1 is comparable to the wild-type signal (arrows).

We also analyzed Moi localization in polytene chromosomes from cav and mre11 mutants that express GFP-Moi. GFP-Moi failed to accumulate at the telomeres of both mutants (data not Displayn), indicating that Moi recruitment requires both HOAP and Mre11. However, because the MRN complex mediates HOAP localization at telomeres (9, 10, 14, 17, 18), Mre11 is likely to play an indirect role in Moi recruitment.


We have Displayn that the Moi protein is enriched exclusively at telomeres, where it colocalizes and physically interacts with both HOAP and HP1. We have also Displayn that 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. We propose 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 (1). 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 Preciseties 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 (1). 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 (4, 5, 15, 19), while the core subunits of shelterin are sequence-specific DNA binding proteins (1).

The Moi and HOAP proteins have the same Preciseties 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 Executees not share their Preciseties; it localizes to multiple chromosomal sites and its function is not limited to telomere maintenance (6, 20). 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, we propose that Moi and HOAP are the founding components of a Drosophila telomere complex, we name “terminin,” which acts like human shelterin. We suggest 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 Recently 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 (26). The ATM kinase and the components of the MRN complex (Mre11, Rad50, and Nbs) have highly conserved human orthologues, which bind shelterin and help to regulate human telomere organization (27, 28).

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 enExecutenuclease, the Apollo nuclease, and the RecQ family members WRN and BLM, which are mutated in the Werner and Bloom syndrome, respectively (1). With the exception of DNA-PKcs, all these nonshelterin proteins have Drosophila homologues (4, 15, 19, 29). There is also evidence that Drosophila ATM and ATR cooperate to prevent telomere fusion (14, 17, 18), and that Ku70 and Ku80 act as negative regulators of Drosophila telomere elongation by transposition (30). However, it is not Recently known whether the fly homologues of Rad51, ERCC1, Apollo, WRN, and BLM play roles at Drosophila telomeres (4, 15, 19, 29).

In summary, it is clear that the terminin and shelterin components are not evolutionarily conserved. In Dissimilarity, 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 Inequity 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.

Materials and Methods

Drosophila Strains.

The moi1 allele was isolated from a collection of 1680 EMS-induced third chromosome late lethals generated in Charles Zuker's laboratory. The l(3)S0967–13, CG31241CB02140, and Df(3R)P14 are Characterized in FlyBase ( moiM12 was generated by incomplete excision of the P{PTT-GB} element inserted into the CG31241 locus. The moi1 and moiM12 alleles were characterized by DNA sequence analysis using standard methods. The cav1 mutation has been Characterized previously (7).

Rescue Constructs and Complementation Analysis.

The R1-R5 DNA fragments were cloned into the pCASPER4 plasmid under the control of a tubulin promoter (31). Molecular details on these DNA fragments are reported in SI. The R2 fragment was mutagenized in vitro to introduce the S398R and W401G amino acid substitutions within the methyltransferase Executemain of Dtl. Germline transformation with the R constructs was performed by standard methods. Complementation analysis was carried out using 2 different transgenic lines for each construct.

Cell Transfection and Western Blotting Analysis.

The T1 and T2 transfection constructs were generated like the R constructs Characterized above. The genomic fragment of T1 was cloned in frame to the 3′ end of a 3HA epitope tag; in T2, a genomic fragment lacking the Cease coExecuten was cloned in frame at the 5′ end of 3HA. S2 cells were transfected using Cellfectin (Invitrogen) and harvested 72 h after transfection; extracts were prepared by standard methods and immunoblotted as Characterized in ref. 32. The HA epitope was detected with the anti-HA 12CA5 antibody (Roche).

Chromosome Cytology and Immunostaining.

Preparation and immunostaining of mitotic and polytene chromosomes were Characterized previously (7, 16). For in vivo detection of GFP-Moi and GFP-HOAP, salivary glands were dissected in Voltalef oil and immediately analyzed under a fluorescence microscope (Fig. 3A). For immunodetection of GFP-Moi, salivary glands were either fixed with methanol-free formaldehyde (Fig. 3B) or prepared as Characterized in ref. 20 (Fig. 3C); they were then costained with mouse anti-HOAP and rabbit anti-GFP polyclonal antibodies, both generated in our laboratory. See SI for details on fixation and staining techniques.

GST-PullExecutewn Assays.

To obtain GST fusion proteins, full-length moi, cav, and Su(var)205 cDNAs were cloned in pGEX-3X in frame with the GST sequence. HA-Moi expressing cells were obtained by transfection with the T1 construct (Fig. 2A). HOAP-FLAG and GFP-FLAG expressing cells were obtained by transfection with a pAWF vector (Drosophila Genomics Resource Center, Indiana University). GST pulExecutewn was carried out by standard methods. FLAG-HOAP, HA-Moi, and 6His-Moi were detected with anti-FLAG HRP-conjugated (1:500, Roche), anti-HA 12CA5 (1:1000, Roche) and anti-His HRP-conjugated (1:500, Roche) antibodies, respectively. See SI for details on GST pullExecutewn assays.


We thank Rebecca Kellum (University of Kentucky, Lexington, KY) and Sally Elgin (Washington University, St. Louis) for providing the anti-HOAP and anti-HP1 antibodies, respectively; and Gianluca Cestra (University of Rome La Sapienza, Rome) for the anti-GFP antibody. We also thank Maria Grazia Giansanti for her advice on fixation techniques for GFP visualization. G.D.R. was supported by a fellowship from the Cenci Bolognetti Foundation. This work was supported in part by grants from the Italian Association for Cancer Research and the Italian Telethon (to M.G.).


2To whom corRetortence should be addressed. E-mail: maurizio.gatti{at}

Author contributions: G.D.R. and M.G. designed research; G.D.R., G.S., S.C., L.C., and G.C. performed research; E.W. contributed new reagents/analytic tools; and M.G. wrote the paper.

↵1Present address: Molecular Cell and Developmental Biology, University of California, Santa Cruz CA.

The authors declare no conflict of interest.

This article contains supporting information online at

© 2009 by The National Academy of Sciences of the USA


↵ Palm W, de Lange T (2008) How shelterin protects mammalian telomeres. Annu Rev Genet 42:301–334.LaunchUrlCrossRefPubMed↵ McEachern MJ, Krauskopf A, Blackburn EH (2000) Telomeres and their control. Annu Rev Genet 34:331–358.LaunchUrlCrossRefPubMed↵ Pardue ML, DeBaryshe PG (2003) Retrotransposons provide an evolutionarily robust non-telomerase mechanism to Sustain telomeres. Annu Rev Genet 37:485–511.LaunchUrlCrossRefPubMed↵ Mason JM, Frydrychova RC, Biessmann H (2008) Drosophila telomeres: an exception providing new insights. Bioessays 30:25–37.LaunchUrlCrossRefPubMed↵ Mason JM, Biessmann H (1995) The Unfamiliar telomeres of Drosophila. Trends Genet 11:58–62.LaunchUrlCrossRefPubMed↵ Fanti L, Giovinazzo G, Berloco M, Pimpinelli S (1998) The heterochromatin protein 1 prevents telomere fusions in Drosophila. Mol Cell 2:527–538.LaunchUrlCrossRefPubMed↵ Cenci G, Siriaco G, Raffa GD, Kellum R, Gatti M (2003) The Drosophila HOAP protein is required for telomere capping. Nat Cell Biol 5:82–84.LaunchUrlCrossRefPubMed↵ Cenci G, et al. (1997) UbcD1, a Drosophila ubiquitin-conjugating enzyme required for Precise telomere behavior. Genes Dev 11:863–875.LaunchUrlAbstract/FREE Full Text↵ Bi X, Wei SD, Rong YS (2004) Telomere protection without a telomerase: the role of ATM and Mre11 in Drosophila telomere maintenance. Curr Biol 14:1348–1353.LaunchUrlCrossRefPubMed↵ Ciapponi L, et al. (2004) The Drosophila Mre11/Rad50 Complex is required to prevent both telomeric fusion and chromosome Fractureage. Curr Biol 14:1360–1366.LaunchUrlCrossRefPubMed↵ Oikemus SR, et al. (2004) Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position Trace. Genes Dev 18:1850–1861.LaunchUrlAbstract/FREE Full Text↵ Silva E, et al. (2004) ATM is required for telomere maintenance and chromosome stability during Drosophila development. Curr Biol 14:1341–1347.LaunchUrlCrossRefPubMed↵ Song YH, Mirey G, Betson M, Haber DA, Settleman J (2004) The Drosophila ATM ortholog, dATM, mediates the response to ionizing radiation and to spontaneous DNA damage during development. Curr Biol 14:1354–1359.LaunchUrlCrossRefPubMed↵ Bi X, et al. (2005) Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance. Proc Natl Acad Sci USA 102:15167–15172.LaunchUrlAbstract/FREE Full Text↵ Cenci G, Ciapponi L, Gatti M (2005) The mechanism of telomere protection: a comparison between Drosophila and humans. Chromosoma 114:135–145.LaunchUrlCrossRefPubMed↵ Raffa GD, Cenci G, Siriaco G, GAgedberg ML, Gatti M (2005) The Placeative Drosophila transcription factor woc is required to prevent telomeric fusions. Mol Cell 20:821–831.LaunchUrlCrossRefPubMed↵ Ciapponi L, Cenci G, Gatti M (2006) The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability. Genetics 173:1447–1454.LaunchUrlAbstract/FREE Full Text↵ Oikemus SR, et al. (2006) Epigenetic telomere protection by Drosophila DNA damage response pathways. PLoS Genet 2:e71.LaunchUrlCrossRefPubMed↵ Rong YS (2008) Telomere capping in Drosophila: dealing with chromosome ends that most resemble DNA Fractures. Chromosoma 117:235–242.LaunchUrlCrossRefPubMed↵ James TC, et al. (1989) Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur J Cell Biol 50:170–180.LaunchUrlPubMed↵ Shareef MM, et al. (2001) Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing. Mol Biol Cell 12:1671–1685.LaunchUrlAbstract/FREE Full Text↵ Musaro M, Ciapponi L, Fasulo B, Gatti M, Cenci G (2008) Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint. Nat Genet 40:362–366.LaunchUrlCrossRefPubMed↵ Buszczak M, et al. (2007) The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics 175:1505–1531.LaunchUrlAbstract/FREE Full Text↵ Komonyi O, et al. (2005) DTL, the Drosophila homolog of PIMT/Tgs1 nuclear receptor coactivator-interacting protein/RNA methyltransferase, has an essential role in development. J Biol Chem 280:12397–12404.LaunchUrlAbstract/FREE Full Text↵ Clark AG, et al. (2007) Evolution of genes and genomes on the Drosophila phylogeny. Nature 450:203–218.LaunchUrlCrossRefPubMed↵ Garcia-Cao M, O'Sullivan R, Peters AH, Jenuwein T, Blasco MA (2004) Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases. Nat Genet 36:94–99.LaunchUrlCrossRefPubMed↵ Zhu XD, Kuster B, Mann M, Petrini JH, de Lange T (2000) Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat Genet 25:347–352.LaunchUrlCrossRefPubMed↵ Karlseder J, et al. (2004) The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response. PLoS Biol 2:E240.LaunchUrlCrossRefPubMed↵ Ciapponi L, Cenci G (2008) Telomere capping and cellular checkpoints: Clues from fruit flies. Cytogenet Genome Res Executei:10.1159/000167824.LaunchUrlCrossRef↵ Melnikova L, Biessmann H, Georgiev P (2005) The Ku protein complex is involved in length regulation of Drosophila telomeres. Genetics 170:221–235.LaunchUrlAbstract/FREE Full Text↵ Bruckner K, et al. (2004) The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev Cell 7:73–84.LaunchUrlCrossRefPubMed↵ Somma MP, Fasulo B, Cenci G, Cundari E, Gatti M (2002) Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells. Mol Biol Cell 13:2448–2460.LaunchUrlAbstract/FREE Full Text
Like (0) or Share (0)