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RESEARCH PAPER
1 Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA; 2 Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal; 3 Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, 4150 Porto, Portugal; 4 ICBAS, Universidade do Porto, 4000 Porto, Portugal
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[Keywords: Drosophila; telomere; protection; ATM; HP1; chromatin]
Received March 11, 2004; revised version accepted May 27, 2004.
; Blackburn 2001). In most organisms, the reverse-transcriptase telomerase prevents the loss of terminal sequences by adding short repeat sequences during S phase. Sequence-specific DNA-binding proteins that recognize telomere repeat sequences help regulate telomere length and protection (Cooper et al. 1997; Shore 1997; de Lange 2002; Karlseder 2003). DNA damage repair or signaling proteins also regulate telomere function (Chan and Blackburn 2002; Bertuch and Lundblad 2003), possibly by recognizing DNA structures at telomeres that resemble damaged chromosomes.
A family of proteins related to the Ataxia Telangiectasia Mutated (ATM) kinase plays conserved roles in DNA repair and telomere function. Mutations in human ATM cause the inherited cancer predisposition syndrome ataxia telangiectasia (Shiloh 2003). Loss of ATM in humans or mice causes high levels of genomic instability and hypersensitivity to ionizing radiation. ATM phosphorylates multiple substrates, including the Chk2 checkpoint kinase and p53 transcription factor, which promote apoptosis, cell cycle arrest, and DNA repair following DNA damage. Two ATM-related kinases, ATR (ATM and Rad3-related) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit), are also activated by DNA damage and cooperate with ATM to regulate the cellular response to DNA damage (Shiloh 2003). ATM also regulates telomere length and protection (Pandita et al. 1995; Metcalfe et al. 1996; Pandita 2002). ATM function is, at least partly, telomerase independent, as mice mutant for both telomerase and ATM have shorter telomeres and more anaphase bridges than single mutant mice; these mice also exhibit striking defects in stemcell populations and aging (Wong et al. 2003). Because of the intimate linkage between telomere length and telomere protection, it is difficult to determine whether the targets of ATM are enzymes that extend or degrade telomere DNA or proteins that directly mediate telomere protection. Identification of these targets may help define telomerase-independent pathways that regulate telomere function.
Budding and fission yeast have homologs of ATM and ATR. Whereas loss of one ATM-like kinase can affect telomere length and repression of gene expression near telomeres (telomere position effect), loss of both ATM and ATR in yeast causes rapid shortening and fusion of yeast telomeres (Naito et al. 1998; Ritchie et al. 1999; Chan et al. 2001; Craven et al. 2002; Chan and Blackburn 2003). Assays that directly measure telomere protection demonstrate that these proteins prevent telomeres from being fused to double-strand DNA breaks or to other telomeres (DuBois et al. 2002; Chan and Blackburn 2003; Mieczkowski et al. 2003). As in mice, the yeast ATM-like kinases act, at least partly, in a telomerase-independent pathway (Ritchie et al. 1999; Nakamura et al. 2002; Chan and Blackburn 2003). Thus, in both yeast and humans, ATM can regulate telomere function independently of telomerase.
Analysis of telomere function in Drosophila has revealed that an epigenetic mechanism contributes to telomerase-independent protection of telomeres. Drosophila telomeres are composed of two non-LTR retrotransposons, Het-A and TART, as well as more proximal telomere-associated sequences (Biessmann et al. 1992; Karpen and Spradling 1992; Levis et al. 1993; Mason and Biessmann 1995; Cryderman et al. 1999; Fanti and Pimpinelli 1999). Terminally deleted chromosomes, lacking all telomere-specific sequences, can be isolated following DNA damage (Mason et al. 1984, 1997; Tower et al. 1993; Ahmad and Golic 1998). Once obtained, the ends of these chromosomes are protected from end-to-end fusion; this property is stably inherited despite the gradual loss of terminal sequences due to the incomplete replication of chromosome ends by DNA polymerase (Biessmann and Mason 1988). The heritable and sequence-independent protection of the ends of terminally deleted chromosomes indicates that this aspect of Drosophila telomere function can be regulated by an epigenetic mechanism.
The Drosophila heterochromatin protein 1 (HP1) and heterochromatin protein 1/ORC2 associated protein (HOAP) are localized to telomeres and required for telomere function. HP1 proteins play an evolutionarily conserved role in heterochromatin function (Eissenberg and Elgin 2000; Grewal and Elgin 2002; Kellum 2003). In Drosophila, HP1 is prominent at centromeric and telomeric heterochromatin, but is also observed at euchromatic sites (James et al. 1989; Fanti et al. 1998, 2003). HOAP forms a complex with HP1 and is strongly localized to telomeres (Shareef et al. 2001; Badugu et al. 2003). Loss of either HP1 or HOAP leads to striking telomere fusion phenotypes, suggesting that chromatin structure plays a central role in telomere protection (Fanti et al. 1998; Cenci et al. 2003). Chromatin-modifying proteins also regulate telomere position effect (TPE) in Drosophila. TPE at the second and third chromosomes is sensitive to the dose of Polycomb group genes, whereas TPE at the fourth chromosome or at a terminally deleted minichromosome is sensitive to the dose of HP1 (Cryderman et al. 1999; Donaldson et al. 2002; Boivin et al. 2003). These results indicate that the regulation of chromatin structure by HP1 is required for both telomere protection and TPE.
Interestingly, terminally deleted chromosomes have normal levels of HP1–HOAP at their telomeres (Fanti et al. 1998; Cenci et al. 2003). The sequence-independent localization of these proteins to telomeres suggests that a structural feature of telomeres, perhaps chromosome ends, helps establish or reinforce the localization of these telomere-protection proteins. Gatti and colleagues have suggested that DNA damage-detection proteins may help recruit HP1–HOAP complexes to telomeres by recognizing chromosome ends (Cenci et al. 2003).
Drosophila encodes two damage-response proteins in the ATM kinase family, MEI-41, which is most similar to ATR, and CG6535/ATM, which is most similar to human ATM (Hari et al. 1995; Sekelsky et al. 2000; Laurencon et al. 2003; Brodsky et al. 2004). ATR/MEI-41 is required for ionizing radiation (IR)-induced cell cycle arrest, but not p53-dependent apoptosis (Hari et al. 1995; Ahmad and Golic 1999; Brodsky et al. 2000a,b, 2004; Ollmann et al. 2000). Telomere fusions have not been described in mitotic cells lacking ATR/MEI-41 or its binding partner, ATRIP/MUS304 (Gatti 1979; Hari et al. 1995; Brodsky et al. 2000b).
In this study, we characterize the role of Drosophila ATM in telomere function. Drosophila ATM is required for viability and for eye, wing, and bristle development. We find high frequencies of telomere fusions and anaphase bridges in the absence of ATM. We demonstrate that chromosomes mutant for the telomere fusion (tefu) gene (Queiroz-Machado et al. 2001) carry truncation mutations in atm. In atm mutant animals, telomere fusions are accompanied by greatly elevated levels of spontaneous apoptosis during tissue growth. This apoptosis is suppressed by mutations in p53, suggesting that loss of telomere protection induces an ATM-independent, but p53-dependent, apoptotic signal. We demonstrate that ATM is specifically required for normal levels of HP1 and HOAP at telomeres, but not at centric heterochromatin. In addition, atm mutations suppress TPE, demonstrating that atm is required for normal telomere chromatin structure. In situ hybridization with telomere-specific sequences demonstrates that the telomere defects in atm mutant cells are not due to loss of telomere sequences. These results support a model for telomere protection in which the recognition of DNA structures at chromosome termini by the ATM kinase provides a sequence-independent mechanism to help recruit telomere protection proteins that modify chromatin structure.
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The genomic structure of atm/CG6535 is shown in Figure 1A. On the basis of cDNA sequencing and transgene rescue, we have identified a functional atm cDNA (see Supplemental Material). Comparison of the predicted peptide sequence to mammalian checkpoint kinases indicates that it is the Drosophila homolog of ATM (see Supplemental Material). To characterize atm function, a deletion mutant (
356) was used that disrupts part of atm and all of the adjacent gene hsc70-4 (Bronk et al. 2001; Fig. 1A). Animals homozygous for this deletion and carrying a transgene containing the hsc70-4 gene (Hing et al. 1999) can be used to study the function of atm alone (Fig. 1A). We will refer to this combination as atm356. A second deletion, 16, includes hsc70-4 but not atm, and serves as an isogenic control (Bronk et al. 2001). As described below, we have also identified two mutations, atmtefu and atmred31 that are predicted to truncate the ATM protein (Fig. 1A–C).
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356 animals die shortly before or after eclosion with a rough eye phenotype, misshapen wings, and missing or abnormal bristles. atm356 produces similar defects in combination with a deficiency, Df(3R)PG4, that includes atm (Hing et al. 1999). atm356/atmtefu animals exhibit similar morphological phenotypes (Fig. 1D–I), but survive longer as adults. Defects were observed in 75% of bristles and 90% of wings (Supplementary Tables 1, 2). atmred31 homozygotes are also viable with rough eyes, but weaker wing and bristle phenotypes (data not shown). Similar eye and bristle phenotypes have been previously observed in Drosophila following DNA damage, or in mutant strains with high levels of genomic instability (Engels et al. 1987; Ashburner 1989; Ahmad and Golic 1999; Brodsky et al. 2000b). Extensive aneuploidy is predicted to disrupt bristle morphology, because bristles are sensitive to haploinsufficiency of ribosomal genes, which are present on all major chromosome arms (Ashburner 1989). In our experiments, the specific bristles affected varied between individuals, consistent with aneuploidy in a variable subset of cells, rather than a specific defect in the pattern of bristle cell specification.
To confirm that these phenotypes were due to loss of ATM, the Gal4–UAS system (Brand and Perrimon 1993) was used to demonstrate that an atm cDNA could rescue the observed atm phenotypes (Supplemental Material). In addition, these experiments indicate that overexpression of atm does not disrupt normal development. Together, these results demonstrate that Drosophila atm has an essential role during normal development and identify a cDNA sequence sufficient to rescue ATM function.
atm mutant tissues have high levels of spontaneous apoptosis
Because human ATM is required for damage-induced apoptosis, apoptosis was examined in animals mutant for Drosophila atm. Late third-instar larvae were irradiated with 0 or 4000 rads of ionizing radiation (IR), and wing imaginal discs were stained for apoptotic cells using either the vital dye acridine orange (Fig. 2A–E) or an antibody specific for an activated form of Caspase 3 (Fig. 2F–O). In apical sections of discs stained for activated Caspase (Fig. 2F–J), apoptotic cells are dispersed among living cells. Basal sections (Fig. 2K–O) highlight apoptotic cells with pyknotic nuclei, suggesting that these basal cells had progressed to late apoptotic stages.
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We considered the possibility that the spontaneous apoptosis in atm- discs is due to activation of a DNA damage-response pathway. Because Drosophila p53 is required for DNA damage-induced apoptosis, the effect of a null mutation in p53 (Rong et al. 2002; Brodsky et al. 2004) on the spontaneous apoptosis and lethality due to loss of atm was examined. Compared with atm single-mutant animals, atm356, p531 double-mutant animals exhibit substantially reduced levels of acridine orange staining (Fig. 2E), activated caspase staining (Fig. 2J,O), and pyknotic nuclei (data not shown). Although most apoptosis is suppressed by removal of p53, the levels of spontaneous apoptosis in atm, p53 mutant discs are still elevated compared with wild type (Fig. 2, cf. A,F,K and E,J,O), revealing some p53-independent apoptosis. Loss of p53 did not rescue atm lethality, as double-mutant animals still died as pharate adults with missing or defective bristles (data not shown). Thus, most of the spontaneous apoptosis in atm- animals is p53 dependent, but suppression of that apoptosis is not sufficient to restore normal development or viability.
atm is not required for damage-induced cell cycle arrest
Because human cells lacking ATM are defective in cell cycle arrest following ionizing irradiation (IR), the role of Drosophila ATM in damage-induced cell cycle arrest was tested. The wing imaginal disc exhibits a G2/M DNA damage checkpoint following X-irradiation, which is dependent on Drosophila homologs of ATR and ATRIP (Hari et al. 1995; Brodsky et al. 2000b). Both wild-type and atm- wing discs exhibit a reduction in the number of mitotic cells at time points from 1 to 8 h following X-irradiation (Fig. 3A–C,E–G; data not shown). Thus, Drosophila ATM is not essential for G2 arrest following DNA damage.
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atm is required to prevent telomere fusions and is mutated in telomere fusion animals
To examine chromosome structure in atm- animals, metaphase and anaphase chromosomes were prepared from wild-type and atm- larval neuroblasts. atm- neuroblasts exhibit a high frequency of chromosome end fusions and anaphase bridges (Fig. 3I–L; Supplementary Fig. 1; Supplementary Tables 3, 4). The aberrations observed indicate that Drosophila ATM is required to protect telomeres from fusion, and that these unprotected telomeres can undergo at least one round of a fusion-bridge-break cycle.
The telomere fusion phenotype observed in atm- neuroblasts resembles the mutant phenotype of the previously described tefu gene (Queiroz-Machado et al. 2001). tefu maps near atm by meiotic recombination and fails to complement a chromosome with a deficiency [Df(3R)red31] in cytological region 88B5-C. Although atm is in 88E3, atm356 mutations fail to complement both tefu and Df(3R)red31 for multiple phenotypes. tefu and Df(3R)red31 also fail to complement Df(3R)PG4, which encompasses the atm locus. The phenotypes of atm356/tefu animals can be rescued by expression of the atm cDNA. Sequencing reveals that both the tefu and Df(3R)red31 chromosomes contain stop mutations in atm (Fig. 1). To demonstrate that the phenotype associated with the Df(3R)red31 is not associated with the described deletion, the atm mutation was separated from the deletion by meiotic recombination. Thus, atm and telomere fusion are the same gene. There are some minor differences between the phenotypes previously described for homozygous tefu animals and those described in this study; these could be due to a closely linked second mutation on the tefu chromosome.
The effects of different alleles of atm on anaphase bridge frequencies were compared (Supplementary Table 4). Animals homozygous for atm356 or heterozygous for atm356 and a deficiency that encompasses atm exhibited similar frequencies of anaphase bridges, suggesting that this allele is a strong, loss-of-function allele. Animals heterozygous for an atm deficiency and either tefu or Df(3R)red31 have similar frequencies of bridges.
atm mutations reduce HP1 and HOAP levels at telomeres and suppress telomere position effect
The phenotypes described above indicate that Drosophila atm is required to protect telomeres from fusion. HP1 and HOAP localize to the telomeres of polytene chromosomes, as well as other sites, and are required for telomere protection in mitotic cells (James et al. 1989; Fanti et al. 1998; Shareef et al. 2001; Cenci et al. 2003). Immunostaining was used to examine the distribution of HP1 and HOAP on wild-type and atm- polytene chromosomes (Fig. 4). Wild-type and atm- polytene chromosomes were prepared in parallel, all samples were treated with the same antibody solutions, and all images were captured using the same exposure times. For each genotype, chromosomes were prepared from 10 different animals and immunostained with antibodies to both proteins. DNA was detected by DAPI staining. HP1 staining at the chromocenter, fourth chromosome, and several euchromatic sites is unaffected by loss of atm, whereas HP1 staining is reduced at most atm- telomeres (Fig. 4C,D,G,H; Supplementary Table 5). At the tip of chromosome 2R, similar levels of HP1 staining at an internal site (cytological position 60F, arrows in Fig. 4 insets) can be observed in wild-type and mutant chromosomes, whereas HP1 is specifically reduced at the telomere of the mutant chromosome (asterisks in Fig. 4 insets). Similar HP1 staining at wild-type and mutant chromosomes was observed using a second, rabbit polyclonal HP1 antibody (data not shown). The normal levels of HP1 at sites other than the telomere indicates that the lack of telomere staining at atm- chromosomes is not due to differences in chromosome preparations or to global changes in chromatin structure in atm- cells. Rather, atm is specifically required to recruit or maintain HP1 to chromosome ends.
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356/ Df(3R)PG4 (Fig. 4) and atmtefu/Df(3R)PG4 (data not shown) animals, indicating that this phenotype is not allele specific. Quantification of the fluorescence intensity associated with HOAP and HP1 staining further demonstrates that there is a reproducible reduction at atm- telomeres compared with wild type (Fig. 4I,J). In contrast, HP1 staining at an internal chromosomal site (60F) is not reduced. Because HP1 and HOAP are required to prevent telomere fusions in mitotic cells, but not salivary glands, localization of these proteins in wild-type neuroblasts was examined. However, the widespread distribution of HP1 on metaphase chromosomes prevents the unambiguous identification of telomeric HP1. HOAP staining was observed at the telomeres of both wild-type and mutant neuroblasts, but the staining is too variable to conclude whether it is reduced as seen in salivary glands (data not shown).
HP1 promotes heterochromatin formation, in part, by recruiting histone-modifying enzymes. To probe whether atm mutations alter chromatin at the telomeres of mitotic cells, telomere position effect (TPE) at three telomeres was examined (Fig. 5). When a white reporter gene is placed adjacent to telomere-associated sequences (TAS), gene expression is silenced (Cryderman et al. 1999; Wallrath 2000). At each site tested, TPE is partially suppressed by mutations in atm (Fig. 5A–C,E–G; Supplementary Fig. 2). In transgenes inserted at nontelomeric genomic positions, placement of the TAS from the telomere of chromosome arm 2L next to the white reporter gene is sufficient to silence white expression (Kurenova et al. 1998). Unlike TAS in their normal location adjacent to telomeres, silencing by a nontelomeric TAS was not affected by atm mutations (Fig. 5D,H; Supplementary Fig. 2). These results indicate that the suppression of TPE is due to the specific action of atm on gene expression near telomeres.
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; Maser and DePinho 2002). In Drosophila, it is possible to recover terminal deletions that remove all telomere-specific sequences. However, these observations do not rule out the possibility that telomere-specific sequences contribute to telomere protection or TPE at normal Drosophila telomeres. In fact, the number of telomere repeats has been shown to influence some forms of TPE (Mason et al. 2003). To test whether the telomere defects in atm- animals could be due to loss of telomere sequences, fluorescent in situ hybridization was performed using a probe to the Het-A retrotransposon, which is specific to telomere DNA. Hybridization was performed with wild-type and atm- diploid and polytene chromosomes. In mitotic chromosomes from diploid neuroblast cells, the levels of Het-A hybridization are variable, but not significantly different between wild-type and atm mutant cells (Fig. 6A–C). In polytene chromosomes, HeT-A sequences are strongly detected at two telomeres of both wild-type and atm- chromosomes (Fig. 6F,G). Previous analysis of HP1 mutants demonstrated that telomere-specific sequences were still present at chromosome fusion sites (Fanti et al. 1998). In atm mutant cells, Het-A hybridization is also detected at sites of chromosome fusion (Fig. 6D,E). These results indicate that the reduction of telomeric HP1–HOAP and the fusion of telomeres in atm- cells is not a direct or indirect result of telomere sequence loss.
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; Fanti et al. 1998; Cenci et al. 2003). These results indicate that sequence-independent mechanisms can recruit and maintain telomere protection complexes to chromosome ends. Here, we demonstrate that Drosophila atm/tefu is required to prevent chromosome end fusions, to regulate levels of HP1 and HOAP at telomeres, and to promote telomereposition effect. We also find that atm is required for induction of apoptosis by ionizing radiation. Given the conserved role of ATM family proteins in recognizing DNA breaks, we suggest that Drosophila ATM protects telomeres by recognizing chromosome ends and recruiting chromatin-modifying proteins to those ends.
We have not, to date, directly detected ATM protein at Drosophila telomeres (S.R. Oikemus and N. McGinnis, unpubl.). However, on the basis of results in mammalian cells, it may be necessary to develop antibodies specific for activated forms of ATM to probe ATM activity at telomeres (Bakkenist and Kastan 2003). However, several observations presented here indicate that Drosophila ATM acts at telomeres to prevent chromosome fusions. First, the chromosome rearrangements observed are consistent with a defect in telomere protection rather than translocations due to defective DNA repair or replication. Most chromosome fusions occur near the ends of chromosome arms, and we demonstrate that the fused chromosomes still contain telomeric DNA sequences. Second, we do not observe a high frequency of acentric chromosome fragments during metaphase. In animals mutant for other damage-signaling genes such as the Drosophila homologs of ATR and ATRIP, acentric chromosome fragments are often observed during metaphase, suggesting that these mutations cause a defect in DNA repair or replication that is not observed in atm- animals (Gatti 1979; Brodsky et al. 2000b). Third, circular chromosomes do not undergo rearrangements in atmtefu mutant animals (Queiroz-Machado et al. 2001), strongly indicating that chromosome fusions are due to fusion of existing chromosome ends rather than the creation of new chromosome breaks. Fourth, we show that ATM is specifically required for localization of HP1 to telomeres but not centromeric or euchromatic sites. Finally, we demonstrate that loss of atm suppresses silencing by telomere-associated sequences when they are adjacent to telomeres, but not when they are at euchromatic sites.
The telomere fusion defect seen in atm- animals is consistent with a partial defect in telomere protection. Whereas 
80% of atm- metaphases contain a chromosome fusion, >95% of metaphases from animals lacking HP1 or HOAP contain a fusion (Fanti et al. 1998; Cenci et al. 2003). Furthermore, in some cells lacking HP1 or HOAP, nearly all telomeres appear to be fused. We have not observed this extreme phenotype in atm mutant nuclei. Consistent with a partial defect in telomere protection, we find that the levels of HP1 and HOAP at polytene telomeres are reduced, but not eliminated, in atm- animals. In mitotic cells, formation of repressive chromatin is also partially disrupted. Our interpretation of these results is that reduced and variable levels of HP1 at the telomeres of atm- animals are sufficient to protect some, but not all telomeres from fusion. Our results also indicate that another pathway, possibly involving other DNA damage-response proteins, must contribute to HP1 and HOAP localization, TPE, and telomere protection.
The direct target of ATM at telomeres is unclear. The decrease in HP1 and HOAP levels at atm- telomeres is not due to a loss of telomere sequences; wild-type and atm- chromosomes exhibit similar levels of a telomere-specific retrotransposon sequence as assayed by FISH, and even sites of fusion retain this sequence. This result is consistent with previous demonstrations that the sequences at chromosome ends are not required for telomere protection or for telomeric localization of HP1 and HOAP (Biessmann et al. 1990; Fanti et al. 1998; Cenci et al. 2003). Instead, ATM is likely to affect the interaction of HP1 and HOAP with telomeres by regulating the formation of the HP1–HOAP complex or by modification of telomeric chromatin. Other proteins in the DNA damage-response pathway may act with ATM to maintain telomere protection. Although Chk1, Chk2, and p53 are targets of mammalian ATM during the DNA damage response (Shiloh 2003), Drosophila homologs of these proteins do not appear to be required for telomere protection, as animals lacking one or more of these genes do not exhibit the high levels of apoptosis associated with loss of ATM (Brodsky et al. 2004). Mutations in homologs of other ATM targets such as NBS1 or SMC1 have not been described in Drosophila.
Recruitment of HP1 and HOAP by ATM is likely to alter chromatin structure at telomeres. HP1 plays a conserved role in heterochromatin formation, histone modification, and gene silencing (Eissenberg and Elgin 2000; Grewal and Elgin 2002). In Drosophila, both HP1 and HOAP are required for gene silencing at pericentric heterochromatin (Eissenberg et al. 1990; Eissenberg and Elgin 2000; Badugu et al. 2003). In addition, HP1 is required for gene silencing near fourth chromosome and terminally deleted telomeres, and for repression of P-element transposition by subtelomeric P-element insertions (Ronsseray et al. 1996, 1998; Cryderman et al. 1999; Donaldson et al. 2002). HP1 homologs are also associated with telomere function in other eukaryotes. In mammals, all three HP1 homologs are found at telomeres, and loss of histone H3 methylases leads to reduced levels of HP1 homologs at telomeres as well as elongated telomeres (Garcia-Cao et al. 2004). In contrast, overexpression of mammalian HP1 homologs is associated with decreased telomere length (Sharma et al. 2003). The fission yeast homolog of HP1 is not required for telomere protection, but does regulate telomere length, telomere clustering, and telomeric gene silencing (Allshire et al. 1995; Ekwall et al. 1995; Koering et al. 2002; Garcia-Cao et al. 2004). Interestingly, as in Drosophila telomere protection, some aspects of telomere function in fission yeast are controlled by an epigenetic mechanism (Sadaie et al. 2003). Together, these observations indicate that a requirement for HP1 in telomere function and chromatin structure is conserved, but that its precise role at the telomere may differ among organisms.
Regulation of telomere chromatin structure is also a conserved function of ATM-like kinases. Fission yeast Rad3 and budding yeast Mec1 are required for gene silencing at telomeres (Matsuura et al. 1999; Craven and Petes 2000) and mutations in human ATM are associated with altered nucleosomal periodicity at telomeres (Smilenov et al. 1999). The conserved role of ATM-kinases in telomere protection (see beginning of this paper) and telomeric chromatin structure suggests that these functions might be linked. Our finding that Drosophila ATM is required for TPE and HP1–HOAP localization to telomeres demonstrates one mechanism by which ATM can influence telomere chromatin. It is possible that in organisms that utilize sequence-specific binding proteins such as TRF2 to protect telomeres, regulation of telomeric heterochromatin by ATM and HP1 plays a minor role in protection of normal telomeres, but a more important role at short telomeres that cannot recruit sufficient levels of TRF2. Such a model might explain the synergistic telomere defects seen in cells lacking both telomerase and ATM (Ritchie et al. 1999; Tsukamoto et al. 2001; Chan and Blackburn 2003; Wong et al. 2003). The lack of an obvious TRF2 homolog may explain why ATM and HP1 play such striking roles in the protection of Drosophila telomeres.
In addition to preventing chromosome end fusion by DNA repair enzymes, telomere protection is required to prevent activation of DNA damage responses, including the induction of p53-dependent apoptosis and senescence (Chin et al. 1999; Karlseder et al. 1999; d'Adda di Fagagna et al. 2003; Takai et al. 2003). Our analysis of the cellular effects of ATM loss indicates that induction of p53-dependent apoptosis is a conserved consequence of unprotected telomeres in metazoans. Because these unprotected telomeres lead to anaphase bridges and chromosome breaks, p53 may be directly activated by unprotected telomeres or may be activated by subsequent chromosome breaks. Drosophila ATM is required for the induction of apoptosis following IR. Because the spontaneous apoptosis in atm- animals is, by definition, ATM independent, a different pathway must be able to activate Drosophila p53 in response to unprotected telomeres. Similarly, loss of mammalian ATM reduces, but does not eliminate p53-dependent apoptosis in response to unprotected telomeres (van Steensel et al. 1998; Takai et al. 2003; Wong et al. 2003). Other DNA damage-response pathways may activate Drosophila p53 in the absence of ATM.
In yeast, insects, and mammals, ATM-kinases are required to activate cellular responses to DNA ends generated by exogenous DNA damage, but also to suppress activation of these pathways by telomeres. Specific recognition of telomere sequences by telomere repeat-binding proteins provides one means to distinguish telomeric DNA ends from damage-induced DNA breaks. However, this mechanism is not sufficient to explain the epigenetic regulation of telomere protection in Drosophila. The requirement of ATM to recruit HP1 and HOAP to Drosophila telomeres suggests that recognition of chromosome ends contributes to chromatin-mediated telomere protection. This model may help explain how terminally deleted chromosomes can be stably inherited without any telomere-specific sequences. Future studies should reveal which other damage response proteins help ATM protect telomeres, what their targets are at telomeres, and how these proteins distinguish between damage-induced DNA ends and the natural ends of chromosomes.
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All animals were raised at 25°C. The deletion mutants, atm356 and hsc70-416 have been previously described (Bronk et al. 2001). Df(3R)PG4 and the transgenic rescue P[hsc70-4+] strains were described by Hing et al. (1999). The tefu mutant was described previously (Queiroz-Machado et al. 2001). A strain carrying the Df(3R)red31 chromosome was obtained from the Bloomington Stock Center. The P[hsc70-4+]; atm356 strain was created by Saeko Takada. The P[hsc70-4+]; atm356, p531 strain was generated by recombination of the atm356 and p531 mutations (Rong et al. 2002). Sequence analysis was performed using genomic DNA obtained from tefu/Df(3R)PG4, Df(3R)red31/Df(3R)PG4, and w1118 animals. atm genomic DNA was amplified by PCR and directly sequenced using gene-specific primers.
Apoptosis and checkpoint assays
Apoptosis and cell cycle arrest was induced in developing wing discs by irradiation of late third-instar larvae with 4000 rads X-rays in a Faxitron RX650 irradiator. Apoptotic cells were detected 4 h following irradiation by staining with the vital dye acridine orange (Abrams et al. 1993; Hay et al. 1994) or by fixation and staining with DAPI and a polyclonal rabbit antibody raised against cleaved human Caspase 3 (Cell Signaling Technology; 1:100 dilution) and counterstained with DAPI. Mitotic cells were visualized 1–4 h after irradiation using an anti-phospho-histone H3 antibody (Upstate Biotechnology; 1:500 dilution). Secondary antibodies were used at a 1:200 dilution. Fixation and staining was performed as described previously (Brodsky et al. 2000b).
Neuroblast preparations
Mitotic chromosome preparations were obtained from third-instar larval brains according to Queiroz-Machado et al. (2001). Briefly, for metaphase spreads, brains were dissected in PBS, incubated in 0.05 mM colchicine for 30 min, then transferred to a hypotonic solution (0.5% sodium citrate) for 10 min. Brains were fixed in 45% acetic acid for 1 min and squashed in 60% acetic acid. The squashed brains were stained with 0.2 µg/mL DAPI and mounted in Vectashield. Anaphase spreads were obtained similarly except that colchicine treatment was omitted and brains were incubated in hypotonic solution for 2 min.
Immunostaining of polytene chromosomes
Polytene chromosomes were prepared for immunostaining as described by Shareef et al. (2001). Chromosomes were incubated with rabbit polyclonal anti-HOAP (gift from R. Kellum, University of Kentucky, Lexington, KY; 1:500 dilution), mouse monoclonal C1A9 anti-HP1 (Developmental Studies Hybridoma Bank; 1:50), or rabbit polyclonal anti-HP1 (a gift from R. Kellum; 1:1000) antibodies at 4°C overnight, followed by a 2-h incubation at room temperature with DAPI, Cy3-conjugated sheep anti-rabbit and FITC-conjugated sheep anti-mouse (Jackson Immunoresearch Laboratories Inc; 1:200) secondary antibodies. For each genotype, salivary glands from 10 larvae were prepared and 10 chromosomes with good morphology and distinct telomeres were analyzed from each preparation. Stained chromosomes were analyzed using a Zeiss Axioskop 2 Plus microscope equipped with an ORCA-ER digital camera (Hamamatsu) and Openlab software (Improvision). Camera exposure times were held constant within an experiment.
Fluorescence intensity was quantified by performing a z series scan on the tip of chromosome 2R using a Leica TCS SP2 confocal microscope. Leica software was used to calculate the total fluorescence intensity for three regions of interest, the telomere, cytological band 60F, and a proximal chromosome region approximately midway between the telomere and 60F (background). Five 2R telomeres were analyzed for each genotype.
In situ hybridization to polytene chromosomes
The DIG Labeling DNA Kit (Roche) was used to make a probe from 1 µg of purified PCR product using a Het-A clone (Danilevskaya et al. 1994). A total of 1 µL of probe in 20 µL of hybridization solution was used per slide. Salivary gland polytene chromosome squashes were prepared for hybridization by standard methods (Pardue 1994), except that the RNase treatment was not included. Slides were prehybridized at 58°C in a moist chamber for 1 h in hybridization solution (0.6 mg/mL Fish Sperm DNA, 7.5 mM MgCl2, 3% 50x Denhardt's solution, 75 mM NaPO4 at pH 7.0, 1 M NaCl). Chromosome squashes were hybridized with the DIG-labeled probe overnight at 58°C. Hybridized probe and DNA were detected with sheep anti-DIG FITC (Roche; 1:200 dilution) and 0.5 µg/mL DAPI. Images were acquired as described for HP1 and HOAP staining above.
In situ hybridization to mitotic chromosomes
In situ hybridization to mitotic chromosomes was performed as described (Carmena et al. 1993). Het-A probe was labeled with biotin-14-dUTP using the Bionick Translation System (GIBCO-BRL). Slides were mounted in Vectashield containing 5 mg/mL of propidium iodide as a DNA counterstain. Quantification of the Het-A fluorescence intensity was performed using the Image J software package. For each Het-A signal selected, average pixel intensity was determined and multiplied by the corresponding area.
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Article published online ahead of print. Article and publication date are at http://www.genesdev.org/cgi/doi/10.1101/gad.1202504.
5 Corresponding author. E-MAIL michael.brodsky{at}umassmed.edu; FAX (508) 856-5460. 
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