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ATM Is Required for Telomere Maintenance and Chromosome Stability during Drosophila Development
Current Biology, Vol. 14, 1341–1347, August 10, 2004, 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j .c ub . 20 04 . 06 .0 5 6
ATM Is Required for Telomere Maintenance and Chromosome Stability during Drosophila Development Elizabeth Silva,1,2 Stanley Tiong,1 Michael Pedersen,1 Ellen Homola,1 Anne Royou,3 Barbara Fasulo,3 Giorgia Siriaco,3 and Shelagh D. Campbell1,* 1 Department of Biological Sciences University of Alberta Edmonton, Alberta, T6G 2E9 Canada 2 Developmental Patterning Lab London Research Institute London, WC2A 3PX United Kingdom 3 Sinsheimer Laboratories Molecular, Cell, and Developmental Biology University of California Santa Cruz, CA 95060
Summary ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints [1]. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs [2–4]. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia. Results and Discussion Eukaryotic cells rely on a core set of conserved DNA damage responses that maintain genome fidelity and ensure survival [5, 6]. In metazoans, ATM and the related protein kinases ATR and DNA-PK play partially overlapping roles in response to genotoxic stress to coordinately regulate DNA repair, cell cycle progression, and apoptosis, ensuring that damaged cells are either re*Correspondence: [email protected]
paired or eliminated [7]. Loss of ATM function causes an inherited human disease called ataxia-telangiectasia (A-T), with symptoms including progressive neurodegeneration, chromosomal instability, and pre-disposition to certain forms of cancer [8, 9]. Our functional analysis of Drosophila ATM, as well as that of the accompanying papers [10, 11], reveals conserved roles for dATM that are essential for normal development. Drosophila ATM Is Essential for Normal Adult Development Genome sequence analysis identified a gene called CG6535 that encodes a protein with highly significant sequence similarity to human ATM [12], referred to as atm in this report. To determine the function of atm, we conducted a genetic screen that isolated eight recessive lethal alleles (Experimental Procedures and Supplemental Data available with this article online). Seven of these alleles (atm1-7) have a nonconditional, pupal, lethal phenotype and one, atm8, is a temperature-sensitive hypomorphic allele. Hemizygous atm1–7 mutants die before emerging as adults, with variable morphological defects affecting the antennae, eyes, bristles, wings, and legs. The completely restrictive temperature for atm8 lethality is approximately 25⬚C. When atm8 mutants are raised at a semirestrictive temperature (24⬚C ⫹/⫺ 1oC), many can survive to adulthood with a similar range of morphological defects (Figures 1A–1D). The morphological defects, as well as atm mutant lethality, are rescued by introduction of a transgenic construct, P{atm⫹}, confirming that these phenotypes are specifically due to loss of atm function (see Experimental Procedures). To determine when dATM activity is required for normal development, we took advantage of the temperaturesensitive allele atm8 in a reciprocal temperature-shift experiment [13] that established the temperature-sensitive period (TSP) for selected atm8 mutant phenotypes (Experimental Procedures). The TSP for adult viability falls between the early third-larval instar and early pupal stage, the TSP for eye development is during the thirdlarval instar, and the TSP for wing and thoracic bristle development is during the pupal stage. These TSPs correspond to critical developmental stages of eye, wing, and bristle development. Requirements for dATM activity appear particularly stringent in developing neural tissues, because the structures most obviously affected in the atm mutants (eye, wing margin, thoracic bristles) are all involved in sensory perception. Locomotor Defects in Drosophila atm Mutants Because movement disorders (ataxia) are a prominent symptom in patients with A-T, we examined whether atm8 mutants exhibited signs of locomotor defects. We measured climbing ability in atm8 mutant and control flies for this purpose (Experimental Procedures and Supplemental Data). Replicate tests were conducted for each fly, and the averages were used for deriving a ratio (mutant/control) that provided a measure of how well
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Figure 1. Eye and Wing Defects Observed in Temperature-Sensitive atm8 Mutant Adults and Developing Imaginal Discs The adult mutant and control flies were raised at the atm8 semirestrictive temperature (24⬚C) and imaged using an environmental scanning electron microscope (A–D). (A) Normal adult eye. (B) atm8 mutant eye. (C) Normal adult wing. (D) atm8 mutant wing. (E–H) Cell patterning and apoptosis defects in third-instar atm8 mutant discs from larvae raised at the restrictive temperature (25⬚C). (E and F) Eyeantennal discs (oriented so that posterior is to the left) were labeled with antibodies to activated Caspase-3 (green), a marker for apoptosis [48] and Elav (red), a marker for differentiating neuronal cells [49]. (E) In a normal eye disc, apoptotic cells (green) are rarely seen at this stage of development and differentiating neuronal cells (red) form organized “preclusters” (inset). (F) Extensive apoptosis (green) occurs in the anterior region of an atm8 mutant disc, accompanied by obvious disorganization of the differentiating neuronal cells (red, inset). (G and H) Third-larval instar wing discs stained with antibodies to Cut [17]. (G) A normal wing disc showing Cut-positive myoblasts [19] associated with the presumptive thorax region of the disc (to the left) and a stripe of Cut-positive cells along the developing wing margin [17]. (H) An atm8 mutant wing disc showing loss of Cut labeling along the presumptive wing margin, although Cut expression appears normal in myoblasts adjoining the thorax region of the wing disc.
atm8 mutants climb, relative to matched controls [14]. The average climbing ability of atm8 mutant males was markedly lower than normal (ratio of mutants/controls ⫽ 0.11). Interestingly, the atm8 mutant females performed even worse in this assay (mutants/controls ⫽ 0.01). A movie illustrating the locomotor defects of atm mutants can be found in the Supplemental Data. The relative differences in climbing ability between mutants and controls did not change over a period of 4 weeks when the flies were maintained at the restrictive temperature of 29⬚C (data not shown). Thus, the locomotor defects in atm mutants appear to be strictly developmental. Apoptosis and Patterning Defects in Developing atm Tissues Because we had established that the temperature-sensitive period for normal eye development in atm8 mutants is during the third-larval instar, we further analyzed eye-antennal discs at this stage of development. A structure called the morphogenetic furrow (MF) sweeps across the eye disc during the late-third instar, marking a transition between proliferating, undifferentiated cells (anterior to the furrow) and nonproliferating cells that are differentiating into distinct neuronal cell types, posterior to the furrow [15]. The atm8 mutant-eye antennal discs are generally smaller than comparably aged controls (data not shown). Conspicuously, atm8 mutant eye discs had large numbers of cells undergoing inappropriate apoptosis, marked by antibody staining for activated caspase-3 (Figures 1E and 1F). There were striking regional differences in the distribution of apoptotic cells
in the mutant eye discs; these differences were also seen in discs stained with acridine orange staining as another marker of apoptosis (data not shown). These apoptotic cells were primarily restricted to the anterior, proliferating region of the disc (Figure 1F, green). There were relatively few apoptotic cells in the posterior region of the mutant discs, containing differentiating neuronal cells (Figure 1F, red). Early neuronal patterning was markedly disrupted in mutant discs, presumably because so many cells were eliminated by earlier apoptosis that normal precluster formation was precluded (Figures 1E and 1F, inset). The adult wing is also affected by loss of atm function, so we examined atm8 mutant third-larval instar wing discs for developmental defects. At this stage, cells in the wing disc are proliferating asynchronously except for a stripe of nondividing, differentiating cells along the presumptive wing margin of the disc [16]. These cells express Cut, a Notch-induced transcription factor required for proper differentiation of the wing margin [17]. We observed partial to complete loss of Cut expression along the presumptive wing margin of atm8 mutant discs, a patterning defect that anticipates the wing margin defects seen in atm mutants later in development (Figures 3G and 3H). Intriguingly, Cut expression was not obviously affected in the myoblast cells adjoining the presumptive dorsal thorax region of the atm mutant wing discs [18, 19]. The Cut expression defect in the developing wing margin could be due to inappropriate cell death in the proliferating imaginal cells, because spontaneous apoptosis also occurs throughout the atm8 mu-
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Figure 2. Early Developmental Defects in atm6 Mutants Derived from Germline Mosaic Females (A) Normal egg, oriented with dorsal appendages to the right. (B) atm6 mutant egg, showing fused dorsal appendages and chorion defects. (C, D) Cycle 11 embryos, stained with antibodies to Centrosomin (red, centrosomes), antibodies to alpha-tubulin (green, microtubules), and Hoescht 33258 (blue, DNA). These images were deconvolved using Volocity Restoration software. (C) Normal metaphase 11. (D) Representative field from a metaphase 11 atm6 mutant, showing extensive mitotic defects (isolated centrosomes, missing nuclei, aberrant mitotic spindles, and chromosome bridges).
tant wing discs at this developmental stage (data not shown; [11]). The spontaneous apoptosis and patterning defects in atm eye and wing imaginal discs suggest that cells are experiencing DNA damage, which is an established trigger for p53-dependent apoptosis in Drosophila [20, 21]. Early Developmental Requirements for Drosophila ATM Oogenesis and early embryogenesis are regulated by maternally acting genes in Drosophila. To determine if dATM activity is required during early stages of development, we examined eggs derived from atm6 mutantgermline mosaic females, generated using the hs-FLP/ FRT ovoD system [22]. atm6 was molecularly characterized as a functional null allele (Experimental Procedures). Maternal mutant atm6 eggs commonly have dorsal appendage defects (Figures 2A and 2B), resembling a phenotype of homologous DSB repair-defective female-sterile mutants [23]. The eggshells of atm6 mutant eggs also appear to be thinner than normal, suggesting that follicle cell development is compromised. Developing atm6 mutant embryos show dramatic mitotic defects during the rapid syncytial divisions of early embryogenesis. These mitotic defects included frequent spindle fusions and chromosomal bridges (Figures 2C and 2D) that could also be observed much earlier in development before the nuclei reach the surface of the embryo (data not shown). Another frequently observed defect involved nuclei detaching from centrosomes and falling from the cortex (Figures 2C and 2D and data not shown).
The centrosome and nuclear fallout defect in atm mutants resembles a local DNA damage-induced response called “centrosome inactivation,” indicating that loss of dATM is associated with spontaneous DNA damage [24, 25]. Chk2 is an established transducer of ATM signaling in mammalian cells [26] and was previously identified as a positive regulator of centrosome inactivation in Drosophila [25]. Because centrosome inactivation occurs spontaneously in atm mutants, this result suggests that either a dATM-independent mechanism for activating Chk2 exists or a Chk2-independent mechanism for promoting centrosome inactivation was deployed. Loss of dATM Causes Extreme Sensitivity to Ionizing Radiation In mammalian cells, the ATM and ATR signaling pathways negatively regulate Cdk1 in response to ionizing radiation, blocking mitosis while DNA is being repaired [27]. Mei-41, the Drosophila ATR ortholog, is required for this response to DNA damage [28–31]. To examine if dATM might also be required for this response, we labeled mitotic cells in atm mutant and control wing discs, with or without exposure to 40 Gy of ␥ irradiation (Figures 3A–3D). Mitotic cells were essentially absent 1 hr after ionizing radiation in both atm mutant and control discs, implying that dATM is dispensable for this premitotic-checkpoint response to acute DNA damage. An accompanying paper reports that dATM operates in a premitotic checkpoint, which is activated earlier in response to ionizing radiation than our measurements but concurs with our findings that the checkpoint response is fully engaged by 1 hr after irradiation [11].
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Figure 3. atm8 Mutants Have a Functional Premitotic Checkpoint Response but Are Extremely Sensitive to Ionizing Radiation (A–D) Third-larval instar wing discs, stained with antibodies to phospho-histone H3 to detect mitotic cells. (A) Unirradiated control (atm8 /TM6B) wing disc. (B) Irradiated control wing disc, fixed 1 hr after the larvae were exposed to 40 Gy of ␥ irradiation. (C) Unirradiated atm8 mutant wing disc. (D) Irradiated atm8 mutant wing disc, fixed 1 hr after exposure to 40 Gy. (E) Genetic crosses were made that were expected to generate hemizygous atm8 mutants as approximately one third of the progeny (raised at fully permissive temperature). The graph shows the atm8/ Df(3R)PG4 survival frequency of adults that were either raised at 22⬚C (permissive temperature: gray bars) or at 24⬚C (semirestrictive temperature: solid bars), relative to the dosage of ionizing radiation received by the respective third-instar larvae.
We also observed increased numbers of apoptotic cells in both controls and atm mutant wing discs, after larvae were exposed to ionizing radiation (data not shown). Because the apoptosis response to ionizing radiation in Drosophila is thought to depend on p53 [20, 21], this result implies that p53-dependent apoptosis is functional in atm mutants. An accompanying paper reports that expression of a dominant-negative p53 transgene suppresses spontaneous apoptosis in atm mutant wing discs, supporting our conclusion that the p53-dependent apoptosis response is intact [11]. We found that atm mutants are extremely sensitive to ionizing radiation. We assayed radiation sensitivity by comparing the adult viability of atm8 mutants, raised at either semirestrictive (24⬚C) or permissive (22⬚C) temperatures, after third-instar larvae were exposed to different doses of ␥ radiation. After taking into account the loss of adult viability caused by raising the mutants at 24⬚C, our data show that atm mutant viability was drastically compromised by exposure to ionizing radiation, even at the lowest dose of 1 Gy (Figure 3E). Thus, we conclude that dATM is required for cells to survive both spontaneous and induced DNA damage.
Telomere Fusions and Chromosome Instability in atm Mutant Neuroblasts ATM has been implicated in two processes that are fundamental to chromosome integrity: repair of DNA double-strand breaks (DSBs) and telomere maintenance [1, 32]. To examine chromosome stability, we analyzed larval neuroblasts from atm mutant and control larvae (Experimental Procedures) and observed an astonishingly high rate of spontaneous chromosomal aberrations in the mutants (compare Figures 4A with 4B–4I). Similar findings are reported in an accompanying paper [10]. The most frequent aberrations were telomere fusions, affecting ⬎50% of metaphase spreads (N ⬎ 100). Both single- and double-telomere associations (TA) were observed, including sister and nonsister fusions of homologous chromosomes (Figures 4B–4H), as well as fusions between nonhomologous chromosomes (Figures 4C– 4F). Acentric chromosome fragments were also observed on rare occasions (Figures 4G and 4H, arrows), and chromosomal transpositions involving the loss of whole chromosome arms (Figure 4I). Dicentric chromosomes resulting from telomere fusions are inherently unstable, because the kinetochores can be captured
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Figure 4. Mutations Affecting atm Cause Spontaneous Telomere Associations and Other Major Chromosomal Abnormalities Shown are mitotic neuroblasts stained with DAPI to visualize DNA. (A) Control (atm/ TM6B) showing a normal male metaphase chromosome configuration. TheTM6B balancer chromosome is labeled with a small capital letter “B”. (B) atm3/atm6 mutant metaphase showing a single telomere association (STA) between two chromosomes 3 (arrow). (C) atm3/atm6 metaphase showing a double telomere association (DTA) between two chromosomes 3 (arrow). (D) atm3/atm6 metaphase showing a DTA between chromosomes 2 and 3 (arrowhead) and between chromosomes 4 and X (arrow). (E) atm3/atm6 metaphase showing a DTA between chromosomes 3 and X (arrowhead), and two STAs (arrows) between chromosome 3, as well as a dicentric made up of two chromsomes 2. (F) atm3/atm6 metaphase showing a DTA between two chromosomes 3 (arrow) and a multi-centric chromosome made up of TAs between two X chromosomes (arrow) and two chromosomes 4 (arrowhead). (G) atm3/atm6 metaphase showing a TA between sister chromatids of the X chromosome (arrowhead). The arrow points to a broken chromosome, probably originating from chromosome 3 (asterisk). (H) atm3/atm6 metaphase showing a TA between sister chromatids of chromosome 3 (arrowhead). The arrow points to a broken chromosome 3. (I) atm3/atm6 metaphase showing two broken arms from chromosome 3 (asterisk), fused to the ends of the other chromosome 3 (arrow). (J) Control atm/TM6B anaphase. (K) atm3/atm6 mutant anaphase, showing a single chromatin bridge. (L) atm3/atm6 mutant anaphase showing multiple chromatin bridges.
by microtubules from opposite spindle poles, causing chromosome bridges that eventually rupture, triggering apoptosis [33]. Consistent with this expectation, we observed frequent chromosomal bridges in atm mutant anaphase spreads (compare Figure 4J with Figures 4K and 4L). Collectively, our data and that of an accompanying paper [10] implicate dATM in normal telomere maintenance and chromosome stability. Conclusions Our results indicate that the primary function of dATM is in chromosome structural maintenance, without which proliferating cells are vulnerable to apoptosis. Data in an accompanying paper further implicate dATM in an early premitotic checkpoint response to ionizing radiation [11], suggesting that ATM and ATR carry out temporally distinct cell cycle checkpoint functions as they do in mammals [27]. Also in mammals, the DNA repair functions of ATM overlap with those of a functionally redundant, ATM-related kinase called DNA-PK [34]. DNA-PK is not conserved in Drosophila [12], perhaps explaining why ATM is essential for morphological development in Drosophila but not in mammals. Inappropriate apoptosis occurs predominantly in proliferating atm mutant cells, suggesting that dATM is re-
quired for repairing spontaneous DSBs arising during DNA replication [35]. The extraordinary frequency of spontaneous telomere fusions in atm mutant neuroblasts also suggests a critical role for dATM in telomere maintenance in cycling cells. Drosophila telomeres are unusual because they are maintained by retrotransposition rather than by telomerase activity, as they are in other systems [36]. Given this difference, it is very intriguing that ATM has now been linked to telomere maintenance in eukaryotic organisms as distantly related as yeast, Drosophila, and humans [32]. The molecular mechanisms of ATM-dependent telomere maintenance have not yet been established in any organism, however, studies in yeast suggest that ATM/TEL1 activity prevents spontaneous chromosomal rearrangements involving telomeres during the repair of DSBs [2–4]. The conserved MRN protein complex (comprised of Mre11, Rad50, and Nbs1) is required for all known ATM-dependent functions including telomere maintenance [32, 37, 38]. Mutations affecting Drosophila mre11 and rad50 also result in pupal lethality and telomere fusions, consistent with these genes being involved in common DNA repair and chromosome structural maintenance functions (M. Gatti and W. Engels, personal communication). Recent genetic screens have identified a number of Dro-
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sophila genes of unknown function that are required for preventing spontaneous telomere fusions, many of which are probably also involved in ATM-dependent telomere maintenance functions [39–41]. Drosophila atm mutants recapitulate major symptoms of ataxia telangiectasia including locomotor defects, sensitivity to ionizing radiation, and chromosome instability [8, 9]. Curiously, loss of ATM seems to affect developing neuronal tissues more than non-neuronal tissues, in both flies and humans. As discussed above, this correlation may reflect a relationship between telomere dysfunction and cell proliferation. Alternatively, specific developmental events may render certain cells more sensitive to loss of ATM function. Chromatin remodelling has been shown to activate ATM in cultured mammalian cells [42], making it tempting to speculate that neuronspecific chromatin remodelling processes might normally elicit ATM activity. Drosophila promises to be an excellent model for investigating the basic mechanisms of chromosome structural maintenance involving ATM, allowing these possibilities to be studied in a meaningful developmental context. Experimental Procedures Genetic Screen for atm Alleles and Rescue with a P{atm⫹} Transgene We followed standard protocols to identify eight ethyl methane sulfonate-induced lethal mutations of atm, by using chromosomal deletion Df(3R)PG4 to target the atm locus [43]. Further details on the screen can be found in Supplemental Data. To confirm that the described mutant phenotypes were due to loss of dATM function, we made a rescue construct by subcloning a 14.5 kilobase PvuIApaI restriction fragment that included the entire atm coding region and adjacent flanking DNA, from genomic DNA clone BACR32A03, into a P{mini-w⫹} vector. We then used standard methods to obtain the P{atm⫹,w⫹} insertion allele [44]. Genomic sequence analysis of the atm6 allele identified a molecular lesion converting a tryptophan codon (W977) to a stop codon, eliminating all conserved regions of dATM that are essential for function. Determining the Temperature-Sensitive Periods for dATM Function A series of parallel genetic crosses were set up, each producing marked, hemizygous atm8/Df(3R)PG4 mutant progeny. Half of the vials were initially raised at 22⬚C (permissive temperature), then shifted up to 29⬚C (nonpermissive temperature), whereas the remaining vials were initially raised at 29⬚C before being shifted to 22⬚C. One vial among each set was shifted each day of the experiment. Flies were scored after development was completed (see Results and Discussion), and the data was graphed relative to the time of the temperature shift for each vial, to identify the temperature-sensitive period for each mutant phenotype [13]. Adult Climbing Assays The genotype of atm mutants used in these assays was atm8/ Df[3R]PG4, controls were P{atm⫹}; atm8/Df[3R]PG4). Flies were raised at the semipermissive temperature for the atm8 allele (24⬚C ⫹/⫺ 1⬚C). Except where noted in the text, climbing assays were conducted on young adult flies within 2 days of pupal eclosion. For the individual climbing assays, we individually tested ten mutant adults and ten controls (of each sex) for their climbing activity, by tapping each fly to the bottom of a graduated pipette and measuring how high it climbed in 15 s, with five replicates. A Microsoft Excel spreadsheet showing the raw data from this experiment can be found in the Supplemental Data, as well as a movie illustrating the atm mutant locomotor defects. Antibody and BrdU Labeling We followed published procedures for BrdU labeling, antibody staining, and electron microscopy [45, 46]. Antibodies, sources, and
dilutions used for the imaginal disc and embryo labeling experiments were: rabbit anti-phospho-histone H3 (Upstate Biochemicals, 1:4000), mouse anti-Cut (DSHB: Developmental Studies Hybridoma Bank, NICHD and University of Iowa, 1:200), mouse anti-Elav (DSHB, 1:100), mouse anti-BrdU (Pharmigen, 1:20), rabbit anti-activated caspase 3 (Cell Signaling Technologies, 1:800), rabbit anti-Centrosomin [47] (T. Kaufmann, 1:500), and mouse anti-␣ tubulin (Sigma, 1:100). Fluorescent secondary antibodies were from Molecular Probes (conjugated with Alexa Fluor 488 and 568, 1:1000). Neuroblast Chromosome Cytology atm3/atm6 mutants (atm3 and atm6 are both null alleles) and their sibling heterozygous control larvae were distinguished using the Tb allele on the TM6B balancer chromosome. For obtaining metaphase chromosome preparations of larval neuroblasts, larval brains were dissected in 0.7% sodium chloride, incubated with 10⫺5 colchicine for 30 min, and then treated with a hypotonic solution (0.5% sodium citrate) for 5 min. Brains were subsequently fixed for 5 min in 2% formaldehyde, 45% acetic acid and then squashed in the same fixative. Slides were frozen in liquid nitrogen, left to dry after flipping the coverslip, and mounted with DAPI-containing Vectashield (Vector Labs). Anaphase preparations were obtained in the same way except that the colchicine and hypotonic treatments were omitted. Supplemental Data Supplemental Data including Supplemental Experimental Procedures, a movie, and a table are available at http://www.currentbiology.com/cgi/content/full/14/15/1341/DC1/. Acknowledgments Many thanks to Rakesh Bhatnagar and Jack Scott for help with microscopy, to Jackie Vogel for image deconvolution, and to Bill Sullivan for his hospitality. Mel Feany, Gabrielle Boulianne, Trudi Schupbach, Jackie Vogel, Michael Weinfeld, Andrew Simmonds, Greg Gloor, and Brian Lee helped with the manuscript. Research funding for the Campell lab was provided by the A-T Children’s Project, the Canadian Institutes of Health Research (CIHR), and the National Science and Engineering Research Council of Canada (NSERC). Grant support for A.R and B.F. (GM46409, W. Sullivan) and for G.S. (GM49883, J. Tamkun) is also gratefully acknowledged. Received: March 11, 2004 Revised: May 27, 2004 Accepted: May 28, 2004 Published online: July 2, 2004 References 1. Shiloh, Y. (2003). ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer 3, 155–168. 2. Chan, S.W., and Blackburn, E.H. (2003). Telomerase and ATM/ Tel1p protect telomeres from nonhomologous end joining. Mol. Cell 11, 1379–1387. 3. Craven, R.J., Greenwell, P.W., Dominska, M., and Petes, T.D. (2002). Regulation of genome stability by TEL1 and MEC1, yeast homologs of the mammalian ATM and ATR genes. Genetics 161, 493–507. 4. Myung, K., Datta, A., and Kolodner, R.D. (2001). Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae. Cell 104, 397–408. 5. Durocher, D., and Jackson, S.P. (2001). DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr. Opin. Cell Biol. 13, 225–231. 6. Yang, J., Yu, Y., Hamrick, H.E., and Duerksen-Hughes, P.J. (2003). ATM, ATR, and DNA-PK: initiators of the cellular genotoxic stress responses. Carcinogenesis 10, 1571,1580. Published online Aug 14, 2003. 7. Shiloh, Y. (2001). ATM and ATR: networking cellular responses to DNA damage. Curr. Opin. Genet. Dev. 11, 71–77. 8. Gatti, R.A., Becker-Catania, S., Chun, H.H., Sun, X., Mitui, M., Lai, C.H., Khanlou, N., Babaei, M., Cheng, R., Clark, C., et al.
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31. Jaklevic, B.R., and Su, T.T. (2004). Relative contribution of DNA repair, cell cycle checkpoints, and cell death to survival after DNA damage in Drosophila larvae. Curr. Biol. 14, 23–32. 32. Pandita, T.K. (2002). ATM function and telomere stability. Oncogene 21, 611–618. 33. Ahmad, K., and Golic, K.G. (1999). Telomere loss in somatic cells of Drosophila causes cell cycle arrest and apoptosis. Genetics 151, 1041–1051. 34. Gurley, K.E., and Kemp, C.J. (2001). Synthetic lethality between mutation in Atm and DNA-PK(cs) during murine embryogenesis. Curr. Biol. 11, 191–194. 35. Costanzo, V., Robertson, K., Bibikova, M., Kim, E., Grieco, D., Gottesman, M., Carroll, D., and Gautier, J. (2001). Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication. Mol. Cell 8, 137–147. 36. Pardue, M.L., and DeBaryshe, P.G. (2003). Retrotransposons provide an evolutionarily robust non-telomerase mechanism to maintain telomeres. Annu. Rev. Genet. 37, 485–511. 37. Usui, T., Ogawa, H., and Petrini, J.H. (2001). A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7, 1255–1266. 38. Uziel, T., Lerenthal, Y., Moyal, L., Andegeko, Y., Mittelman, L., and Shiloh, Y. (2003). Requirement of the MRN complex for ATM activation by DNA damage. EMBO J. 22, 5612–5621. 39. Cenci, G., Siriaco, G., Raffa, G.D., Kellum, R., and Gatti, M. (2003). The Drosophila HOAP protein is required for telomere capping. Nat. Cell Biol. 5, 82–84. 40. Cenci, G., Rawson, R.B., Belloni, G., Castrillon, D.H., Tudor, M., Petrucci, R., Goldberg, M.L., Wasserman, S.A., and Gatti, M. (1997). UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior. Genes Dev. 11, 863–875. 41. Queiroz-Machado, J., Perdigao, J., Simoes-Carvalho, P., Herrmann, S., and Sunkel, C.E. (2001). tef: a mutation that causes telomere fusion and severe genome rearrangements in Drosophila melanogaster. Chromosoma 110, 10–23. 42. Bakkenist, C.J., and Kastan, M.B. (2003). DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506. 43. Hing, H.K., Bangalore, L., Sun, X., and Artavanis-Tsakonas, S. (1999). Mutations in the heatshock cognate 70 protein (hsc4) modulate Notch signaling. Eur. J. Cell Biol. 78, 690–697. 44. Spradling, A.C. (1986). P element-mediated transformation. In Drosophila: a practical approach., D.B. Roberts, ed. (Oxford: IRL). 45. Wolff, T. (2000). Histological techniques for the Drosophila eye Part I: Larva and Pupa. In Drosophila Protocols, W. Sullivan, M. Ashburner and R.S. Hawley, eds. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 201–227. 46. Wolff, T. (2000). Histological techniques for the Drosophila eye Part II: Adult. In Drosophila Protocols, W. Sullivan, M. Ashburner and R.S. Hawley, eds. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 229–244. 47. Li, K., and Kaufman, T.C. (1996). The homeotic target gene centrosomin encodes an essential centrosomal component. Cell 85, 585–596. 48. Baker, N.E., and Yu, S.Y. (2001). The EGF receptor defines domains of cell cycle progression and survival to regulate cell number in the developing Drosophila eye. Cell 104, 699–708. 49. Robinow, S., and White, K. (1991). Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development. J. Neurobiol. 22, 443–461. Note Added in Proof After the IEP (immediate early publication) version of this paper was published online July 2, 2004, the authors realized the following errors, which have since been corrected in this version. A calculation made in the conversion from rads to grays was off by one order of magnitude; therefore, in Figure 3E online, the lowest dose of radiation on the X axis should have been 1 Gy rather than 0.1 Gy. The text referring to these data was likewise incorrect online. Also, the legend for Figure 3E incorrectly stated that the gray bars represent the semirestrictive temperature and the solid bars represent the permissive temperature. The reverse is correct.
DOI 10.1016/j .c ub . 20 04 . 06 .0 5 6
ATM Is Required for Telomere Maintenance and Chromosome Stability during Drosophila Development Elizabeth Silva,1,2 Stanley Tiong,1 Michael Pedersen,1 Ellen Homola,1 Anne Royou,3 Barbara Fasulo,3 Giorgia Siriaco,3 and Shelagh D. Campbell1,* 1 Department of Biological Sciences University of Alberta Edmonton, Alberta, T6G 2E9 Canada 2 Developmental Patterning Lab London Research Institute London, WC2A 3PX United Kingdom 3 Sinsheimer Laboratories Molecular, Cell, and Developmental Biology University of California Santa Cruz, CA 95060
Summary ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints [1]. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs [2–4]. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia. Results and Discussion Eukaryotic cells rely on a core set of conserved DNA damage responses that maintain genome fidelity and ensure survival [5, 6]. In metazoans, ATM and the related protein kinases ATR and DNA-PK play partially overlapping roles in response to genotoxic stress to coordinately regulate DNA repair, cell cycle progression, and apoptosis, ensuring that damaged cells are either re*Correspondence: [email protected]
paired or eliminated [7]. Loss of ATM function causes an inherited human disease called ataxia-telangiectasia (A-T), with symptoms including progressive neurodegeneration, chromosomal instability, and pre-disposition to certain forms of cancer [8, 9]. Our functional analysis of Drosophila ATM, as well as that of the accompanying papers [10, 11], reveals conserved roles for dATM that are essential for normal development. Drosophila ATM Is Essential for Normal Adult Development Genome sequence analysis identified a gene called CG6535 that encodes a protein with highly significant sequence similarity to human ATM [12], referred to as atm in this report. To determine the function of atm, we conducted a genetic screen that isolated eight recessive lethal alleles (Experimental Procedures and Supplemental Data available with this article online). Seven of these alleles (atm1-7) have a nonconditional, pupal, lethal phenotype and one, atm8, is a temperature-sensitive hypomorphic allele. Hemizygous atm1–7 mutants die before emerging as adults, with variable morphological defects affecting the antennae, eyes, bristles, wings, and legs. The completely restrictive temperature for atm8 lethality is approximately 25⬚C. When atm8 mutants are raised at a semirestrictive temperature (24⬚C ⫹/⫺ 1oC), many can survive to adulthood with a similar range of morphological defects (Figures 1A–1D). The morphological defects, as well as atm mutant lethality, are rescued by introduction of a transgenic construct, P{atm⫹}, confirming that these phenotypes are specifically due to loss of atm function (see Experimental Procedures). To determine when dATM activity is required for normal development, we took advantage of the temperaturesensitive allele atm8 in a reciprocal temperature-shift experiment [13] that established the temperature-sensitive period (TSP) for selected atm8 mutant phenotypes (Experimental Procedures). The TSP for adult viability falls between the early third-larval instar and early pupal stage, the TSP for eye development is during the thirdlarval instar, and the TSP for wing and thoracic bristle development is during the pupal stage. These TSPs correspond to critical developmental stages of eye, wing, and bristle development. Requirements for dATM activity appear particularly stringent in developing neural tissues, because the structures most obviously affected in the atm mutants (eye, wing margin, thoracic bristles) are all involved in sensory perception. Locomotor Defects in Drosophila atm Mutants Because movement disorders (ataxia) are a prominent symptom in patients with A-T, we examined whether atm8 mutants exhibited signs of locomotor defects. We measured climbing ability in atm8 mutant and control flies for this purpose (Experimental Procedures and Supplemental Data). Replicate tests were conducted for each fly, and the averages were used for deriving a ratio (mutant/control) that provided a measure of how well
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Figure 1. Eye and Wing Defects Observed in Temperature-Sensitive atm8 Mutant Adults and Developing Imaginal Discs The adult mutant and control flies were raised at the atm8 semirestrictive temperature (24⬚C) and imaged using an environmental scanning electron microscope (A–D). (A) Normal adult eye. (B) atm8 mutant eye. (C) Normal adult wing. (D) atm8 mutant wing. (E–H) Cell patterning and apoptosis defects in third-instar atm8 mutant discs from larvae raised at the restrictive temperature (25⬚C). (E and F) Eyeantennal discs (oriented so that posterior is to the left) were labeled with antibodies to activated Caspase-3 (green), a marker for apoptosis [48] and Elav (red), a marker for differentiating neuronal cells [49]. (E) In a normal eye disc, apoptotic cells (green) are rarely seen at this stage of development and differentiating neuronal cells (red) form organized “preclusters” (inset). (F) Extensive apoptosis (green) occurs in the anterior region of an atm8 mutant disc, accompanied by obvious disorganization of the differentiating neuronal cells (red, inset). (G and H) Third-larval instar wing discs stained with antibodies to Cut [17]. (G) A normal wing disc showing Cut-positive myoblasts [19] associated with the presumptive thorax region of the disc (to the left) and a stripe of Cut-positive cells along the developing wing margin [17]. (H) An atm8 mutant wing disc showing loss of Cut labeling along the presumptive wing margin, although Cut expression appears normal in myoblasts adjoining the thorax region of the wing disc.
atm8 mutants climb, relative to matched controls [14]. The average climbing ability of atm8 mutant males was markedly lower than normal (ratio of mutants/controls ⫽ 0.11). Interestingly, the atm8 mutant females performed even worse in this assay (mutants/controls ⫽ 0.01). A movie illustrating the locomotor defects of atm mutants can be found in the Supplemental Data. The relative differences in climbing ability between mutants and controls did not change over a period of 4 weeks when the flies were maintained at the restrictive temperature of 29⬚C (data not shown). Thus, the locomotor defects in atm mutants appear to be strictly developmental. Apoptosis and Patterning Defects in Developing atm Tissues Because we had established that the temperature-sensitive period for normal eye development in atm8 mutants is during the third-larval instar, we further analyzed eye-antennal discs at this stage of development. A structure called the morphogenetic furrow (MF) sweeps across the eye disc during the late-third instar, marking a transition between proliferating, undifferentiated cells (anterior to the furrow) and nonproliferating cells that are differentiating into distinct neuronal cell types, posterior to the furrow [15]. The atm8 mutant-eye antennal discs are generally smaller than comparably aged controls (data not shown). Conspicuously, atm8 mutant eye discs had large numbers of cells undergoing inappropriate apoptosis, marked by antibody staining for activated caspase-3 (Figures 1E and 1F). There were striking regional differences in the distribution of apoptotic cells
in the mutant eye discs; these differences were also seen in discs stained with acridine orange staining as another marker of apoptosis (data not shown). These apoptotic cells were primarily restricted to the anterior, proliferating region of the disc (Figure 1F, green). There were relatively few apoptotic cells in the posterior region of the mutant discs, containing differentiating neuronal cells (Figure 1F, red). Early neuronal patterning was markedly disrupted in mutant discs, presumably because so many cells were eliminated by earlier apoptosis that normal precluster formation was precluded (Figures 1E and 1F, inset). The adult wing is also affected by loss of atm function, so we examined atm8 mutant third-larval instar wing discs for developmental defects. At this stage, cells in the wing disc are proliferating asynchronously except for a stripe of nondividing, differentiating cells along the presumptive wing margin of the disc [16]. These cells express Cut, a Notch-induced transcription factor required for proper differentiation of the wing margin [17]. We observed partial to complete loss of Cut expression along the presumptive wing margin of atm8 mutant discs, a patterning defect that anticipates the wing margin defects seen in atm mutants later in development (Figures 3G and 3H). Intriguingly, Cut expression was not obviously affected in the myoblast cells adjoining the presumptive dorsal thorax region of the atm mutant wing discs [18, 19]. The Cut expression defect in the developing wing margin could be due to inappropriate cell death in the proliferating imaginal cells, because spontaneous apoptosis also occurs throughout the atm8 mu-
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Figure 2. Early Developmental Defects in atm6 Mutants Derived from Germline Mosaic Females (A) Normal egg, oriented with dorsal appendages to the right. (B) atm6 mutant egg, showing fused dorsal appendages and chorion defects. (C, D) Cycle 11 embryos, stained with antibodies to Centrosomin (red, centrosomes), antibodies to alpha-tubulin (green, microtubules), and Hoescht 33258 (blue, DNA). These images were deconvolved using Volocity Restoration software. (C) Normal metaphase 11. (D) Representative field from a metaphase 11 atm6 mutant, showing extensive mitotic defects (isolated centrosomes, missing nuclei, aberrant mitotic spindles, and chromosome bridges).
tant wing discs at this developmental stage (data not shown; [11]). The spontaneous apoptosis and patterning defects in atm eye and wing imaginal discs suggest that cells are experiencing DNA damage, which is an established trigger for p53-dependent apoptosis in Drosophila [20, 21]. Early Developmental Requirements for Drosophila ATM Oogenesis and early embryogenesis are regulated by maternally acting genes in Drosophila. To determine if dATM activity is required during early stages of development, we examined eggs derived from atm6 mutantgermline mosaic females, generated using the hs-FLP/ FRT ovoD system [22]. atm6 was molecularly characterized as a functional null allele (Experimental Procedures). Maternal mutant atm6 eggs commonly have dorsal appendage defects (Figures 2A and 2B), resembling a phenotype of homologous DSB repair-defective female-sterile mutants [23]. The eggshells of atm6 mutant eggs also appear to be thinner than normal, suggesting that follicle cell development is compromised. Developing atm6 mutant embryos show dramatic mitotic defects during the rapid syncytial divisions of early embryogenesis. These mitotic defects included frequent spindle fusions and chromosomal bridges (Figures 2C and 2D) that could also be observed much earlier in development before the nuclei reach the surface of the embryo (data not shown). Another frequently observed defect involved nuclei detaching from centrosomes and falling from the cortex (Figures 2C and 2D and data not shown).
The centrosome and nuclear fallout defect in atm mutants resembles a local DNA damage-induced response called “centrosome inactivation,” indicating that loss of dATM is associated with spontaneous DNA damage [24, 25]. Chk2 is an established transducer of ATM signaling in mammalian cells [26] and was previously identified as a positive regulator of centrosome inactivation in Drosophila [25]. Because centrosome inactivation occurs spontaneously in atm mutants, this result suggests that either a dATM-independent mechanism for activating Chk2 exists or a Chk2-independent mechanism for promoting centrosome inactivation was deployed. Loss of dATM Causes Extreme Sensitivity to Ionizing Radiation In mammalian cells, the ATM and ATR signaling pathways negatively regulate Cdk1 in response to ionizing radiation, blocking mitosis while DNA is being repaired [27]. Mei-41, the Drosophila ATR ortholog, is required for this response to DNA damage [28–31]. To examine if dATM might also be required for this response, we labeled mitotic cells in atm mutant and control wing discs, with or without exposure to 40 Gy of ␥ irradiation (Figures 3A–3D). Mitotic cells were essentially absent 1 hr after ionizing radiation in both atm mutant and control discs, implying that dATM is dispensable for this premitotic-checkpoint response to acute DNA damage. An accompanying paper reports that dATM operates in a premitotic checkpoint, which is activated earlier in response to ionizing radiation than our measurements but concurs with our findings that the checkpoint response is fully engaged by 1 hr after irradiation [11].
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Figure 3. atm8 Mutants Have a Functional Premitotic Checkpoint Response but Are Extremely Sensitive to Ionizing Radiation (A–D) Third-larval instar wing discs, stained with antibodies to phospho-histone H3 to detect mitotic cells. (A) Unirradiated control (atm8 /TM6B) wing disc. (B) Irradiated control wing disc, fixed 1 hr after the larvae were exposed to 40 Gy of ␥ irradiation. (C) Unirradiated atm8 mutant wing disc. (D) Irradiated atm8 mutant wing disc, fixed 1 hr after exposure to 40 Gy. (E) Genetic crosses were made that were expected to generate hemizygous atm8 mutants as approximately one third of the progeny (raised at fully permissive temperature). The graph shows the atm8/ Df(3R)PG4 survival frequency of adults that were either raised at 22⬚C (permissive temperature: gray bars) or at 24⬚C (semirestrictive temperature: solid bars), relative to the dosage of ionizing radiation received by the respective third-instar larvae.
We also observed increased numbers of apoptotic cells in both controls and atm mutant wing discs, after larvae were exposed to ionizing radiation (data not shown). Because the apoptosis response to ionizing radiation in Drosophila is thought to depend on p53 [20, 21], this result implies that p53-dependent apoptosis is functional in atm mutants. An accompanying paper reports that expression of a dominant-negative p53 transgene suppresses spontaneous apoptosis in atm mutant wing discs, supporting our conclusion that the p53-dependent apoptosis response is intact [11]. We found that atm mutants are extremely sensitive to ionizing radiation. We assayed radiation sensitivity by comparing the adult viability of atm8 mutants, raised at either semirestrictive (24⬚C) or permissive (22⬚C) temperatures, after third-instar larvae were exposed to different doses of ␥ radiation. After taking into account the loss of adult viability caused by raising the mutants at 24⬚C, our data show that atm mutant viability was drastically compromised by exposure to ionizing radiation, even at the lowest dose of 1 Gy (Figure 3E). Thus, we conclude that dATM is required for cells to survive both spontaneous and induced DNA damage.
Telomere Fusions and Chromosome Instability in atm Mutant Neuroblasts ATM has been implicated in two processes that are fundamental to chromosome integrity: repair of DNA double-strand breaks (DSBs) and telomere maintenance [1, 32]. To examine chromosome stability, we analyzed larval neuroblasts from atm mutant and control larvae (Experimental Procedures) and observed an astonishingly high rate of spontaneous chromosomal aberrations in the mutants (compare Figures 4A with 4B–4I). Similar findings are reported in an accompanying paper [10]. The most frequent aberrations were telomere fusions, affecting ⬎50% of metaphase spreads (N ⬎ 100). Both single- and double-telomere associations (TA) were observed, including sister and nonsister fusions of homologous chromosomes (Figures 4B–4H), as well as fusions between nonhomologous chromosomes (Figures 4C– 4F). Acentric chromosome fragments were also observed on rare occasions (Figures 4G and 4H, arrows), and chromosomal transpositions involving the loss of whole chromosome arms (Figure 4I). Dicentric chromosomes resulting from telomere fusions are inherently unstable, because the kinetochores can be captured
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Figure 4. Mutations Affecting atm Cause Spontaneous Telomere Associations and Other Major Chromosomal Abnormalities Shown are mitotic neuroblasts stained with DAPI to visualize DNA. (A) Control (atm/ TM6B) showing a normal male metaphase chromosome configuration. TheTM6B balancer chromosome is labeled with a small capital letter “B”. (B) atm3/atm6 mutant metaphase showing a single telomere association (STA) between two chromosomes 3 (arrow). (C) atm3/atm6 metaphase showing a double telomere association (DTA) between two chromosomes 3 (arrow). (D) atm3/atm6 metaphase showing a DTA between chromosomes 2 and 3 (arrowhead) and between chromosomes 4 and X (arrow). (E) atm3/atm6 metaphase showing a DTA between chromosomes 3 and X (arrowhead), and two STAs (arrows) between chromosome 3, as well as a dicentric made up of two chromsomes 2. (F) atm3/atm6 metaphase showing a DTA between two chromosomes 3 (arrow) and a multi-centric chromosome made up of TAs between two X chromosomes (arrow) and two chromosomes 4 (arrowhead). (G) atm3/atm6 metaphase showing a TA between sister chromatids of the X chromosome (arrowhead). The arrow points to a broken chromosome, probably originating from chromosome 3 (asterisk). (H) atm3/atm6 metaphase showing a TA between sister chromatids of chromosome 3 (arrowhead). The arrow points to a broken chromosome 3. (I) atm3/atm6 metaphase showing two broken arms from chromosome 3 (asterisk), fused to the ends of the other chromosome 3 (arrow). (J) Control atm/TM6B anaphase. (K) atm3/atm6 mutant anaphase, showing a single chromatin bridge. (L) atm3/atm6 mutant anaphase showing multiple chromatin bridges.
by microtubules from opposite spindle poles, causing chromosome bridges that eventually rupture, triggering apoptosis [33]. Consistent with this expectation, we observed frequent chromosomal bridges in atm mutant anaphase spreads (compare Figure 4J with Figures 4K and 4L). Collectively, our data and that of an accompanying paper [10] implicate dATM in normal telomere maintenance and chromosome stability. Conclusions Our results indicate that the primary function of dATM is in chromosome structural maintenance, without which proliferating cells are vulnerable to apoptosis. Data in an accompanying paper further implicate dATM in an early premitotic checkpoint response to ionizing radiation [11], suggesting that ATM and ATR carry out temporally distinct cell cycle checkpoint functions as they do in mammals [27]. Also in mammals, the DNA repair functions of ATM overlap with those of a functionally redundant, ATM-related kinase called DNA-PK [34]. DNA-PK is not conserved in Drosophila [12], perhaps explaining why ATM is essential for morphological development in Drosophila but not in mammals. Inappropriate apoptosis occurs predominantly in proliferating atm mutant cells, suggesting that dATM is re-
quired for repairing spontaneous DSBs arising during DNA replication [35]. The extraordinary frequency of spontaneous telomere fusions in atm mutant neuroblasts also suggests a critical role for dATM in telomere maintenance in cycling cells. Drosophila telomeres are unusual because they are maintained by retrotransposition rather than by telomerase activity, as they are in other systems [36]. Given this difference, it is very intriguing that ATM has now been linked to telomere maintenance in eukaryotic organisms as distantly related as yeast, Drosophila, and humans [32]. The molecular mechanisms of ATM-dependent telomere maintenance have not yet been established in any organism, however, studies in yeast suggest that ATM/TEL1 activity prevents spontaneous chromosomal rearrangements involving telomeres during the repair of DSBs [2–4]. The conserved MRN protein complex (comprised of Mre11, Rad50, and Nbs1) is required for all known ATM-dependent functions including telomere maintenance [32, 37, 38]. Mutations affecting Drosophila mre11 and rad50 also result in pupal lethality and telomere fusions, consistent with these genes being involved in common DNA repair and chromosome structural maintenance functions (M. Gatti and W. Engels, personal communication). Recent genetic screens have identified a number of Dro-
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sophila genes of unknown function that are required for preventing spontaneous telomere fusions, many of which are probably also involved in ATM-dependent telomere maintenance functions [39–41]. Drosophila atm mutants recapitulate major symptoms of ataxia telangiectasia including locomotor defects, sensitivity to ionizing radiation, and chromosome instability [8, 9]. Curiously, loss of ATM seems to affect developing neuronal tissues more than non-neuronal tissues, in both flies and humans. As discussed above, this correlation may reflect a relationship between telomere dysfunction and cell proliferation. Alternatively, specific developmental events may render certain cells more sensitive to loss of ATM function. Chromatin remodelling has been shown to activate ATM in cultured mammalian cells [42], making it tempting to speculate that neuronspecific chromatin remodelling processes might normally elicit ATM activity. Drosophila promises to be an excellent model for investigating the basic mechanisms of chromosome structural maintenance involving ATM, allowing these possibilities to be studied in a meaningful developmental context. Experimental Procedures Genetic Screen for atm Alleles and Rescue with a P{atm⫹} Transgene We followed standard protocols to identify eight ethyl methane sulfonate-induced lethal mutations of atm, by using chromosomal deletion Df(3R)PG4 to target the atm locus [43]. Further details on the screen can be found in Supplemental Data. To confirm that the described mutant phenotypes were due to loss of dATM function, we made a rescue construct by subcloning a 14.5 kilobase PvuIApaI restriction fragment that included the entire atm coding region and adjacent flanking DNA, from genomic DNA clone BACR32A03, into a P{mini-w⫹} vector. We then used standard methods to obtain the P{atm⫹,w⫹} insertion allele [44]. Genomic sequence analysis of the atm6 allele identified a molecular lesion converting a tryptophan codon (W977) to a stop codon, eliminating all conserved regions of dATM that are essential for function. Determining the Temperature-Sensitive Periods for dATM Function A series of parallel genetic crosses were set up, each producing marked, hemizygous atm8/Df(3R)PG4 mutant progeny. Half of the vials were initially raised at 22⬚C (permissive temperature), then shifted up to 29⬚C (nonpermissive temperature), whereas the remaining vials were initially raised at 29⬚C before being shifted to 22⬚C. One vial among each set was shifted each day of the experiment. Flies were scored after development was completed (see Results and Discussion), and the data was graphed relative to the time of the temperature shift for each vial, to identify the temperature-sensitive period for each mutant phenotype [13]. Adult Climbing Assays The genotype of atm mutants used in these assays was atm8/ Df[3R]PG4, controls were P{atm⫹}; atm8/Df[3R]PG4). Flies were raised at the semipermissive temperature for the atm8 allele (24⬚C ⫹/⫺ 1⬚C). Except where noted in the text, climbing assays were conducted on young adult flies within 2 days of pupal eclosion. For the individual climbing assays, we individually tested ten mutant adults and ten controls (of each sex) for their climbing activity, by tapping each fly to the bottom of a graduated pipette and measuring how high it climbed in 15 s, with five replicates. A Microsoft Excel spreadsheet showing the raw data from this experiment can be found in the Supplemental Data, as well as a movie illustrating the atm mutant locomotor defects. Antibody and BrdU Labeling We followed published procedures for BrdU labeling, antibody staining, and electron microscopy [45, 46]. Antibodies, sources, and
dilutions used for the imaginal disc and embryo labeling experiments were: rabbit anti-phospho-histone H3 (Upstate Biochemicals, 1:4000), mouse anti-Cut (DSHB: Developmental Studies Hybridoma Bank, NICHD and University of Iowa, 1:200), mouse anti-Elav (DSHB, 1:100), mouse anti-BrdU (Pharmigen, 1:20), rabbit anti-activated caspase 3 (Cell Signaling Technologies, 1:800), rabbit anti-Centrosomin [47] (T. Kaufmann, 1:500), and mouse anti-␣ tubulin (Sigma, 1:100). Fluorescent secondary antibodies were from Molecular Probes (conjugated with Alexa Fluor 488 and 568, 1:1000). Neuroblast Chromosome Cytology atm3/atm6 mutants (atm3 and atm6 are both null alleles) and their sibling heterozygous control larvae were distinguished using the Tb allele on the TM6B balancer chromosome. For obtaining metaphase chromosome preparations of larval neuroblasts, larval brains were dissected in 0.7% sodium chloride, incubated with 10⫺5 colchicine for 30 min, and then treated with a hypotonic solution (0.5% sodium citrate) for 5 min. Brains were subsequently fixed for 5 min in 2% formaldehyde, 45% acetic acid and then squashed in the same fixative. Slides were frozen in liquid nitrogen, left to dry after flipping the coverslip, and mounted with DAPI-containing Vectashield (Vector Labs). Anaphase preparations were obtained in the same way except that the colchicine and hypotonic treatments were omitted. Supplemental Data Supplemental Data including Supplemental Experimental Procedures, a movie, and a table are available at http://www.currentbiology.com/cgi/content/full/14/15/1341/DC1/. Acknowledgments Many thanks to Rakesh Bhatnagar and Jack Scott for help with microscopy, to Jackie Vogel for image deconvolution, and to Bill Sullivan for his hospitality. Mel Feany, Gabrielle Boulianne, Trudi Schupbach, Jackie Vogel, Michael Weinfeld, Andrew Simmonds, Greg Gloor, and Brian Lee helped with the manuscript. Research funding for the Campell lab was provided by the A-T Children’s Project, the Canadian Institutes of Health Research (CIHR), and the National Science and Engineering Research Council of Canada (NSERC). Grant support for A.R and B.F. (GM46409, W. Sullivan) and for G.S. (GM49883, J. Tamkun) is also gratefully acknowledged. Received: March 11, 2004 Revised: May 27, 2004 Accepted: May 28, 2004 Published online: July 2, 2004 References 1. Shiloh, Y. (2003). ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer 3, 155–168. 2. Chan, S.W., and Blackburn, E.H. (2003). Telomerase and ATM/ Tel1p protect telomeres from nonhomologous end joining. Mol. Cell 11, 1379–1387. 3. Craven, R.J., Greenwell, P.W., Dominska, M., and Petes, T.D. (2002). Regulation of genome stability by TEL1 and MEC1, yeast homologs of the mammalian ATM and ATR genes. Genetics 161, 493–507. 4. Myung, K., Datta, A., and Kolodner, R.D. (2001). Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae. Cell 104, 397–408. 5. Durocher, D., and Jackson, S.P. (2001). DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr. Opin. Cell Biol. 13, 225–231. 6. Yang, J., Yu, Y., Hamrick, H.E., and Duerksen-Hughes, P.J. (2003). ATM, ATR, and DNA-PK: initiators of the cellular genotoxic stress responses. Carcinogenesis 10, 1571,1580. Published online Aug 14, 2003. 7. Shiloh, Y. (2001). ATM and ATR: networking cellular responses to DNA damage. Curr. Opin. Genet. Dev. 11, 71–77. 8. Gatti, R.A., Becker-Catania, S., Chun, H.H., Sun, X., Mitui, M., Lai, C.H., Khanlou, N., Babaei, M., Cheng, R., Clark, C., et al.
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