A-T is an autosomal recessive disorder characterized by progressive cerebellar degeneration, immunodeficiency, cancer predisposition, gonadal atrophy, growth retardation, premature aging, and hypersensitivity to IR (Lavin and Shiloh, 1997; McKinnon, 2004). Cells from A-T patients generally have short telomeres and are also highly sensitive to IR (Kishi and Lu, 2002; Metcalfe, et al., 1996; Meyn, 1995; Pandita et al., 1995; Shiloh, 1995; Smilenov et al., 1997). The molecular cloning of the gene responsible for A-T, ATM, has allowed a better understanding of both ATM function and the A-T pleiotropic phenotypes (Savitsky et al., 1995a; Savitsky et al., 1995b;
Taylor, 1998). The ATM gene encodes a nuclear phospho-protein (Chen and Lee, 1996; Scott et al., 1998), with serine/threonine protein kinase activity for which many downstream molecules, such as p53, Chk2, Mdm2, NBS1, BRCA1, 53BP1 SMC1, FANC2, H2AX, Pin2/TRF1, and TRF2, which control cell cycle check points, DNA double-strand break or repair pathway, and telomere metabolism, have been identified as substrates (Kastan and Lim, 2000; Kishi and Lu, 2002; Kishi et al., 2001; Pandita, 2002; Shiloh, 2003; Tanaka et al., 2005). ATM functions as a potent protein kinase that is activated by DNA damage, such as IR, to phosphorylate target substrates. The identification of these potential substrates places ATM in a signal transduction pathway, through which it functions to regulate cell-cycle checkpoints that mediate the DNA damage response and telomere home-ostasis (Verdun et al., 2005).
As a model system, ATM-knockout mice have been created in several laboratories by specific germline inacti-vation of the ATM gene (Barlow et al., 1996; Elson et al., 1996; Herzog et al., 1998; Xu et al., 1996). Fibroblasts isolated from ATM-knockout mice display similar cellular phenotypes to those observed in cells from A-T patients (Elson et al., 1996). Also, phenotypically, ATM-deficient mice display a variety of growth defects, meiotic defects, immunological abnormalities, radiation hypersensitivity and cancer predisposition, similar to those seen in A-T patients, confirming the most common pleiotropic roles of ATM. Early resistance to apoptosis in the developing central nervous system (CNS) of ATM-knockout mice has been observed after IR, especially in diverse regions of the CNS including the cerebellum (Herzog et al., 1998), which is markedly affected in A-T. Interestingly, the neurological defects in A-T become apparent early in life, suggesting that they likewise originate during development (Herzog et al., 1998). However, little is known about ATM expression and function during early development in lower vertebrates, such as zebrafish.
To develop a zebrafish A-T model, we first isolated zebrafish ATM (zATM) cDNA. We recently provided the zATM cDNA sequence as a predicted primary structure (Imamura and Kishi, 2005). At the amino acid level, the overall identity was 58% between zATM and hATM. The high degree of conservation between zATM and hATM especially within the kinase domains (81% identity) strongly suggests that the catalytic activity of these proteins is conserved. The decreasing levels of identity outside the catalytic domains may reflect potential differences in regulation of these proteins between species, though this remains to be elucidated. Importantly, a number of amino acids corresponding to sites that are found to be mutated in A-T patients are highly conserved between hATM and zATM (Gilad et al., 1996). In actuality, Garg et al. recently reported that a kinase-inactive form of the zATM catalytic domain exhibited dominant-negative activity in human and zebrafish cell lines (Garg et al., 2004). The presence of these conserved amino acid sites further attests that corresponding zATM mutants from chemically point-mutated zebrafish genomes can probably be identified.
We also outlined zATM expression patterns during early development of zebrafish embryos. We observed that zATM mRNA was ubiquitously expressed during gastrulation and early neurulation. Differential tissue expression was evidenced by increased mRNA levels at later developmental stages in the eye, brain, and somites, with relatively weak expression in the trunk and tail. The profile of zATM expression is consistent with previous observations in Xenopus (Hensey et al., 2000) and mice (Chen and Lee, 1996; Soares et al., 1998), which also show increased expression of ATM in CNS, suggesting a common importance in early development of the nervous system among vertebrates.
By using antisense-morpholino oligonucleotides (MOs) to achieve in vivo elimination of zATM expression, we observed that loss of zATM leads to abnormal development and increased lethality in the early stages of development upon IR-induced DNA damage. Our results strongly support a model for structural and functional conservation of ATM's role in the DNA damage response among vertebrates from fish to human.
Our reported results also support the notion that ATM heterozygous phenotypes resemble those caused by protein dosage reduction, in genomic chromosomal instability and cancer susceptibility, which may all be due to diminished cell-cycle checkpoint function. We suspect that this hypothesis would be relevant not only to human A-T carriers, but also to heterozygous A-T zebrafish. However, there are conflicting data on the role of ATM heterozygosity in cancer risk (Concannon, 2002; FitzGerald et al., 1997; Laposa et al., 2004; Spring et al., 2002). On the one hand, Barlow et al. argued that ATM heterozygous mice showed no evidence of increased acute radiation toxicity, although the mice intriguingly displayed premature greying and decreased survival at higher sublethal doses of irradiation (Barlow et al., 1999b), suggesting progressive senescence with DNA damage.
ATM function is essential for telomere metabolism as well as DNA damage response. ATM is required for telo-mere maintenance and chromosome stability because inactivation of ATM causes telomere shortening (Metcalfe et al., 1996; Pandita, 2002). Importantly, terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In zebrafish as well as in mammals, ATM can be activated by DNA damage and could act through a telomerase-independent mechanism to regulate telomere length, protection, and homeostasis. It has been demonstrated that the Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene (Bi et al., 2004; Oikemus et al., 2004; Queiroz-Machado et al.,
2001; Silva et al., 2004; Song et al., 2004). In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present in Drosophila. Highly spontaneous apoptosis is observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates ATM-independent but p53-dependent apoptosis through an alternative DNA damage-response mechanism. Furthermore, loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect, suggesting that recognition of chromosome ends by ATM prevents telomerc fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres (Oikemus et al., 2004).
Epigenetic control apparently provides a mechanism not only for the position effect at telomeres on the transcription of nearby genes but also for the reversible silencing of telomerase expression that occurs as a natural consequence of cellular proliferation and differentiation. There exists significant overlap between indirect telomeric regulation pathways and cell-cycle checkpoint pathways, suggesting that these discrete genetic elements—namely, ATM, p53, p21, and TERT—synergistically cooperate to inhibit tumorigenesis and to govern aging. Mutations in these pathways have been known to contribute to cancer formation and senescence occurence in mammals. Besides genetic control, the incorporation of epigenetic regulatory mechanisms provides another line of defense against these negative occurrences. Although the debate still continues, there is significant evidence to view the process of cellular senescence as an in vitro model for human aging. In addition to A-T, other disorders such as Werner's syndrome, dyskeratosis congenita, ulcerative colitis, and atherosclerosis have been linked to aberration of telomere homeostasis, and other aging-related lesions could be related to changes in cellular microenvironment resulting from the presence of senescent cells. Therefore, simply restoring direct telomerase activity as a putative therapeutic strategy rather than fine-tuning telomere homeostasis necessitates further study to elucidate the link between genetic and epigenetic modulations of telo-merase in humans (Lai et al., 2005). This is a relevant argument considering our observation of zebrafish telomerase and telomere regulation in vivo because as mentioned above, their telomere metabolism is not simply regulated by detectable telomerase activity, particularly very late in life with stochastic telomere shortening in the presence of constitutive telomerase activity.
In any case, once a stable line of zebrafish with ATM disruption or inactivation has been obtained, it will be possible to use the zebrafish A-T model to identify candidate therapeutic interventions. On a broad scope, zebrafish additionally couple the power of genetics and functional genomics for efficient mutant screening. As such, a zebrafish model system for A-T will be amenable to studies directed towards identifying functional modifier genes that affect the ATM signaling pathway, as well as ultimately identifying small molecules that can ameliorate functional disorders caused by ATM mutations.
With respect to a future direction of this study, pilot screens of genetic mutants have been conducted using F2 heterozygous embryos under genotoxic or oxidative stress. Developmental defects and premature senescence phenotype have been confirmed in F3 homozygous recessive embryos. These results imply that a large-scale screen using a chemical mutagen will allow us to identify physiologically relevant players in the stress-associated signaling pathway as targets for the interventions in aging and age-associated diseases. However, positional cloning of the point mutations of a target gene will still be time consuming for identifying whether ATM or the other related gene is critical for readout of the zebrafish mutants.
Further studies of DNA damage response and ATM function in zebrafish may serve to unravel the regulation of genomic integrity and pleiotropic features, including progeroid manifestation, as the functional roles of ATM seem to have been commonly conserved or have commonly evolved among vertebrates from fish to humans.
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