Altered Telomere Nuclear Matrix Interactions and Nucleosomal Periodicity in Ataxia Telangiectasia Cells before and after Ionizing Radiation Treatment

Ataxia telangiectasia (A-T) is a rare autosomal human recessive disorder characterized by progressive neurological degeneration, growth retardation, premature aging, telangiectasia, specific immunodeficiencies, high sensitivity to ionizing radiation, gonadal atrophy, genomic instability, and predisposition to cancer (9, 26, 65). Cells derived from A-T individuals exhibit a variety of abnormalities in culture such as a higher requirement for serum factors, hypersensitivity to ionizing radiation, and cytoskeletal defects (50, 72). Primary fibroblasts from humans and mice with a defective ATM gene (see below for description) grow slowly in culture and appear to undergo premature senescence in culture (4, 26, 50, 72). They also show a prominent chromatin defect at chromosome ends in the form of chromosome end-to-end associations seen at different phases of the cell cycle (63, 64, 74), and these associations are enhanced by stress such as ionizing radiation treatment (74). Chromosome end associations involve telomeres composed of repetitive DNA sequences of TTAGGG arrays concealed by a complex of specialized proteins that protect ends from exonucleolytic attack, fusion, and incomplete replication. Telomeric associations correlate with genomic instability and carcinogenesis (15, 63, 64). Telomeres shorten as a function of age in cells derived from normal human blood, skin, and colonic mucosa (1, 17, 27, 43, 80). As a result of this shortening, it is thought that critical genes at the ends of the chromosomes either become deleted or are activated, leading to cell growth arrest (41, 57, 58, 86). Recovery of proper telomere length by the activation of telomerase prolongs the life span of a cell (8, 81). Shortening or loss of telomeres in a variety of cancers and immortalized cell lines is correlated with chromosome end associations that could be the cause of genomic instability and gene amplification (15, 46, 55, 61, 63, 75, 77). There is growing evidence suggesting that both the shielding of telomeric ends and their elongation by telomerase are dependent on telomere binding proteins. Mammalian telomeres are packaged in telomere-specific chromatin (76). Human and mouse cell lines have their telomeric tracts attached to the nuclear matrix, which is a proteinaceous subnuclear fraction (16, 44). There is a difference in nucleosomal organization of telomeres compared to bulk DNA, and telomeric histone H4 is hypoacetylated (40, 47, 59, 78). Telomere length homeostasis in yeast requires the binding of a RAP1p molecule along the telomeric tract (36, 45, 48), and change in the telomeric matrix binding site occurs at least once in every kilobase of the telomeric tract in tumor-derived cell lines (44). It has been suggested that mammalian telomeres have frequent multiple interactions with the nuclear matrix over a large domain that encompasses the entire telomeres of most of the chromosome ends (44). Whether the ATM gene influences the interaction of telomeres with the nuclear matrix is not yet known. The gene that mediates the disease A-T has been designated ATM (A-T, mutated), and its product shares the phosphatidylinositol 3′-kinase signature of a growing family of proteins involved in the control of cell cycle progression, processing of DNA damage, and maintenance of genomic stability (28, 31, 68, 69). The protein shows similarity to several yeast and mammalian proteins involved in meiosis of fission yeast and to the TOR proteins of yeast and mammals (54, 69). In mitotic cells, ATM is required for a DNA damage-dependent signal transduction cascade that activates multiple cell cycle checkpoints (50, 52, 72). The presence of a leucine zipper (LZ) in the ATM protein suggests possible dimerization of the protein or interaction with additional proteins (68, 69). The only known proteins that interact with ATM are p53, c-Abl, and β-adaptin (5, 32, 42, 71). The ATM protein has been shown to contribute to the induction of c-Abl activity (5, 71), a tyrosine kinase activated by ionizing radiation, and certain other DNA-damaging agents (33). Hawley and Friend (28) have suggested that an ATM-like protein has a critical role in maintaining chromosome condensation in the vicinity of recombination intermediates. In support of the role of the ATM gene in chromosome condensation, it has been reported that mei-41 (homologue of the ATM gene)-bearing oocytes exhibit diffused chromatin (25). Because of the homology of ATM to TEL1 mutants of yeast (23), it has further been suggested that mutations in ATM could lead to defective telomere maintenance. Mammalian telomeres have gained importance because of their possible link with carcinogenesis (15, 17, 27, 63). Since patients with A-T are prone to develop cancer and the ATM gene influences telomere metabolism (74), we have studied the differences among A-T and normal cells in telomere-nuclear matrix interactions, telomere DNA structure, and nucleosomal periodicity before and after ionizing radiation treatment. To directly test whether the ATM gene product influences telomere-nuclear matrix interactions, we examined cells with dominant-negative as well as complementing activity with respect to ATM function. Expression of the dominant-negative ATM fragments in RKO cells leads to decreased clonogenic survival, increased chromosomal aberrations, radioresistant DNA synthesis after treatment with ionizing radiation, and defective telomere metabolism (53, 74). The ATM protein or fragment containing the kinase domain complemented radiosensitivity, the S-phase checkpoint, irradiation-induced activation of c-Abl, reduced chromosome aberrations after treatment with gamma rays, and reduced frequency of cells with telomere fusions in simian virus 40-transformed fibroblasts derived from A-T individuals (5, 53, 71, 87).