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Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G1, G2, and metaphase. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. We examined these interactions in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affected the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, we examined such interactions in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, we found that A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene.
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).
Derivations of the cell strains used are given in Table Table1.1. All fibroblast strains were maintained according to procedures described earlier (62, 63, 74). Cell viability was monitored by the trypan blue exclusion test, and cell population densities were determined by hemacytometer and electronic counting (Coulter Electronics Inc., Hialeah, Fla.).
The colorectal carcinoma (RKO) cells, with and without the ENA/FB2F-expressing ATM fragment which contains the LZ motif, were grown as described previously (53, 74). A-T cells with and without full-length cDNA (AT22IJE-T, AT22IJE-TpEBS7, and AT22IJE-TpEBS7-YZ5) were obtained from Yossi Shiloh, Tel Aviv University, Israel, and the conditions for maintaining the clones were the same as described previously (87). Expression of the ATM LZ fragments in the RKO cell line and the ATM full-length cDNA was determined as described earlier (53, 87). Relevant characteristics of the isogenic cells used are summarized in Table Table2.2.
DNA was isolated from plateau-phase cells by a procedure described earlier (64, 74). For measuring terminal restriction fragment lengths, DNA was digested with restriction enzyme RsaI or HinfI, which do not cut TTAGGG sequences, processed for fractionation, and hybridized with a 32P-labeled (TTAGGG)5 probe. Detection and measurement for terminal restriction fragment length were performed as described earlier (63, 64). The mean length of the telomere terminal restriction fragment was measured by using ImageQuant (version 1.2, build 039; Molecular Dynamics.
Plateau-phase cells were used to prepare the nuclear matrix halos, which were isolated by removing histones and other loosely bound proteins. Nuclear halos are morphologically defined as nuclear structures that remain after the selective removal of perinuclear components with ionic detergent. The halos are thought to represent relaxed lengths of loops of DNA with periodical attachment to the nuclear matrix, which is a residual framework of nucleoskeletal proteins. The procedure used for the isolation of lithium diiodosalicylate (LIS)-generated halo structures is a modification of the LIS technique described by Mirkovitch et al. (51), Dijkwel and Hamlin (18), Luderus et al. (44), Berezney and Coffey (6), and Berezney et al. (7). Cells were trypsinized, washed twice with cold phosphate-buffered saline (PBS) and twice with 25 ml of cold cell wash buffer (CWB; 50 mM KCl, 0.5 mM EDTA, 0.05 mM spermidine, 0.05 mM spermine, 0.25 mM phenylmethylsulfonyl fluoride [PMSF], 0.5% thiodiglycol, 5 mM Tris-HCl [pH 7.4]), pelleted at 1,000 × g for 5 min, and then suspended in 12 ml of CWB containing 0.1% digitonin (Boehringer Mannheim). The cells were passed through a 20-gauge needle, and lysis was monitored by phase-contrast microscopy. The 2-ml suspension was loaded on 3 ml of 10% glycerol cushion in CWB and spun for 10 min at 800 × g; the nuclei were washed with CWB containing 0.1% digitonin, suspended in CWB with 0.1% digitonin and 0.5 mM CuSO4 but without EDTA, and incubated for 20 min at 37°C. About 19 volumes of LIS solution (10 mM LIS, 100 mM lithium acetate, 0.1% digitonin, 0.05 mM spermine, 0.125 mM spermidine, 0.25 mM PMSF, 20 mM HEPES-KOH [pH 7.4]) was added, and the mixture was incubated for 10 min at room temperature. Halos were collected by centrifugation for 10 min at 2,800 rpm in a benchtop Eppendorf centrifuge (model 5403) and washed three times with matrix wash buffer (MWB; 20 mM KCl, 70 mM NaCl, 10 mM MgCl2, 10 mM Tris HCl [pH 7.4]) with 0.1% digitonin. The resulting halo structures contain naked chromosomal DNA and the nuclear matrix. The nuclear halos were then washed with a restriction enzyme buffer, 6 × 106 halos were cleaved in a volume of 0.5 ml containing 1,000 U of restriction enzyme StyI for 3 h at 37°C, and the nuclear matrices were pelleted by centrifugation. To purify released and attached DNA fragments to the nuclear matrix, both fractions were treated with proteinase K in a solution containing 10 mM EDTA, 0.5% sodium dodecyl sulfate, and 10 mM Tris-HCl (pH 7.4) and incubated overnight at 37°C. DNA was purified as described previously (64, 74). Agarose gel electrophoresis was performed for the fractionation of DNA (64). For Southern blot analysis, equal volumes from about 106 halos were fractionated on 0.8% agarose gels. Prior to DNA loading, RNase was added to a final concentration of 10 μg/ml. Fractionation of DNA, transfer to a Hybond-N membrane, slot blotting of DNA, hybridization with a 32P-labeled (TTAGGG)5 probe, and detection were done as described previously (64). Quantitation and comparison of the telomeric DNA among total, released, and telomeric DNA fragments attached to the nuclear matrix were achieved by phosphorimaging.
Cells were grown to plateau phase, trypsinized, and washed twice with growth medium. Cell viability was monitored by trypan blue exclusion, and cell counts were determined by hemacytometer and electronic counting (Coulter). Cells were kept on ice and were used immediately for chromatin preparation. All manipulations were done at 4°C. Cells were suspended in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 3 mM MgCl2, 1 mM CaCl2, and 0.5 mM PMSF at 2 × 106 cells per ml and washed three times with the same buffer. Cells were lysed by addition NP-40 to a final concentration of 0.5% and incubation on ice for 15 min. Lysis was monitored by visualizing the nuclei under a microscope. Nuclei were washed twice with buffer to remove the detergent and aliquoted in 100-μl volumes containing 5 × 106 nuclei. Micrococcal nuclease (MNase) (Nuclease S7; Boehringer Mannheim) was added to aliquots of nuclei at 30°C for 5 min to give final concentrations ranging from 0 to 8,000 U/ml, and digestion was terminated by the addition of an equal volume of solution containing 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.1% sodium dodecyl sulfate, and proteinase K at a final concentration of 50 μg/ml. The solutions were incubated for 2 h at 37°C, and DNA was purified by phenol-chloroform extraction, precipitated with isopropanol in the presence of 0.3 M sodium acetate (pH 5.5), and dissolved in 200 μl of 10 mM Tris-HCl. DNA (5 μg) was loaded into a 1.5% agarose gel, and blotting and hybridization for TTAGGG were performed as described previously (64).
Total RNA was isolated from fibroblasts by using an RNeasy kit (Qiagen, Santa Clarita, Calif.). cDNA was prepared from equal amounts of RNA as described previously (73). The primers used for amplification of the human telomere binding factor (hTRF1 and hTRF2) genes were designed from cDNA sequences (10, 14). Primer information will be provided on request. Equal amounts of cDNA from different cell types were used for PCR amplification and quantification of the TRF1 and TRF2 products.
To determine the mutations in the hTRF1 and hTRF2 genes, we analyzed cDNA by the cold single-strand conformational polymorphism (SSCP) protocol (24, 29). RNA isolation and cDNA preparation were done as described above. Five sets of primers covering the hTRF1 cDNA and six sets of primers covering the hTRF2 cDNA were made. Primer information will be provided on request. PCR conditions were the same as described previously (19, 24). Cold SSCP was performed according to the instructions of Novex (San Diego, Calif.). Electrophoresis was carried in ready-made precast polyacrylamide gels (Novex), using a ThermoFlow cold SSCP Novex unit. A positive control that has one allele mutated and produces four bands was also run.
Antibodies against TRF1 and TRF2 proteins obtained as a kind gift from Titia de Lange (Rockefeller University, New York, N.Y.) were used. Cells were grown on coverslips, washed with PBS, fixed in 2% paraformaldehyde in PBS for 10 min, permeabilized for 20 min in 0.5% NP-40, and then stained with the primary antibodies and anti-rabbit Cy-3-labeled donkey antibody (Jackson Research Laboratories).
We used human Atlas cDNA expression arrays (Clontech Laboratories, Palo Alto, Calif.) to determine the differences in gene expression among A-T and normal fibroblasts, using the procedure described in the Clontech manual. The Atlas cDNA membrane contains 588 known human genes. In brief, poly(A)+ RNA was isolated from the fibroblasts by using an Oligotex Direct mRNA mini kit (Qiagen). The poly(A)+ RNA was treated with RNase-free DNase I (Boehringer Mannheim) and then used for cDNA synthesis and labeled with α-32P. The labeled cDNA was hybridized to the Atlas arrays, followed by washing and exposure of the membrane in a PhosphorImager (Molecular Dynamics). Image analysis and quantification were done individually for each dot by using the Scion Image program (Scion Corp., Frederick, Md.).
Studies of telomere-nuclear matrix interactions and telomere nucleosomal periodicity in A-T and normal individuals were carried out on primary fibroblasts. The telomeres of these cells shorten during proliferation in culture. It is possible that the interactions of telomeres with the nuclear matrix depend on the length of the terminal restriction fragment (telomere length) of the chromosomes. Therefore, we determined the mean telomere length of each cell type at the time their nuclear matrix interactions and nucleosomal periodicity were examined. To determine the size of the terminal restriction fragment length, we digested the DNA with restriction enzyme RsaI or HinfI, which do not cut TTAGGG sequences. First, we examined several cell strains of A-T and normal individuals to select those with comparable telomere lengths; five strains were selected for study. The mean telomere lengths of A-T primary cells (GM5823 and GM2052; 7.0 ± 1.0 and 10.3 ± 1.7, respectively) were comparable to those of the controls (AG1522, AG6234, and C21F; 8.6 ± 1.3, 9.8 ± 0.9, and 7.2 ± 0.6, respectively). The mean telomere length observed in cells at different passages (Fig. (Fig.1)1) used for further experiments did not show any significant changes.
To characterize the nature of telomere anchorage to the nuclear matrix of different cell types, plateau-phase cells were processed by the LIS procedure (6, 18, 44) and the resulting nuclear matrix halos were cleaved with StyI. The nuclear matrix halos are the insoluble nonchromatin scaffolding of the interphase nuclei. The nuclear remnant and associated DNA were isolated by centrifugation and suspended in MWB. For genomic blotting analysis, equal volumes representing DNA from the identical numbers of halos were fractionated side by side on 1.5% agarose gels. The amount of telomeric sequence in each sample was determined by storage phosphorimage analysis. The normal fibroblasts have about 52 to 60% of the telomeric DNA associated with the nuclear matrix (attached) (P) fraction and 40% to 48% in the soluble (free) (S) fraction (Fig. (Fig.2a;2a; Table Table3).3). Summation of the P and S values is equal to total telomeric DNA (T), suggesting that no telomeric DNA was lost during the extraction procedure. Figures Figures2b2b and c show comparison among A-T and control cells for the P and S fractions of telomeric DNA. In A-T cells (GM2052), more than 92% of the telomeric DNA is attached to the nuclear matrix, whereas in control cells (AG6234) about 52% is attached (Fig. (Fig.2b;2b; Table Table3).3). In another A-T cell strain (GM5823) more than 95% of the telomeric DNA is attached to the nuclear matrix, whereas in control cells (AG1522) about 60% is attached (Fig. (Fig.2c;2c; Table Table3).3). The ratio between the S and P fractions of telomeric DNA is about 1:19 to 1:11.5 in A-T cells, compared to 1:1.5 to 1:1.1 in normal cells (Table (Table3).3). These results suggest that the major portion of telomeres in A-T cells is associated with the nuclear matrix.
To determine whether altered interactions of telomeres with the nuclear matrix are due to ATM function, we examined isogenic cells with and without normal ATM function. Two different approaches were used to determine the influence of ATM function on telomere associations with the nuclear matrix. In the first approach, we determined P and S values for RKO cells with and without expression of the dominant-negative ATM fragment. We found RKO cells expressing dominant-negative fragments have 87% of telomeric DNA in the P fraction and 13% in the S fraction, whereas parental RKO cells have 71% in the P fraction and 29% in the S fraction (Fig. (Fig.3a;3a; Table Table4).4). When ratios between means of P and S were determined (Table (Table4)4) and compared between RKO and RKOFB2F7 (with expression of dominant-negative fragment) cells, it was found that RKOFB2F7 cells have 3.1-fold higher ratio than RKO cells, suggesting that inactivation of ATM influences the telomere associations with the nuclear matrix. However, the difference in P values of RKO cells with and without expression of dominant negative ATM fragment was lower than the differences in P values between primary cells derived from A-T and normal individuals. The possible reason for this could be that RKO cells are derived from tumors and thus may have other factors that could partly rescue the ATM phenotype. In the second approach, we determined whether wild-type ATM could correct the altered interactions between telomeres and the nuclear matrix by examining A-T (AT22IJE-T) cells with and without expression of the wild-type ATM protein. Two different techniques were used to determine the values of P and S. By slot blot analysis, differences in the values of P and S were distinct between the parental cell line (ATT221JE-T) and the derivative cells with the wild-type ATM gene (AT221JE-TpEBS7-YZ5) (Fig. (Fig.3b;3b; Table Table4).4). Similar results were obtained by using Southern analysis (Fig. (Fig.3c;3c; Table Table4).4). AT221JE-TpEBS7-YZ5 cells with a wild-type ATM gene have lower amounts of P fraction compared to AT221JE-TpEBS7 cells that contain an empty vector. These results reveal that the expression of the wild-type ATM gene in A-T cells restored the normal telomere-nuclear matrix interactions, as is evident by the decrease in the amount of the P fraction (Fig. (Fig.3;3; Table Table4).4).
The results presented above suggested that untransformed A-T cells have altered telomere-nuclear matrix associations. By using the isogenic cells, we further demonstrated that ATM function influences telomere-nuclear matrix associations. Since gamma irradiation triggers telomere associations in A-T cells (74), it was important to determine the effects of ionizing radiation on the interactions of telomeres with the nuclear matrix. Plateau-phase cells were treated with a dose of 5 Gy of ionizing radiation, and proportions of S and P fractions of telomeric DNA were determined. As shown in Fig. Fig.4,4, no change in the ratio of S versus P fractions of telomeric DNA was seen immediately after treatment with ionizing radiation in either the control or the A-T cells. However, an increase in telomeric DNA in the S fraction was seen in A-T cells 1 h after treatment, whereas no such change was found in normal cells. The ratio of S versus P fractions of telomeric DNA changed from 1:19 at 0 min to 1:5 at 60 min postirradiation. Since we did not see any change in this ratio in normal cells 1 h postirradiation, we wished to determine whether there are any changes immediately after irradiation. Therefore, we examined the S and P fractions of telomeric DNA in normal cells at 0, 15, 30, and 60 min postirradiation and found no differences (data not shown). These observations suggest that the interactions of telomeric DNA in normal cells are not influenced by exposure to 5 Gy of gamma rays.
To determine the nucleosomal organization of the telomeric arrays of TTAGGG, we digested the nuclei with different concentrations of MNase and detected telomere repeats by Southern analysis using the TTAGGG, probe. Cells were grown to plateau phase and trypsinized, and the nuclei were prepared as described recently (73). Equal numbers of nuclei from each cell line were digested with MNase, and the isolated DNA fragments were fractionated on an agarose gel. We compared the organization of bulk and telomeric chromatin by determining the nucleosome periodicity in both samples. This was achieved by simultaneously digesting nuclei from all cell strains with different concentrations of MNase and fractionating the isolated DNA fragments on agarose (Fig. (Fig.5).5). Bulk nucleosome arrays were detected by ethidium bromide staining, and telomeric arrays were detected by filter hybridization with the TTAGGG, probe. All cell lines showed a ladder pattern upon ethidium bromide staining (Fig. (Fig.5).5). Interestingly, we found higher MNase digestion of chromatin in A-T cells than of normal cells (compare Fig. Fig.5a5a with Fig. Fig.5c5c and e). This suggests that the chromatin in A-T cells might be more loosely condensed than that in normal cells. As shown in Fig. Fig.5b,5b, normal (AG1522) cells have a telomere ladder containing partial digestions of up to seven subunits. In contrast, the telomeric pattern in A-T (GM5823 and GM2052) cells revealed a less extensive MNase-dependent nucleosomal periodicity (Fig. (Fig.5d5d and f) and telomeric nucleosomal arrays of up to three subunits. Further, when MNase digestion products were run in parallel for a longer time, the differences in nucleosomal band positions between A-T and normal cells became apparent (Fig. (Fig.5g).5g). Normal cells have a telomere ladder containing digestions of up to seven bands, whereas A-T cells have only three. These results suggest that the telomeric nucleosome arrays in A-T cells might be less uniformly spaced and extend over a smaller region than the arrays of normal cells.
To determine how the altered nucleosomal periodicity in telomeres seen in A-T cells responded to ionizing radiation, we examined the influence of radiation treatment on nucleosomal compaction in telomeres. Normal (C21F) fibroblasts in plateau phase were irradiated with 5 Gy of gamma rays, collected at 0 and 60 min after treatment, and subjected to MNase digestion. There was no change in the appearance of nucleosomal bands of bulk chromatin, as revealed by ethidium bromide staining in normal cells (data not shown). Similar to bulk nucleosomes, there was no change in the nucleosome banding pattern of telomere in normal cells (Fig. (Fig.6a).6a). However we observed the disappearance of the nucleosomal bands in the telomere region of A-T cells 1 h after gamma ray treatment (Fig. (Fig.6b),6b), whereas no such change was seen in normal cells (Fig. (Fig.6a).6a). The disappearance of nucleosomal bands in A-T cells suggests that ATM influences the response of telomere chromatin to radiation.
To determine whether the abnormalities in telomere-nuclear matrix interactions and nucleosomal periodicity seen in A-T cells are correlated with alterations in telomere binding factors, we first analyzed the expression of TRF1 and TRF2 in A-T fibroblasts. Using the reverse transcription-PCR approach, we found comparable levels of expression of TRF1 and TRF2 in A-T and normal control cells (data not shown). Despite this finding, mutations in these genes could lead to altered interactions of telomeres with the nuclear matrix. Therefore, we carried mutational analysis of TRF1 and TRF2 genes in A-T and control cells. Analysis of TRF1 and TRF2 cDNA in A-T cells by the cold SSCP protocol detected no mutations (data not shown).
To test whether TRF1 and TRF2 were localized correctly in the A-T cells, we performed immunostaining of the cells and found that both proteins were localized in the nuclei of both A-T and control cells (data not shown). These observations suggest that alterations in the structure or expression of TRF1 and TRF2 are not the cause of altered interactions of the telomeres with the nuclear matrix and nucleosomal periodicity changes in A-T cells.
In an attempt to identify gene products that might be involved with the altered interactions of telomeres with the nuclear matrix in A-T cells, we used the Atlas cDNA microarray to analyze the expression of genes. The expression profiles of primary fibroblasts of A-T and normal control were compared by using poly(A)+ RNA for synthesizing 32P-labeled cDNA, subsequently hybridized separately to array membranes (data not shown). No significant differences in the expression of the 588 genes on the array were found between A-T and normal control cells.
Cells derived from A-T individuals show a prominent chromatin defect at chromosome ends in the form of chromosome end-to-end associations seen at G1, G2, and metaphase (34, 63, 64). A-T cells also show an accelerated loss of telomeres (49, 63, 64, 74, 82). Whether the chromosome end associations are the cause or the effect of the accelerated loss of telomeres is unclear. With the cloning of the gene for A-T, it has been suggested that defective telomere maintenance could be due to the ATM gene because of its homology to TEL1 mutants of yeast and mei-41 mutants of Drosophila (23, 25, 54, 68, 69). Since these genes have a phosphatidylinositol 3′-kinase domain, the chromosome end association defect could be due to a defective kinase activity. Recently, we reported that the ATM gene influences chromosome end associations as well as telomere length (74); however, it is not clear how chromosome end associations are formed in cells derived from A-T patients.
What other factors influence chromosome end association? One possible factor is loss or shortening of telomeres, as suggested by Counter et al. (15); another is altered chromatin structure. In our previous studies, we reported that the frequency of cells with chromosome end associations is higher in G1 phase than in G2 phase followed by metaphase, and for each phase of the cell cycle, the frequency of cells with end associations was significantly higher in A-T than in normal cells (63, 64, 74). It is probable that the end associations seen at mitosis reflect a continuation of interphase chromosome behavior, perhaps indicating interactions or linkages between chromosome ends and the nuclear matrix. Since the telomeric signals are seen at the chromosome end association sites (64), it is possible that in the absence of ATM function, the chromosome end associations are the consequences of the failure of the nuclear matrix with holding the telomeres together. The telomeric signals at the chromosome end association sites in A-T cells suggest that chromosome end associations could be the primary event that subsequently lead to the shortening of telomeres. This interpretation is consistent with the recent findings of van Steensel et al. (79), who also reported that the telomeric signals were present at sites of chromosome end associations and that shortening of telomeres is not a prerequisite for chromosome end associations. Telomeric signals at the chromosome end association sites and changes in the frequency of cells with chromosome end associations through the cell cycle raise the possibility that A-T cells have an altered nuclear matrix, leading to defective interactions between telomeric DNA and the nuclear matrix.
Telomeres are important components of chromosomes, as they have been implicated in several cellular functions involved in aging and cancer development. Telomeres have been shown cytologically as well as biochemically to be tethered to the nuclear matrix. The nuclear matrix is a proteinaceous scaffold in the interphase nucleus isolated by removing most of the nuclear DNA and RNA, along with histones and loosely bound proteins (6, 7). Our present study shows that telomeres of primary fibroblasts are associated with the nuclear matrix, and such observations are consistent with the previous observations of de Lange (16) and Luderus et al. (44). However, we found a significant difference in the ratio of the P versus S fractions of telomeric DNA between A-T and normal control cells. This difference could be attributed to alterations in the interactions between telomeric DNA and the nuclear matrix. Our studies demonstrate that changes in the lengths of the telomeric DNA were not involved in these differences. The present results are consistent with the hypothesis that the telomere nucleoprotein structure or nuclear matrix structure is different in A-T cells. The fact that telomere binding to the matrix is greater in A-T cells and is specifically influenced by irradiation shows that changes in telomere-matrix association could be involved in the chromosome-destabilizing function of the ATM gene. The role of ATM function in telomere nuclear matrix interactions is further strengthened by the fact that cells expressing dominant-negative fragment of the ATM gene have altered telomere nuclear matrix interactions. The altered telomere nuclear matrix interactions seen in A-T cells were reversed by expression of the wild-type ATM gene. An influence of the ATM gene product on the interactions of telomeres with the nuclear matrix might be an important modulator of cellular processes influencing cellular senescence and cellular transformation.
Genomic DNA is compacted within the nucleus as chromatin (nucleoprotein complex), and the basic unit of chromatin is the nucleosome that consists of 146 bp of DNA wrapped around an octamer of histones (35). The nucleosomal sizes in bulk as well as telomeric DNA are similar between A-T and normal cells. The differences lie in the periodicity of the nucleosomes in the telomeric region in A-T versus normal cells. This is further indicative of altered nuclear matrix composition. Nucleosomes in telomeres of A-T cells are loosely spaced, and this state of nucleosomal periodicity in telomeres could not be attributed to the length of telomeres, as the telomeres of the untransformed A-T and normal fibroblasts examined were similar in size.
Chromatin structure is an important factor in determining protein-DNA interactions, with consequences for DNA metabolism and transcription control (60, 67, 83). Since the nucleosomal model emerged, there has been considerable progress in elucidating how chromatin structure at the level of nucleosome organization can either repress or potentiate transcription (2, 39, 84, 85). It has been demonstrated that nuclear structure is very important for the site-specific initiation of DNA replication (22). ATM is a nuclear protein (11, 13, 20, 37) that also colocalizes with chromatin associated proteins on meiotic chromosomes (30, 66). Recently, Gately et al. (20) have provided biochemical evidence that ATM is associated with chromatin in somatic cells. Our studies demonstrate that the altered telomere-nuclear matrix interactions seen in A-T cells could be the reason for aberrant radioresistent DNA synthesis in A-T cells. The genes involved in signal transduction could influence chromatin structure, and that may explain the basis of the cell cycle checkpoint defect in A-T cells (38, 56, 70) and the prevalence of chromosome damage. Since a chromatin defect in A-T cells is pronounced at telomeres, their interactions with the nuclear matrix may influence chromatin structure and thus the function of neighboring genes.
The different response of A-T compared to normal cells after ionizing radiation exposure could partly be attributed to altered telomere chromatin organization, as is evident from the differences in nucleosomal periodicity and nucleosomal compaction after ionizing radiation treatment. When nucleosomal compaction was examined in A-T and normal fibroblasts after ionizing radiation exposure, bulk chromatin did not show any distinct difference; however, nucleosomal compaction was influenced in the telomeric region of A-T cells but not in normal fibroblasts. An explanation for altered nucleosomal compaction in A-T cells could be that the major portions of telomeric DNA are attached to the nuclear matrix, whereas only a fraction of the bulk DNA is associated with the matrix. Therefore, an altered nuclear matrix could influence specifically the matrix-associated nucleosomes. The results presented here suggest that the altered telomere chromatin responds to DNA damage in a different way and thus influences the nucleosomal compaction of telomeres only in A-T cells. Although it is clear that ATM influences the interaction of telomeric DNA with the nuclear matrix and nucleosomal periodicity, it is not clear how it does so. Information about the interactions of the ATM gene with other genes is limited. It has been shown that ATM interacts with c-Abl, p53, and β-adaptin (3, 5, 12, 32, 42, 71). However, it remains to be established if such gene products can influence telomere interactions with the nuclear matrix and chromosome stability.
This work was supported by NIH grant NS34746.
Thanks are due to W. E. Wright, H. B. Liebermann, A. S. Balajee, C. R. Geard, W. N. Hittelman, and M. D. Story for critical discussion of the manuscript. Thanks are also due to S. G. Sawant, W. Mellado, and R. K. Pandita for technical assistance.