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Previous Article | Next Article 
Molecular and Cellular Biology, June 2004, p. 5050-5059, Vol. 24, No. 11
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.11.5050-5059.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Haeyoung Kim,2 Amalia Dutia,3 Evgenia Pak,3 Stephanie Smith,1 and Eric A. Hendrickson2*
Genome Instability Section, Genetics & Molecular Biology Branch,1 Cytogenetic and Confocal Microscopy Core, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892,3 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota 554552
Received 11 September 2003/ Returned for modification 24 October 2003/ Accepted 6 February 2004
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ABSTRACT |
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INTRODUCTION |
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Telomeres are the terminal structures of linear chromosomes. Telomeres appear to perform at least two functions: (i) they allow for the replication of the ends of chromosomes, and (ii) they stabilize chromosomes by keeping them from recombining with one another (reviewed in references 17 and 24). Telomeric DNA consists of a repetitive motif with the general form TxAyGz, which in mammals is T2A1G3. At the ends of the chromosomes, the G-rich strand is often extended over the C-rich strand for a variable number of nucleotides (reviewed in reference 7). Many of the genes involved in telomere biogenesis and stability have been identified, and their subsequent characterization has led to the identification of even more genes, leaving the field with a rich, yet complicated, picture. In yeast, for example, mutation of any of more than 25 different genes can deleteriously affect telomere length and/or structure (17). The mammalian counterparts of some of these genes have been identified, and these include the ribonucleoprotein complex consisting of TERT (telomerase reverse transcriptase) (35) and TR (telomerase RNA) (8), which is responsible for the synthesis of the T2A1G3 repeat; TRF1 and TRF2 (telomere recognition factors 1 and 2, respectively) (12, 18), which bind to the double-strand portion of the T2A1G3 repeat; and Pot1 (protection of telomeres 1) (3), which binds to the single-stranded, G-rich strand overhang. In addition, a variety of DNA repair proteins are also associated, directly or indirectly, with telomeres.
The DNA-dependent protein kinase catalytic subunit DNA-PKcs, together with the heterodimeric Ku protein (Ku86 and Ku70), comprises a complex, DNA-PK, that is critically involved in DNA DSB (double-strand break) repair and V(D)J recombination in mammalian cells (reviewed in references 41 and 50). Animals mutant at the DNA-PKcs locus are IRs (ionizing radiation sensitive), defective in DNA DSB repair, and immunodeficient. Moreover, DNA-PKcs appears to regulate telomere length (28, 36, 69) and prevent GCRs (2, 31, 32). Ku is a heterodimeric protein of 70- and 86-kDa subunits that is conserved from prokaryotes (21, 82) to humans. Ku binds in a sequence-nonspecific fashion to all double-stranded DNA ends, including 5' and 3' overhangs, blunt ends, duplex DNA ending in stem-loop structures, and telomeres (reviewed in references 39 and 75). Mice containing targeted disruptions of Ku86 (59, 84), Ku70 (49, 61), or DNA-PKcs (10, 30, 43, 73) are IRs and defective for DNA DSB repair and have the anticipated immune defects. In addition, inactivation of the murine Ku86 gene results in cells with severe growth retardation (60), premature senescence (79), and a marked increase in chromosomal aberrations (20, 25, 44) that show elevated telomeric fusions (2, 67). Thus, DNA-PK is an important mammalian DNA repair complex, and mutations in Ku or DNA-PKcs result in DNA DSB repair defects and immunodeficiencies.
Despite the relative uniformity of phenotypes of eukaryotic cell lines with defects in DNA-PK concerning repair and recombination, there are some glaring differences concerning telomere biology. In Saccharomyces cerevisiae, Ku86 and Ku70 mutant strains exhibit temperature-sensitive lethality and aberrant telomere shortening (11, 33, 63) or telomeric repeat maintenance (26, 29, 34, 74). Ku-null Schizosaccharomyces pombe (4, 52) and trypanosome (19) cells also show telomere shortening but no exacerbated telomere degradation and lethality. In contrast to these organisms, the majority of Ku-null chicken DT40 cells exhibit telomeres of the parental length although some telomeric expansions have been observed in independent subclones (81). In contrast to the sporadic telomeric expansions seen in the chicken—and in sharp contrast to what has been observed with yeast and trypanosomes—Ku-null Arabidopsis thaliana plants show consistent, massive telomeric expansions (65). In rodents, multiple contradictory studies have reported telomeric shortening (20), telomeric expansions—some slight (27, 67), some large (36, 69)—and/or no discernible effects (28, 31, 32) in Ku- or DNA-PKcs-null cell lines and animals. The fact that all three of the DNA-PK mutant knockout mouse lines are fertile and viable, however, suggests that if there are telomeric defects, they cannot be as severe as those caused by the loss of telomerase activity, which results in senescent and infertile animals by the sixth generation (9). Given the glaring lack of uniformity between—and even within—the various model systems, it has been unclear which model system is most applicable to humans. Moreover, because DNA-PK activity in humans appears to be essential (48), the direct effect of DNA-PK mutations on telomere function has not been reported.
In the present report, we demonstrate that the inactivation of a single allele of Ku86 in human somatic cells results in dramatic telomere shortening. This telomeric shortening is accompanied by chromosomal fusions, aneuploidy, and GCRs. Thus, human Ku86 appears to be a critical suppressor of genomic instability.
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MATERIALS AND METHODS |
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Immunoblotting. Levels of Ku86 protein in the various cell lines were determined by Western blotting, which was performed exactly as previously described (48). Ku86 and ß-actin were detected with antibodies SC-5280 (Santa Cruz) and A5441 (Sigma), respectively.
Telomeric TRF and G-strand overhang analyses. TRF (terminal restriction fragment) and G-strand overhang assays were performed exactly as previously described (45, 77), with the restriction enzymes MboI and AluI (New England Biolabs). Oligonucleotides used for probes were obtained from Operon/Qiagen. Exonuclease I (ExoI) was purchased from Amersham.
FISH and SKY analyses. Fluorescence in situ hybridization (FISH) analysis for the presence of telomeres was performed with metaphase-arrested cells with a protein-nucleic acid telomere-specific probe [Cy3 conjugated to (T2AG3)3] in accordance with a protocol provided by the manufacturer (DAKO) (66). Individual rearrangements in mammalian cells were analyzed by spectral karyotyping (SKY) analysis as previously described (14).
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RESULTS |
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30 generations of cell growth, these subclones were subjected to a TRF analysis, which demonstrated that the telomeres of clones A6 and 10 (Fig. 2B, lanes 2 and 5, respectively) were partially complemented and migrated at a size intermediate between those of wild-type HCT116 (Fig. 2B, lane 1) and the heterozygous clone #70 cell line (Fig. 2B, lane 3). In contrast, there was no complementation of the telomere length in clones C7 and C5, which contained only the empty vector (Fig. 2B, lanes 7 and 8, respectively). When all of these cell lines were grown for an additional 30 generations, another TRF analysis was carried out. The complemented clones did not show any additional lengthening of their telomeres (Fig. 2B, lanes 4 and 6). Thus, the partially complemented phenotype was stable. These data strongly reinforced the contention that a reduction in Ku86 expression in the heterozygous cell lines was directly responsible for their short-telomere phenotype.
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DISCUSSION |
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Haploinsufficiency of Ku86 results in short telomeres. Here we have demonstrated that the functional inactivation via gene targeting of a single allele of human Ku86 results in profound telomere shortening. One possibility is that Ku might regulate TRF1 and/or TRF2. Ku is known to physically interact with TRF1 (40) and TRF2 (71). Moreover, overexpression of TRF1 (76) or TRF2 (70) results in telomeric shortening in human cells. Thus, if Ku negatively regulates TRF1 or TRF2, then a Ku86-heterozygous cell could be functionally equivalent to a TRF1- or TRF2-overexpressing cell. This hypothesis, while attractive, would not explain the increase in the frequency or length of the G-strand overhangs (Fig. 3 and Table 1). Thus, we favor a model in which Ku acts as a physical barrier to nucleases, probably by binding to the double-stranded-to-single-stranded transition—a structure to which Ku is known to tightly bind (75)—present at the telomeric end. The absence of Ku at this transition could generally reduce protection of the telomere from nucleases and could also account for the increase in the frequency or length of the G-strand overhangs (Fig. 3) by allowing preferential resection of the C-rich strand (42). Restoration of the G-strand overhang to the wild-type length upon reintroduction of a Ku86 cDNA (Table 1) strongly supports this hypothesis. In Ku86-null yeast strains, the elongation of the G-strand overhang can be upwards of an order of magnitude greater (6, 33, 72). In Ku86-heterozygous HCT116 cells, the effect we observed, 1.5- to 3-fold, was much smaller. While this difference may be simply due to the comparison between null and heterozygous cell lines, respectively, it is also possible that human cells possess alternative or additional end protection activities.
Complementation experiments suggest the existence of different mechanisms of Ku86-dependent telomere maintenance. Reintroduction of a Ku86 cDNA into Ku86-heterozygous cells resulted in significant, albeit partial, complementation of the overall telomere length (Fig. 2B). If Ku86 only functioned in end protection as postulated above, there was no a priori reason to expect that the telomeres should be re-extended at all by reintroduction of Ku86. The fact that the telomeres did elongate suggests that Ku86 may also participate in telomere elongation. One possibility is that Ku86 is required for telomerase activity and that the short-telomere phenotype is an indirect effect of reduced telomerase activity. This is consistent with a report that Ku physically interacts with telomerase and that telomerase activity is slightly reduced in Ku86-heterozygous cells (16). A second possibility is that Ku86 is required for hTR biogenesis. Biochemical and genetic data demonstrate that—in yeast—Ku86 can bind to the RNA component of telomerase (6, 62, 72). If human Ku86 possesses a similar hTR binding activity, it is likely that telomere length maintenance would be aberrantly effected in Ku86-heterozygous cell lines. A third possibility is that Ku86 may be required for recruiting telomerase to the telomere. In its role as a DNA DSB repair protein, Ku normally binds to a broken double-stranded end and recruits DNA-PKcs (50). At a telomere, Ku may perform an analogous role by recruiting telomerase. Whichever, if any, of these three hypotheses is correct, it is clear that Ku86 is not essential for these functions since only partial complementation of the short-telomere phenotype was observed upon restoration of Ku86 expression (Fig. 2B). Last, it should be emphasized that in human cells, Ku86 is often associated with the DNA-PK complex. Thus, the short-telomere phenotype could also be due to the reduced DNA-PK activity in these cells (48). A model consistent with all of our results is that the Ku heterodimer alone normally provides end-blocking activity but that Ku86, as part of the DNA-PK complex, may also be required for telomere elongation. Many aspects of this model can ultimately be addressed by the construction of conditionally null Ku86 and DNA-PKcs cell lines in isogenic backgrounds.
Ku86 protects cells from genomic instability. The telomeres observed in a Ku86-heterozygous cell, while shortened, appear relatively stable, as some of these cell lines have been grown in continuous culture for more than a year. One possibility is that the residual levels of Ku are sufficient to keep the telomeres in this short-but-stable configuration. This would be consistent with the observations that further reductions in Ku86 levels by gene targeting (48) or RNA interference (data not shown; I. Jaco, P. Munoz, and M. A. Blasco, personal communication) result in cell death. Alternatively, although Ku may be important for proper telomere length maintenance, a Ku-independent mechanism may exist to protect or maintain short telomeres as a last defense against genomic instability. Presumably, when this final barrier, perhaps mediated through TRF2 (45), is overcome, the cells begin a GCR process that is either lethal or oncogenic or leads to senescence. This model is consistent with observations in the mouse that suggest that it is not the average telomere length that regulates genomic stability but the frequency of chromosomes containing very short or no telomeres (27, 37). Similarly, while our TRF analysis (Fig. 1) showed that the average telomere length of all chromosomes in a Ku86-heterozygous cell is reduced, the FISH experiments (Fig. 4) demonstrated that only 1.4% of the chromosomes contained no detectable telomere sequences (Table 2). Indeed, this value may actually be an overestimation of the number of chromosomes lacking telomeres since some chromosomes with very short, albeit functional, telomeres may have escaped detection. If 1.4 out of 100 chromosomes were completely lacking telomeres and each telomereless chromosome led to a detectable GCR, then maximally one out of every two cells would be expected to contain a GCR. By FISH (Fig. 4 and Table 2) and SKY (Fig. 5 and Table 3) analyses, we observed that 26 and 25%, respectively, of the human Ku86+/– cells had a GCR, which is in fairly good agreement with the expected frequency. Consistent with this model is the observation that the p53-null Ku86+/+ cell line had, on average, slightly shorter telomeres than the parental control cell line (Fig. 1). However, by FISH analysis, we did not observe any telomere loss (data not shown) and this cell line is not prone to GCRs (data not shown; 14). In summary, Ku86 levels appear to critically regulate telomere shortening, which, in turn, is a key step in the production of the nonfunctional telomeric ends that generate GCRs and cellular catastrophe.
Where are all of the human Ku mutants? In the preceding decade, mutations of many DNA repair genes have been linked to human pathologies including, predominately, cancer predisposition (38). This is not the case for Ku and DNA-PK. To our knowledge, not a single case study of a Ku- or DNA-PKcs-null or heterozygote patient has ever been reported. The demonstration that the functional inactivation of both alleles of Ku86 in a somatic cell line is lethal provides a partial explanation for this discrepancy (48). If Ku86 is essential in somatic cells, it is likely that human Ku86-null individuals are inviable. Our current demonstration that Ku86-heterozygous cells have profound telomere defects and genomic instability suggests that even haploinsufficiency of Ku86 in humans may be lethal. Alternatively, while the genomic instability of the heterozygous cell lines is significant, their radiosensitivity is rather slight (48). Thus, the target group of radiosensitive, immunodeficient patients among whom Ku and DNA-PK mutant individuals have been sought (see, e.g., reference 22) may, in retrospect, not be the group in which they are most likely to be found. Our data suggest that if Ku and DNA-PKcs patients do exist, (i) they will only be heterozygous or contain hypomorphic alleles, (ii) their chromosomes will have shortened telomeres, and (iii) they will likely present with some clinical feature of genomic instability in a haploinsufficient state. DKC (dyskeratosis congenita) is a rare inherited disorder that encompasses all three of these phenotypes. In particular, while DKC patients usually die an early death from bone marrow failure, they are also afflicted with an increased risk of cancer (reviewed in reference 55). The disease is defined by multiple complementation groups, and two of the relevant genes have been cloned and identified. One is the RNA component of telomerase, hTR (80), while the other is in dsykerin (58). Dyskerin is a nucleolar protein that binds to snoRNAs (small nucleolar RNAs) and appears to be critical for proper formation of the telomerase ribonuclear protein particle. DKC patients with mutations in either hTR or dsykerin have lower levels of hTR, produce lower levels of telomerase activity, and have shortened telomeres (55). Intriguingly, some of the DKC-hTR patients are heterozygotes and transmit the disease in what appears to be an autosomal dominant fashion that is due to haploinsufficiency (80). Last, in yeast, Ku86 has been shown to bind to the telomeric RNA (6, 62, 72). Together, these observations suggest that patients with Ku or DNA-PK mutations may present with the clinical features that are encompassed by DKC. Future research should clarify this issue.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address: The Children's Hospital, Harvard Medical School, Boston, MA 02115.
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