Our results demonstrate that the distance over which a telomere exerts its influence differs depending on the endpoint being analyzed. The large decrease in the level of expression of the Neo and HSV-tk transgenes (12 and 7.8-fold, respectively) upon relocation of the telomere immediately adjacent to the pNTIL-100kb plasmid clearly demonstrates a substantial reduction in TPE at a distance of 100 kb from this telomere. The reduced level of expression near the telomere is typical of the reduced expression previously reported for transgenes located near telomeres in other human tumor cells (4
) and mouse ES cells (43
). Our results are also consistent with studies using telomeric transgenes in yeast, which demonstrate that TPE is dependent upon the distance from a telomere (55
). However, the analysis of endogenous genes in yeast also shows that the influence of a telomere on expression can be discontinuous, depending on the presence of specific sequences that can serve as insulators (45
). Thus, it is likely that the distance over which a telomere can influence gene expression in mammalian cells will also vary, depending on the type of sequences present in the subtelomeric DNA. Regardless, our results provide the first evidence that the influence of a telomere on gene expression is relatively limited in range, and therefore only genes relatively close to a telomere are likely to be affected by changes in TPE. Because subtelomeric regions in mammalian cells have been shown to consist of heterochromatin (7
), our results suggest that the heterochromatin does not extend more than 100 kb from this telomere. It is important to note, however, that we have demonstrated that TPE in normal somatic mouse cells involves complete silencing associated with DNA methylation (43
), which could increase the distance over which TPE extends into the chromosome. This complete silencing and DNA methylation have not been observed by us (unpublished observation) or others (4
) in telomeric transgenes in human tumor cells, even though the DNA near the ends of chromosomes in normal human cells is heavily methylated (8
). The mechanisms involved in formation of TPE and the distance that TPE extends from the end of the chromosome may therefore differ between normal human cells and human cancer cells.
Although the region 100 kb from the telomere on chromosome 16p shows diminished TPE compared to the region immediately adjacent to the telomere, it continues to show increased sensitivity to DSBs. In cells constitutively expressing I-SceI, small deletions at the I-SceI site in clones 10-7A and 10-7B were present in 15 and 20% of the cells in the population, respectively. This frequency of small deletions is similar to the frequency observed at both interstitial and telomeric I-SceI sites (65
). This similarity in the frequency of small deletions demonstrates that the production of DSBs by the I-SceI endonuclease is similar at the different locations. We have previously reported that small deletions are by far the most common type of event at I-SceI-induced DSBs at interstitial sites (65
), consistent with the results from a number of other laboratories (27
). However, in clone 10-7, the combined frequency of large deletions and terminal deletions is much greater than the frequency of small deletions. In view of the fact that terminal deletions result in the loss of the telomere and therefore cause GCRs, our results demonstrate that DSBs occurring within 100 kb of a telomere are much more likely to result in chromosome instability than DSBs at interstitial sites.
The demonstration that a portion of the genome is highly sensitive to DSBs has important implications for chromosome instability resulting from oncogene-mediated replication stress and exposure to ionizing radiation. The continuous cell division resulting from oncogene expression results in stalled replication forks at regions that pose problems for DNA replication, known as fragile sites (3
). Stalled replication forks can lead to DSBs, and therefore oncogene-mediated replication stress can result in chromosome rearrangements. Replication forks have been demonstrated to stall near telomeres in yeast (30
) and behave like fragile sites in mammalian cells (53
), and therefore telomeres are likely sites of DSB formation during replication stress. Moreover, while replication stress would result in DSBs at other fragile sites, stalled replication forks near telomeres would be especially deleterious because of the sensitivity of subtelomeric regions to DSBs. Similarly, regions near telomeres would also be susceptible to chromosome instability as a result of DSBs generated by ionizing radiation. In view of the fact that there are 96 telomeres, the total length of the sensitive region is at least 9.6 ×103
kb (96 × 100 kb) and possibly longer, depending on the how far the sensitive region extends from the telomere. Because the total length of the mammalian genome is 6 × 106
kb, the frequency that random DSBs generated by ionizing radiation would occur within this region is 0.0016 (1 in 625 DSBs). Of these, approximately half experience rearrangements leading to inactivation of the HSV-tk gene, and half of these contain terminal deletions. Thus, a minimum of 1 cell in 2,500 experiencing a DSB will be likely to experience GCRs as a result of DSBs near telomeres. Moreover, many of these GCRs will involve the formation of ring chromosomes, dicentric chromosomes, or sister chromatid fusion, which result in chromosome instability (40
The mechanism responsible for the sensitivity of subtelomeric regions to DSBs has yet to be determined. A likely explanation for this sensitivity is a deficiency in DSB repair in subtelomeric regions. Subtelomeric regions in yeast have been shown to be deficient in nonhomologous end joining (NHEJ), and as a result, I-SceI-induced DSBs near telomeres result in an increase in frequency of GCRs (48
). Similarly, the sensitivity in clone 10-7 is also manifest as an increase in GCRs involving both large deletions and terminal deletions. However, these two endpoints, although related, can occur independently of one another since terminal deletions can occur without large deletions and since large deletions can occur without terminal deletions. Therefore, terminal deletions do not occur solely from extensive degradation at the I-SceI site. An increase in large deletions and terminal deletions would be consistent with a deficiency in DSB repair by either NHEJ or homologous recombination repair. Large deletions involving both strands can result from extensive resection when DSBs remain unrepaired (64
). Similarly, deficient DSB repair would also lead to an increase in loss of the terminal fragment containing the telomere, which in turn would lead to GCRs and chromosome instability. However, this deficiency in DSB repair would have a minimal effect on the formation of small deletions, which have been reported to occur primarily through a mechanism involving alternative NHEJ (Alt-NHEJ) (5
). Alt-NHEJ has also been shown to be associated with extensive degradation and chromosome rearrangements (23
) and may therefore be responsible for the formation of large deletions and GCRs in our system.
Although the region 100 kb from the telomere is sensitive to DSBs, the frequency of chromosome healing at this location is significantly diminished relative to the frequency of chromosome healing we previously reported for DSB occurring 3 kb from this telomere (65
). We have now shown that this decreased frequency of chromosome healing at interstitial sites is not due to cell lethality since we have demonstrated that clone 10-7 can survive the loss of the 100-kb terminal fragment using Cre-mediated recombination. Therefore, chromosome healing is either enhanced near telomeres or is inhibited at interstitial sites. In yeast, chromosome healing is inhibited by Pif1 helicase, which has been proposed as a mechanism for preventing de novo
telomere addition from interfering with DSB repair (63
). Moreover, a recent study found that Pif1 is phosphorylated in response to DSBs and that this phosphorylation is required for the inhibition of chromosome healing (37a
). It is therefore likely that chromosome healing is also inhibited at most locations in mammalian chromosomes. However, unlike chromosome healing at interstitial sites, chromosome healing in subtelomeric regions would not result in the loss of substantial amounts of genetic information. The results therefore support our model that chromosome healing serves as a mechanism for stabilizing DSBs near telomeres to compensate for the deficiency of other DSB repair mechanisms in subtelomeric regions. However, our results also show that the region that is sensitive to DSBs extends farther from the telomere than the region in which chromosome healing occurs. In fact, even when DSBs occur within 3 kb of the telomere (17
), chromosome healing is much less efficient in the EJ-30 human tumor cell line than in mouse ES cells, where chromosome healing is the most frequent event resulting from DSBs near telomeres (19
). This diminished chromosome healing could contribute to the increased chromosome instability that results from a high rate of spontaneous telomere loss typically observed in human tumor cells (17
In summary, we have demonstrated that DNA methylation-independent TPE extends a relatively short distance from the telomere and therefore would affect only a small number of genes. Similarly, we have also demonstrated that chromosome healing is limited to regions near telomeres. In contrast, the sensitivity of subtelomeric regions extends at least 100 kb from a telomere, making subtelomeric regions a relatively large target for DSB-induced chromosome instability. Moreover, the fact that chromosome healing does not extend as far as the sensitivity to DSBs means that chromosome healing is not available as a mechanism to counteract the instability resulting from DSBs within subtelomeric regions. Finally, the fact that the sensitivity to DSBs extends farther than TPE suggests that the sensitivity does not result from chromatin structure or suppression of transcription but, instead, is associated with other cis-acting telomere functions.