Effect of Telomere Proximity on Telomere Position Effect,Chromosome Healing,and Sensitivity to DNA Double-Strand Breaks in a Human Tumor Cell Line

The ends of chromosomes, called telomeres, are composed of a DNA repeat sequence and associated proteins, which prevent DNA degradation and chromosome fusion. We have previously used plasmid sequences integrated adjacent to a telomere to demonstrate that mammalian telomeres suppress gene expression, called telomere position effect (TPE). We have also shown that subtelomeric regions are highly sensitive to double-strand breaks, leading to chromosome instability, and that this instability can be prevented by the addition of a new telomere to the break, a process called chromosome healing. We have now targeted the same plasmid sequences to a site 100 kb from a telomere in a human carcinoma cell line to address the effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to double-strand breaks. The results demonstrate a substantial decrease in TPE 100 kb from the telomere, demonstrating that TPE is very limited in range. Chromosome healing was also diminished 100 kb from the telomere, consistent with our model that chromosome healing serves as a repair process for restoring lost telomeres. Conversely, the region 100 kb from the telomere was highly sensitive to double-strand breaks, demonstrating that the sensitive region is a relatively large target for ionizing radiation-induced chromosome instability.Telomeres are composed of a six-base pair repeat sequence and associated proteins that together form a cap to protect the ends of chromosomes and prevent chromosome fusion (6). Telomeres are actively maintained by the enzyme telomerase in human germ line cells but shorten with age in most somatic cells due to the low level of expression of telomerase (12). When a telomere shortens to the point that it is recognized as a double-strand break (DSB), it serves as a signal for replicative cell senescence (13). Human cells that lose the ability to senesce continue to show telomere shortening and eventually enter crisis, which involves increased chromosome fusion, aneuploidy, and cell death (11, 15). An important step that is required for continued division of cancer cells is therefore that they possess the ability to maintain telomeres, not only to avoid senescence but also to avoid chromosome fusion brought on by crisis (11, 25).In addition to their role in protecting the ends of chromosomes, telomeres can also inhibit the expression of nearby genes, called telomere position effect (TPE). TPE has been proposed to have a role in the cellular response to changes in telomere length (26); however, the function of TPE remains unknown. TPE has been extensively studied in Saccharomyces cerevisiae using transgenes integrated near telomeres on truncated chromosomes (1, 2, 22, 47). These studies demonstrated that TPE involves changes in chromatin conformation and is dependent upon both the distance from the telomere and telomere length (55). Subsequent studies of endogenous yeast genes, however, revealed that the influence of TPE on gene expression varies depending on the presence of insulator sequences (18, 45). TPE also occurs in mammalian cells and has been implicated in the loss of expression of genes relocated near telomeres in a variety of human syndromes (9, 16, 28, 58, 59). As in yeast, transgenes located near telomeres have been used to study TPE in the C33-A (32) and HeLa (4) human cervical carcinoma cell lines. We have also studied TPE using transgenes located adjacent to telomeres in mouse embryonic stem (ES) cells, mouse embryo fibroblasts, and transgenic mice (43). However, none of the studies of TPE in mammalian cells has addressed the distance over which TPE extends from the telomere, and so the number of genes whose expression is likely to be affected is not known.The presence of a telomere can also influence the sensitivity of subtelomeric regions to DSBs. We previously demonstrated the sensitivity of subtelomeric regions to DSBs using selectable transgenes and a recognition site for the I-SceI endonuclease that are integrated immediately adjacent to a telomere. Unlike I-SceI-induced DSBs at most locations, which primarily result in small deletions (27, 34, 46, 50), I-SceI-induced DSBs near telomeres commonly result in large deletions, gross chromosome rearrangements (GCRs), and chromosome instability in both mouse ES cells (37) and human tumor cells (65). Therefore, depending on the size of the sensitive region, the combined targets of the subtelomeric regions on all telomeres could contribute significantly to the genomic instability caused by ionizing radiation or other agents that produce DSBs (35). This sensitivity to DSBs may result from a deficiency in DSB repair since regions near telomeres in yeast are deficient in nonhomologous end joining, resulting in an increase in GCRs (48). One possible reason for a deficiency in DSB repair near telomeres is the role of the telomere in preventing chromosome fusion. Telomeric repeat sequences in yeast have been shown to suppress the activation of cell cycle checkpoints in response to DSBs (39). Similarly, the human TRF2 protein, which is required to prevent chromosome fusion, has been demonstrated to inhibit ATM (31), whose activation is instrumental in the repair of DSBs in heterochromatin (20).One mechanism for avoiding the consequences of DSBs near telomeres is through the addition of a new telomere to the site of a DSB, termed chromosome healing (44). Studies in yeast have shown that chromosome healing occurs through the de novo addition of telomeric repeat sequences by telomerase (14, 33, 38). Chromosome healing in S. cerevisiae is inhibited by the 5′-3′ helicase, Pif1 (52), with Pif1-deficient cells showing up to a 1,000-fold increase in chromosome healing (33, 38). The ability of Pif1 to inhibit chromosome healing has been proposed to serve as a mechanism to prevent chromosome healing from interfering with DSB repair (63). Mammalian cells that express telomerase are also capable of performing chromosome healing. We have shown that chromosome healing can also occur following spontaneous telomere loss (17, 49) or DSBs near telomeres in a human cancer cell line (65) or mouse ES cells (19, 54). We have also shown that chromosome healing can prevent the chromosome instability resulting from DSBs near telomeres (19). Because the de novo addition of telomeric repeat sequences has not been observed in mammalian cells at I-SceI-induced DSBs at interstitial sites (27, 34, 46, 50), we have proposed that chromosome healing is inhibited at most locations but serves as an important mechanism for dealing with DSBs near telomeres that would otherwise result in chromosome instability. However, an alternative possibility that has not been ruled out is that chromosome healing also occurs at interstitial sites but that the large terminal deletions that it causes at these sites results in cell death.In the present study, we address several key questions regarding the importance of telomere proximity on TPE, chromosome healing, and sensitivity to DSBs by investigating how telomere proximity affects these processes. The first of these questions involves establishing the distance over which TPE extends from the telomere to gain insights into the numbers of genes that would be affected by changes in TPE. Second, we will investigate whether chromosome healing can occur at a site that is distant from a telomere but in which terminal deletions are known not to be lethal. This will determine for the first time whether chromosome healing is limited to regions near telomeres. Finally, we will investigate the size of the region near a telomere that is sensitive to DSBs, which will address the potential importance of the subtelomeric region as a target for ionizing radiation-induced genomic instability (35). The distance over which a telomere can exert its effects was investigated by comparing TPE, chromosome healing, and the sensitivity to DSBs at a site 100 kb from a telomere with a site immediately adjacent to the same telomere. As a control for the efficiency of generating DSBs at these sites, we have also analyzed the frequency of small deletions, the most common type of I-SceI-induced DNA rearrangement at interstitial sites in mammalian cells (27, 60). Small deletions serve as an excellent internal control for comparing the frequency of other types of rearrangements since we have previously observed a similar frequency of small deletions at telomeric and interstitial sites (65). The results provide important information on the distance over which a telomere can influence TPE, chromosome healing, and the sensitivity to DSBs.