Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.     

A Dynamic Equilibrium between CDKs and PP2A Modulates Phosphorylation of pRB,p107 and p130

We have recently demonstrated that several types of genomic rearrangements (i.e. telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells’ gained ability to protect chromosome ends with eroded telomeres allowing them to escape “end crisis”. However, the possibility that ES cells were more permissive to genomic rearrangements than other cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.     

RTEL1 contributes to DNA replication and repair and telomere maintenance

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.     

Telomere dysfunction and chromosome instability

The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is therefore important for understanding chromosome instability in human cancer.     

Increased sensitivity of subtelomeric regions to DNA double-strand breaks in a human cancer cell line

We previously reported that a single DNA double-strand break (DSB) near a telomere in mouse embryonic stem cells can result in chromosome instability. We have observed this same type of instability as a result of spontaneous telomere loss in human tumor cell lines, suggesting that a deficiency in the repair of DSBs near telomeres has a role in chromosome instability in human cancer. We have now investigated the frequency of the chromosome instability resulting from DSBs near telomeres in the EJ-30 human bladder carcinoma cell line to determine whether subtelomeric regions are sensitive to DSBs, as previously reported in yeast. These studies involved determining the frequency of large deletions, chromosome rearrangements, and chromosome instability resulting from I-SceI endonuclease-induced DSBs at interstitial and telomeric sites. As an internal control, we also analyzed the frequency of small deletions, which have been shown to be the most common type of mutation resulting from I-SceI-induced DSBs at interstitial sites. The results demonstrate that although the frequency of small deletions is similar at interstitial and telomeric DSBs, the frequency of large deletions and chromosome rearrangements is much greater at telomeric DSBs. DSB-induced chromosome rearrangements at telomeric sites also resulted in prolonged periods of chromosome instability. Telomeric regions in mammalian cells are therefore highly sensitive to DSBs, suggesting that spontaneous or ionizing radiation-induced DSBs at these locations may be responsible for many of the chromosome rearrangements that are associated with human cancer.     

PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells

Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1(ΔB) hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1(ΔB) hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing.     

Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.     

Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ

Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462–2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635–1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV–mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.     

Molecular analysis of telomere fusions in Arabidopsis: multiple pathways for chromosome end-joining

End-to-end fusion of critically shortened telomeres in higher eucaryotes is presumed to be mediated by nonhomologous end-joining (NHEJ). Here we describe two PCR-based methods to monitor telomere length and examine the fate of dysfunctional telomeres in Arabidopsis lacking the catalytic subunit of telomerase (TERT) and the DNA repair proteins Ku70 and Mre11. Primer extension telomere repeat amplification relies on the presence of an intact G-overhang, and thus measures functional telomere length. The minimum functional telomere length detected was 300-400 bp. PCR amplification and sequence analysis of chromosome fusion junctions revealed exonucleolytic digestion of dysfunctional ends prior to fusion. In ku70 tert mutants, there was a greater incidence of microhomology at the fusion junction than in tert mutants. In triple ku70 tert mre11 mutants, chromosome fusions were still detected, but microhomology at the junction was no longer favored. These data indicate that both Ku70 and Mre11 contribute to fusion of critically shortened telomeres in higher eucaryotes. Furthermore, Arabidopsis processes critically shortened telomeres as double-strand breaks, using a variety of end-joining pathways.     

Telomerase dependence of telomere lengthening in Ku80 mutant Arabidopsis

We have identified a ku80 mutant of Arabidopsis and show that telomerase is needed to generate the longer telomeres observed in this mutant. Telomeres are specialized nucleoprotein structures at the ends of chromosomes that permit cells to distinguish chromosome ends from double-strand breaks, thus preventing chromosome fusion events. Ku80 deficiency results in the lengthening of telomeres, a phenotype also seen in an Arabidopsis ku70 mutant. Furthermore, homogeneous populations of ku80 mutant cells show a steady increase in the length of telomere tracts, which reach an equilibrium length and then stabilize. In contrast to that in mammals, Ku80 deficiency in Arabidopsis cells does not cause end-to-end fusion of chromosomes. This telomere lengthening is dependent on the presence of telomerase, although it is not attributable to a significant increase in telomerase activity per se. These results demonstrate the essential role of the Ku80 protein as a negative regulator of telomerase function in plant cells.     

Chromosome rearrangements resulting from telomere dysfunction and their role in cancer

Telomeres play a vital role in protecting the ends of chromosomes and preventing chromosome fusion. The failure of cancer cells to properly maintain telomeres can be an important source of the chromosome instability involved in cancer cell progression. Telomere loss results in sister chromatid fusion and prolonged breakage/fusion/bridge (B/F/B) cycles, leading to extensive DNA amplification and large deletions. These B/F/B cycles end primarily when the unstable chromosome acquires a new telomere by translocation of the ends of other chromosomes. Many of these translocations are nonreciprocal, resulting in the loss of the telomere from the donor chromosome, providing a mechanism for transfer of instability from one chromosome to another until a chromosome acquires a telomere by a mechanism other than nonreciprocal translocation. B/F/B cycles can also result in other forms of chromosome rearrangements, including double-minute chromosomes and large duplications. Thus, the loss of a single telomere can result in instability in multiple chromosomes, and generate many of the types of rearrangements commonly associated with human cancer.     

Sudden telomere lengthening triggers a Rad53-dependent checkpoint in Saccharomyces cerevisiae

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.     

Mutant telomere sequences lead to impaired chromosome separation and a unique checkpoint response

Mutation of the template region in the RNA component of telomerase can cause incorporation of mutant DNA sequences at telomeres. We made all 63 mutant sequence combinations at template positions 474-476 of the yeast telomerase RNA, TLC1. Mutants contained faithfully incorporated template mutations, as well as misincorporated sequences in telomeres, a phenotype not previously reported for Saccharomyces cerevisiae telomerase template mutants. Although growth rates and telomere profiles varied widely among the tlc1 mutants, chromosome separation and segregation were always aberrant. The mutants showed defects in sister chromatid separation at centromeres as well as telomeres, suggesting activation of a cell cycle checkpoint. Deletion of the DNA damage response genes DDC1, MEC3, or DDC2/SML1 failed to restore chromosome separation in the tlc1 template mutants. These results suggest that mutant telomere sequences elicit a checkpoint that is genetically distinct from those activated by deletion of telomerase or DNA damage.     

Role of ATM in the telomere response to the G-quadruplex ligand 360A

Telomeres are known to prevent chromosome ends from being recognized as DNA double-strand breaks. Conversely, many DNA damage response proteins, including ATM, are thought to participate to telomere maintenance. However, the precise roles of ATM at telomeres remain unclear due to its multiple functions in cell checkpoints and apoptosis. To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM. We showed, by using Fluorescence in situ hybridization (FISH) and Chromosome Orientation-FISH using telomere PNA probes, that 360A induced specific telomere aberrations occurring during or after replication, mainly consisting in sister telomere fusions and also recombinations that involved preferentially the lagging strand telomeres. We demonstrate that ATM reduced telomere instability independently of apoptosis induction. Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres.     

Telomeres, chromosome instability and cancer

Telomeres are composed of repetitive G-rich sequence and an abundance of associated proteins that together form a dynamic cap that protects chromosome ends and allows them to be distinguished from deleterious DSBs. Telomere-associated proteins also function to regulate telomerase, the ribonucleoprtotein responsible for addition of the species-specific terminal repeat sequence. Loss of telomere function is an important mechanism for the chromosome instability commonly found in cancer. Dysfunctional telomeres can result either from alterations in the telomere-associated proteins required for end-capping function, or from alterations that promote the gradual or sudden loss of sufficient repeat sequence necessary to maintain proper telomere structure. Regardless of the mechanism, loss of telomere function can result in sister chromatid fusion and prolonged breakage/fusion/bridge (B/F/B) cycles, leading to extensive DNA amplification and large terminal deletions. B/F/B cycles terminate primarily when the unstable chromosome acquires a new telomere, most often by translocation of the ends of other chromosomes, thereby providing a mechanism for transfer of instability from one chromosome to another. Thus, the loss of a single telomere can result in on-going instability, affect multiple chromosomes, and generate many of the types of rearrangements commonly associated with human cancer.     

Telomeres and chromosome instability

Genomic instability has been proposed to play an important role in cancer by accelerating the accumulation of genetic changes responsible for cancer cell evolution. One mechanism for chromosome instability is through the loss of telomeres, which are DNA-protein complexes that protect the ends of chromosomes and prevent chromosome fusion. Telomere loss can occur as a result of exogenous DNA damage, or spontaneously in cancer cells that commonly have a high rate of telomere loss. Mouse embryonic stem cells and human tumor cell lines that contain a selectable marker gene located immediately adjacent to a telomere have been used to investigate the consequences of telomere loss. In both cell types, telomere loss is followed by either the addition of a new telomere on to the end of the broken chromosome, or sister chromatid fusion and prolonged breakage/fusion/bridge (B/F/B) cycles that result in DNA amplification and large terminal deletions. The regions amplified by B/F/B cycles can then be transferred to other chromosomes, either through the formation of double-minute chromosomes that reintegrate at other sites, or through end-to-end fusions between chromosomes. B/F/B cycles eventually end when a chromosome acquires a new telomere by one of several mechanisms, the most common of which is translocation, which can involve either nonreciprocal transfer or duplication of all or part of an arm of another chromosome. Telomere acquisition involving nonreciprocal translocations results in the loss of a telomere on the donor chromosome, which subsequently becomes unstable. In contrast, translocations involving duplications do not destabilize the donor chromosome, although they result in allelic imbalances. Thus, the loss of a single telomere can generate a wide variety of chromosome alterations commonly associated with human cancer, not only on the chromosome that originally lost its telomere, but other chromosomes as well. Factors promoting spontaneous telomere loss and the resulting B/F/B cycles are therefore likely to be important in generating the karyotypic changes associated with human cancer.     

Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping

Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.     

Telomere fusion to chromosome breaks reduces oncogenic translocations and tumour formation

Telomeres protect chromosome ends from fusion, degradation and recombination. Loss of telomere function has opposite effects on tumorigenesis: apoptosis, which inhibits tumour growth, and genomic instability, which accelerates tumour formation. Here we describe a new mechanism by which short telomeres inhibit tumorigenesis through interference with oncogenic translocations. In mice that are null for both ataxia-telangiectasia-mutated (Atm) and telomerase RNA (mTR), the first generation (G1) Atm-/- mTR-/- mice have a lower rate of tumour formation than Atm-/- mTR+/+ mice. These Atm-/- mTR-/- G1 tumours show no increase in either apoptosis or overall genomic instability. Strikingly, the tumours show a high fraction of translocations containing telomere signals at the translocation junctions. Translocations of the T-cell receptors on chromosome 14, which initiate tumorigenesis, were interrupted by fusion with telomeres. Telomere repeats were also detected at the translocation junctions in pre-malignant thymocytes. We propose that telomere fusion to DNA double-strand breaks competes with the generation of oncogenic translocations and thus reduces tumour formation.     

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.