Telomeric recombination induced by dysfunctional telomeres

Telomere maintenance is essential for cellular immortality, and most cancer cells maintain their telomeres through the enzyme telomerase. Telomeres and telomerase represent promising anticancer targets. However, 15% of cancer cells maintain their telomeres through alternative recombination-based mechanisms, and previous analyses showed that recombination-based telomere maintenance can be activated after telomerase inhibition. We determined whether telomeric recombination can also be promoted by telomere dysfunction. We report for the first time that telomeric recombination can be induced in human telomerase-positive cancer cells with dysfunctional telomeres.     

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length.     

Telomere lengthening early in development

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.     

Recombination can either help maintain very short telomeres or generate longer telomeres in yeast cells with weak telomerase activity

Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.     

Association of BLM and BRCA1 during Telomere Maintenance in ALT Cells

Fifteen percent of tumors utilize recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. The mechanisms underlying ALT are unclear but involve several proteins involved in homologous recombination including the BLM helicase, mutated in Bloom''s syndrome, and the BRCA1 tumor suppressor. Cells deficient in either BLM or BRCA1 have phenotypes consistent with telomere dysfunction. Although BLM associates with numerous DNA damage repair proteins including BRCA1 during DNA repair, the functional consequences of BLM-BRCA1 association in telomere maintenance are not completely understood. Our earlier work showed the involvement of BRCA1 in different mechanisms of ALT, and telomere shortening upon loss of BLM in ALT cells. In order to delineate their roles in telomere maintenance, we studied their association in telomere metabolism in cells using ALT. This work shows that BLM and BRCA1 co-localize with RAD50 at telomeres during S- and G2-phases of the cell cycle in immortalized human cells using ALT but not in cells using telomerase to maintain telomeres. Co-immunoprecipitation of BRCA1 and BLM is enhanced in ALT cells at G2. Furthermore, BRCA1 and BLM interact with RAD50 predominantly in S- and G2-phases, respectively. Biochemical assays demonstrate that full-length BRCA1 increases the unwinding rate of BLM three-fold in assays using a DNA substrate that models a forked structure composed of telomeric repeats. Our results suggest that BRCA1 participates in ALT through its interactions with RAD50 and BLM.     

Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells

It has been shown previously that some immortalized human cells maintain their telomeres in the absence of significant levels of telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). Cells utilizing ALT have telomeres of very heterogeneous length, ranging from very short to very long. Here we report the effect of telomerase expression in the ALT cell line GM847. Expression of exogenous hTERT in GM847 (GM847/hTERT) cells resulted in lengthening of the shortest telomeres; this is the first evidence that expression of hTERT in ALT cells can induce telomerase that is active at the telomere. However, rapid fluctuation in telomere length still occurred in the GM847/hTERT cells after more than 100 population doublings. Very long telomeres and ALT-associated promyelocytic leukemia (PML) bodies continued to be generated, indicating that telomerase activity induced by exogenous hTERT did not abolish the ALT mechanism. In contrast, when the GM847 cell line was fused with two different telomerase-positive tumor cell lines, the ALT phenotype was repressed in each case. These hybrid cells were telomerase positive, and the telomeres decreased in length, very rapidly at first and then at the rate seen in telomerase-negative normal cells. Additionally, ALT-associated PML bodies disappeared. After the telomeres had shortened sufficiently, they were maintained at a stable length by telomerase. Together these data indicate that the telomerase-positive cells contain a factor that represses the ALT mechanism but that this factor is unlikely to be telomerase. Further, the transfection data indicate that ALT and telomerase can coexist in the same cells.     

Recombination-based telomere maintenance is dependent on Tel1-MRN and Rap1 and inhibited by telomerase, Taz1, and Ku in fission yeast

Fission yeast cells survive loss of the telomerase catalytic subunit Trt1 (TERT) through recombination-based telomere maintenance or through chromosome circularization. Although trt1Δ survivors with linear chromosomes can be obtained, they often spontaneously circularize their chromosomes. Therefore, it was difficult to establish genetic requirements for telomerase-independent telomere maintenance. In contrast, when the telomere-binding protein Taz1 is also deleted, taz1Δ trt1Δ cells are able to stably maintain telomeres. Thus, taz1Δ trt1Δ cells can serve as a valuable tool in understanding the regulation of telomerase-independent telomere maintenance. In this study, we show that the checkpoint kinase Tel1 (ATM) and the DNA repair complex Rad32-Rad50-Nbs1 (MRN) are required for telomere maintenance in taz1Δ trt1Δ cells. Surprisingly, Rap1 is also essential for telomere maintenance in taz1Δ trt1Δ cells, even though recruitment of Rap1 to telomeres depends on Taz1. Expression of catalytically inactive Trt1 can efficiently inhibit recombination-based telomere maintenance, but the inhibition requires both Est1 and Ku70. While Est1 is essential for recruitment of Trt1 to telomeres, Ku70 is dispensable. Thus, we conclude that Taz1, TERT-Est1, and Ku70-Ku80 prevent telomere recombination, whereas MRN-Tel1 and Rap1 promote recombination-based telomere maintenance. Evolutionarily conserved proteins in higher eukaryotic cells might similarly contribute to telomere recombination.     

Telomerase activation in human fibroblasts during escape from crisis

The immortalization of human diploid fibroblasts requires the circumvention of both the senescence (M1) and crisis (M2) mechanisms of growth control. Cells expressing the SV40 T antigen virtually always bypass senescence, but only rarely escape crisis. The low frequency of this latter event indicates that cellular mutations are necessary to escape crisis. Thirteen subpopulations of T antigen-expressing human fibroblasts were cultured into crisis. Colonies that appeared to resume growth were assayed for telomerase activity, telomere maintenance, and the immortal phenotype. Our results show that 33 of 35 colonies were telomerase negative and were not immortal. Two colonies were telomerase positive when assayed in the first approximately 15 population doublings after crisis. The first was strongly positive, maintained telomeres at a stable short length, and was later determined to be immortal. The second initially had a weak telomerase signal, grew extremely slowly, and when examined had greatly elongated telomeres consistent with the ALT (alternative lengthening of telomeres) mechanism of telomere maintenance. These cells eventually grew faster and were later determined to be immortal. Additionally, two subpopulations had initially weak and later strong telomerase activity and the cells never entered a defined crisis period. We observed a perfect correlation between telomere maintenance and escape from crisis, supporting the hypothesis that the lack of stable telomeres causes crisis and that the ability to maintain telomeres abrogates crisis. J. Cell. Physiol. 180:46–52, 1999. © 1999 Wiley-Liss, Inc.     

Short telomeres, even in the presence of telomerase, limit tissue renewal capacity

Autosomal-dominant dyskeratosis congenita is associated with heterozygous mutations in telomerase. To examine the dosage effect of telomerase, we generated a line of mTR+/- mice on the CAST/EiJ background, which has short telomeres. Interbreeding of heterozygotes resulted in progressive telomere shortening, indicating that limiting telomerase compromises telomere maintenance. In later-generation heterozygotes, we observed a decrease in tissue renewal capacity in the bone marrow, intestines, and testes that resembled defects seen in dyskeratosis congenita patients. The progressive worsening of disease with decreasing telomere length suggests that short telomeres, not telomerase level, cause stem cell failure. Further, wild-type mice derived from the late-generation heterozygous parents, termed wt*, also had short telomeres and displayed a germ cell defect, indicating that telomere length determines these phenotypes. We propose that short telomeres in mice that have normal telomerase levels can cause an occult form of genetic disease.     

Disruption of telomere maintenance by depletion of the MRE11/RAD50/NBS1 complex in cells that use alternative lengthening of telomeres

Immortalized human cells are able to maintain their telomeres by telomerase or by a recombination-mediated DNA replication mechanism known as alternative lengthening of telomeres (ALT). We showed previously that overexpression of Sp100 protein can suppress ALT and that this was associated with sequestration of the MRE11/RAD50/NBS1 (MRN) recombination protein complex by Sp100. In the present study, we determined whether MRN proteins are required for ALT activity. ALT cells were depleted of MRN proteins by small hairpin RNA-mediated knockdown, which was maintained for up to 100 population doublings. Knockdown of NBS1 had no effect on the level of RAD50 or MRE11, but knockdown of RAD50 also depleted cells of NBS1, and knockdown of MRE11 depleted cells of all three MRN proteins. Depletion of NBS1, with or without depletion of other members of the complex, resulted in inhibition of ALT-mediated telomere maintenance, as evidenced by decreased numbers of ALT-associated promyelocytic leukemia bodies and decreased telomere length. In some clones there was an initial period of rapid shortening followed by stabilization of telomere length, whereas in others there was continuous shortening at a rate within the reported range for normal human somatic cells lacking a telomere maintenance mechanism. In contrast, depletion of NBS1 in telomerase-positive cells did not result in telomere shortening. A recent study showed that NBS1 was required for the formation of extrachromosomal telomeric circles (Compton, S. A., Choi, J. H., Cesare, A. J., Ozgur, S., and Griffith, J. D. (2007) Cancer Res. 67, 1513-1519), also a marker for ALT. We conclude that the MRN complex, and especially NBS1, is required for the ALT mechanism.     

Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process

Type II survivors arise in Saccharomyces cells lacking telomerase by a recombinational pathway that results in very long and heterogeneous length telomeres. Here we show that type II telomeres appeared abruptly in a population of cells with very short telomeres. Once established, these long telomeres progressively shortened. Short telomeres were substrates for rare, one-step lengthening events. The generation of type II survivors was absolutely Rad50p dependent. In a telomerase-proficient cell, the telomere-binding Rif proteins inhibited telomerase lengthening of telomeres. In a telomerase-deficient strain, Rif proteins, especially Rif2p, inhibited type II recombination. These data argue that only short telomeres are substrates for type II recombination and suggest that the donor for this recombination is not a chromosomal telomere.     

Abrupt telomere losses and reduced end-resection can explain accelerated senescence of Smc5/6 mutants lacking telomerase

The highly conserved Structural Maintenance of Chromosome (SMC) proteins are crucial for the formation of three essential complexes involved in high fidelity chromosome transmission during cell division. Recently, the Smc5/6 complex has been reported to be important for telomere maintenance in yeast and also in cancerous human ALT cells, where it could function in a homologous recombination-based (HR) telomere maintenance pathway. Here, we investigate the possible roles of the budding yeast Smc5/6 complex in maintaining appropriate chromosome end-structures allowing cell survival in absence of telomerase. The results show that cells harbouring mutant alleles of genes encoding Smc5/6-complex proteins rapidly stop growing after telomerase loss. Furthermore, this telomerase-induced growth arrest is much more pronounced as compared to cultures with a functional Smc5/6-complex. Bulk telomere sequence loss is not increased in the mutant cells and the evidence suggests that Smc5/6 slows senescence through a partially HR-independent pathway. We propose that in yeast, the Smc5/6-complex is required for efficient and timely termination of DNA replication and repair at telomeres to avoid stochastic telomere loss during cell division. Consistent with this hypothesis, sequencing of telomeres from telomerase-positive smc5/6 mutant cells revealed a higher frequency of telomere breakage events. Finally, the results also show that on dysfunctional telomeres, the generation of 3'-single stranded DNA is impaired, suggesting that the complex may also participate in the formation of single-stranded overhangs which are thought to be the substrates for telomere repeat replenishment in the absence of telomerase.     

Tel1 kinase and subtelomere-bound Tbf1 mediate preferential elongation of short telomeres by telomerase in yeast

Telomerase enables telomere length homeostasis, exhibiting increasing preference for telomeres as their lengths decline. This regulation involves telomere repeat-bound Rap1, which provides a length-dependent negative feedback mechanism, and the Tel1 and Mec1 kinases, which are positive regulators of telomere length. By analysing telomere elongation of wild-type chromosome ends at single-molecule resolution, we show that in tel1Delta cells the overall frequency of elongation decreases considerably, explaining their short telomere phenotype. At an artificial telomere lacking a subtelomeric region, telomere elongation no longer increases with telomere shortening in tel1Delta cells. By contrast, a natural telomere, containing subtelomeric sequence, retains a preference for the elongation of short telomeres. Tethering of the subtelomere binding protein Tbf1 to the artificial telomere in tel1Delta cells restored preferential telomerase action at short telomeres; thus, Tbf1 might function in parallel to Tel1, which has a crucial role in a TG-repeat-controlled pathway for the activation of telomerase at short telomeres.     

Telomerase enzymatic component hTERT shortens long telomeres in human cells

Telomere lengths are tightly regulated within a narrow range in normal human cells. Previous studies have extensively focused on how short telomeres are extended and have demonstrated that telomerase plays a central role in elongating short telomeres. However, much about the molecular mechanisms of regulating excessively long telomeres is unknown. In this report, we demonstrated that the telomerase enzymatic component, hTERT, plays a dual role in the regulation of telomere length. It shortens excessively long telomeres and elongates short telomeres simultaneously in one cell, maintaining the optimal telomere length at each chromosomal end for efficient protection. This novel hTERT-mediated telomere-shortening mechanism not only exists in cancer cells, but also in primary human cells. The hTERT-mediated telomere shortening requires hTERT’s enzymatic activity, but the telomerase RNA component, hTR, is not involved in that process. We found that expression of hTERT increases telomeric circular DNA formation, suggesting that telomere homologous recombination is involved in the telomere-shortening process. We further demonstrated that shelterin protein TPP1 interacts with hTERT and recruits hTERT onto the telomeres, suggesting that TPP1 might be involved in regulation of telomere shortening. This study reveals a novel function of hTERT in telomere length regulation and adds a new element to the current molecular model of telomere length maintenance.     

Molecular insights into the heterogeneity of telomere reprogramming in induced pluripotent stem cells

Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs). Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage, and suggest that these differences likely result from multiple potential factors, including the telomerase machinery, telomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors. Using a genetic model of telomerase-deficient (Terc(-/-) and Terc(+/-)) cells for derivation and passages of iPSCs, we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs. Further, telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required, but not sufficient. Together, several aspects of telomere biology may account for the variable telomere dynamics in iPSCs. Notably, the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells. These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.     

Mutant Telomeric Repeats in Yeast Can Disrupt the Negative Regulation of Recombination-Mediated Telomere Maintenance and Create an Alternative Lengthening of Telomeres-Like Phenotype

Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent of human ALT cells. In this study, we generated telomeres composed of either of two types of mutant telomeric repeats, Acc and SnaB, that each alter the binding site for the telomeric protein Rap1. We show here that arrays of both types of mutant repeats present basally on a telomere were defective in negatively regulating telomere length in the presence of telomerase. Similarly, when each type of mutant repeat was spread to all chromosome ends in cells lacking telomerase, they led to the formation of telomeres produced by RTE that were much longer than those seen in cells with only wild-type telomeric repeats. The Acc repeats produced the more severe defect in both types of telomere maintenance, consistent with their more severe Rap1 binding defect. Curiously, although telomerase deletion mutants with telomeres composed of Acc repeats invariably showed extreme telomere elongation, they often also initially showed persistent very short telomeres with few or no Acc repeats. We suggest that these result from futile cycles of recombinational elongation and truncation of the Acc repeats from the telomeres. The presence of extensive 3′ overhangs at mutant telomeres suggests that Rap1 may normally be involved in controlling 5′ end degradation.     

Hyper-phosphorylated retinoblastoma protein suppresses telomere elongation

Immortalized cell lines maintain telomeres by the expression of telomerase or by a mechanism designated alternative lengthening of telomeres (ALT). Although DNA polymerase alpha (pol-alpha) is reported to be required for telomere maintenance, the critical role of pol-alpha in telomere maintenance has not been firmly determined. We examined the role of retinoblastoma protein (pRb) and pol-alpha in the regulation of telomere length, using telomere-fiber FISH. Telomere length varied dependent on the intracellular abundance of pol-alpha or pRb in HeLa cells. A proportion of hyper-phosphorylated pRb (ppRb) molecules localized to sites of telomeric DNA replication in HeLa cells. Pol-alpha might thus contribute to telomere maintenance, and might be regulated by ppRb.