Journal articles on the topic 'Telomeres maintenance mechanism'
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1
Lin, Chi-Ying, Hsih-Hsuan Chang, Kou-Juey Wu, Shun-Fu Tseng, Chuan-Chuan Lin, Chao-Po Lin, and Shu-Chun Teng. "Extrachromosomal Telomeric Circles Contribute to Rad52-, Rad50-, and Polymerase δ-Mediated Telomere-Telomere Recombination in Saccharomyces cerevisiae." Eukaryotic Cell 4, no. 2 (February 2005): 327–36. http://dx.doi.org/10.1128/ec.4.2.327-336.2005.
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ABSTRACT Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the telomerase reverse transcriptase. In both tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. By using an in vivo inducible Cre-loxP system to generate and trace the fate of marked telomeric DNA-containing rings, the efficiency of telomere-telomere recombination can be determined quantitatively. We show that the telomeric loci are the primary sites at which a marked telomeric ring-containing DNA is observed among wild-type and surviving cells lacking telomerase. Marked telomeric DNAs can be transferred to telomeres and form tandem arrays through Rad52-, Rad50-, and polymerase δ-mediated recombination. Moreover, increases of extrachromosomal telomeric and Y′ rings were observed in telomerase-deficient cells. These results imply that telomeres can use looped-out telomeric rings to promote telomere-telomere recombination in telomerase-deficient Saccharomyces cerevisiae.
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Royle, Nicola J., Aarón Méndez-Bermúdez, Athanasia Gravani, Clara Novo, Jenny Foxon, Jonathan Williams, Victoria Cotton, and Alberto Hidalgo. "The role of recombination in telomere length maintenance." Biochemical Society Transactions 37, no. 3 (May 20, 2009): 589–95. http://dx.doi.org/10.1042/bst0370589.
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Human telomeres shorten during each cell division, predominantly because of incomplete DNA replication. This eventually results in short uncapped telomeres that elicit a DNA-damage response, leading to cellular senescence. However, evasion of senescence results in continued cell division and telomere erosion ultimately results in genome instability. In the long term, this genome instability is not sustainable, and cancer cells activate a TMM (telomere maintenance mechanism), either expression of telomerase or activation of the ALT (alternative lengthening of telomeres) pathway. Activation of the ALT mechanism results in deregulation of recombination-based activities at telomeres. Thus ALT+ cells show elevated T-SCE (telomere sister-chromatid exchange), misprocessing of t-loops that cap chromosomes and recombination-based processes between telomeres or between telomeres and ECTRs (extrachromosomal telomeric repeats). Some or all of these processes underlie the chaotic telomere length maintenance that allows cells in ALT+ tumours unlimited replicative capacity. ALT activation is also associated with destabilization of a minisatellite, MS32. The connection between the minisatellite instability and the deregulation of recombination-based activity at telomeres is not understood, but analysis of the minisatellite can be used as a marker for ALT. It is known that telomere length maintenance in ALT+ cells is dependent on the MRN [MRE11 (meiotic recombination 11)–Rad50–NBS1 (Nijmegen breakage syndrome 1)] complex, but knowledge of the role of other genes, including the Werner's (WRN) and Bloom's (BLM) syndrome DNA helicase genes, is still limited.
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Chen, W., S. M. Chen, Y. Yu, B. K. Xiao, Z. W. Huang, and Z. Z. Tao. "Telomerase inhibition alters telomere maintenance mechanisms in laryngeal squamous carcinoma cells." Journal of Laryngology & Otology 124, no. 7 (April 20, 2010): 778–83. http://dx.doi.org/10.1017/s0022215109992854.
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AbstractBackground and purpose:Telomere length must be maintained throughout cancer cell progression and proliferation. In most tumours, telomerase activity maintains telomere length. Therefore, telomerase is a target for cancer treatments. However, some cancer cells maintain telomere length through an alternative mechanism termed ‘alternative lengthening of telomeres’. To determine how telomerase inhibition relates to the initiation of the alternative lengthening of telomeres pathway, we investigated telomerase activity and telomere maintenance in Hep-2 cells with and without reduced telomerase activity.Materials and methods:We investigated telomerase activity levels in a normal Hep-2 cell line and in residual cells following telomerase inhibition treatment. Additionally, we looked for expression of a marker protein for the alternative lengthening of telomeres mechanism.Results and conclusions:In the residual cells, telomerase activity was eliminated. However, these cells had higher levels of the alternative lengthening of telomeres biomarker, suggesting an alternative mechanism for telomere maintenance following telomerase inhibition. These results could have a major impact on the design of new cancer treatments.
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Niida, Hiroyuki, Yoichi Shinkai, M. Prakash Hande, Takehisa Matsumoto, Shoko Takehara, Makoto Tachibana, Mitsuo Oshimura, Peter M. Lansdorp, and Yasuhiro Furuichi. "Telomere Maintenance in Telomerase-Deficient Mouse Embryonic Stem Cells: Characterization of an Amplified Telomeric DNA." Molecular and Cellular Biology 20, no. 11 (June 1, 2000): 4115–27. http://dx.doi.org/10.1128/mcb.20.11.4115-4127.2000.
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ABSTRACT Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER −/− cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicatescis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.
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Calado, Rodrigo T., Solomon A. Graf, and Neal S. Young. "Telomeric Recombination in Lymphocytes Implicates ALT, an Alternative Mechanism for Telomere Length Maintenance, in Normal Human Hematopoietic Cells." Blood 110, no. 11 (November 16, 2007): 1332. http://dx.doi.org/10.1182/blood.v110.11.1332.1332.
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Abstract Telomeres are the very ends of chromosomes and protect the genome from recombination, end-to-end-fusion, and recognition as damaged DNA. Telomeres are eroded with each cell division, eventually reaching such critically short length as to cause cell cycle arrest, apoptosis, or genomic instability. In most highly proliferative cells, including hematopoietic stem cells and T lymphocytes, telomere attrition is countered by telomere extension by telomerase reverse transcriptase complex. The majority of cancer cells also express telomerase, which maintains telomere length and allows indefinite cell proliferation. However, about 10% of tumors maintain telomere length in the absence of telomerase by mechanisms collectively termed alternative lengthening of telomeres (ALT). ALT mainly acts through asymmetrical exchange of telomeric material between chromosomes or sister chromatids, producing one daughter-cell with short telomeres and a limited life-span and its sister with long telomeres and higher proliferative capacity. To date, ALT has only been reported in cancer cells or through genetic engineering of mammalian cells. Here we investigated whether ALT mechanisms were active in hematopoietic cells using chromosome orientation fluorescent in situ hybridization (CO-FISH). In standard FISH, a telomeric probe produces fours signals per chromosome, one at each end of the two chromatids. Using CO-FISH, the newly synthesized DNA strand is fragmented by BrdU incorporation and UV light exposure and then digested by exonucleases. In CO-FISH, a telomeric probe produces two signals only, one at each end of the chromosome; in the presence of telomeric recombination, the telomeric signal is split, generating more than two signals per chromosome. Peripheral blood lymphocytes from three healthy volunteers, normal human fibroblasts, K562 cells, telomerase-positive HeLa cells (known to be negative for ALT),and telomerase-negative VA13 cells (known to be positive for ALT) were investigated for telomeric sister chromatid exchange (t-SCE); at least 20 metaphases per cell type were examined. Cultured peripheral blood lymphocytes and VA13 cells both showed increased levels of telomeric sister chromatid exchange in comparison to the other cells (P=0.0001): telomeric probe generated 2.62±0.11 telomeric signals/chromosome in lymphocytes; 2.23±0.04 in VA13 cells; 2.09±0.01 in HeLa cells; 2.02±0.01 in K562 cells; and 2.02±0.01 in human skin fibroblasts. Staining incorporated-BrdU over 24 hours and evaluation of “harlequin” chromosomes point to a similar rate of genomic sister chromatid exchange in lymphocytes, VA13 cells, and HeLa cells, suggesting that high chromatid exchange is confined to the telomeric region. A physical association between promyelocytic leukemia protein (PML) and telomeres is characteristic of some ALT-positive cells, but confocal microscopy failed to co-localize the telomeric probe and anti-PML monoclonal antibody in peripheral blood lymphocytes, suggesting that t-SCE in lymphocytes is not mediated by PML. This is the first demonstration of ALT activation in normal mammalian cells. ALT may be activated in peripheral blood lymphocytes as a complementary mechanism to maintain telomere length, and may explain the differences in age-related telomere shortening observed between lymphocytes and granulocytes.
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Basenko, Evelina, Zeki Topcu, and Michael J. McEachern. "Recombination Can either Help Maintain Very Short Telomeres or Generate Longer Telomeres in Yeast Cells with Weak Telomerase Activity." Eukaryotic Cell 10, no. 8 (June 10, 2011): 1131–42. http://dx.doi.org/10.1128/ec.05079-11.
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ABSTRACT 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.
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Dreesen, Oliver, and George A. M. Cross. "Telomerase-Independent Stabilization of Short Telomeres in Trypanosoma brucei." Molecular and Cellular Biology 26, no. 13 (July 1, 2006): 4911–19. http://dx.doi.org/10.1128/mcb.00212-06.
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ABSTRACT In cancer cells and germ cells, shortening of chromosome ends is prevented by telomerase. Telomerase-deficient cells have a replicative life span, after which they enter senescence. Senescent cells can give rise to survivors that maintain chromosome ends through recombination-based amplification of telomeric or subtelomeric repeats. We found that in Trypanosoma brucei, critically short telomeres are stable in the absence of telomerase. Telomere stabilization ensured genomic integrity and could have implications for telomere maintenance in human telomerase-deficient cells. Cloning and sequencing revealed 7 to 27 TTAGGG repeats on stabilized telomeres and no changes in the subtelomeric region. Clones with short telomeres were used to study telomere elongation dynamics, which differed dramatically at transcriptionally active and silent telomeres, after restoration of telomerase. We propose that transcription makes the termini of short telomeres accessible for rapid elongation by telomerase and that telomere elongation in T. brucei is not regulated by a protein-counting mechanism. Many minichromosomes were lost after long-term culture in the absence of telomerase, which may reflect their different mitotic segregation properties.
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Amato, Roberta, Martina Valenzuela, Francesco Berardinelli, Erica Salvati, Carmen Maresca, Stefano Leone, Antonio Antoccia, and Antonella Sgura. "G-quadruplex Stabilization Fuels the ALT Pathway in ALT-positive Osteosarcoma Cells." Genes 11, no. 3 (March 13, 2020): 304. http://dx.doi.org/10.3390/genes11030304.
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Most human tumors maintain telomere lengths by telomerase, whereas a portion of them (10–15%) uses a mechanism named alternative lengthening of telomeres (ALT). The telomeric G-quadruplex (G4) ligand RHPS4 is known for its potent antiproliferative effect, as shown in telomerase-positive cancer models. Moreover, RHPS4 is also able to reduce cell proliferation in ALT cells, although the influence of G4 stabilization on the ALT mechanism has so far been poorly investigated. Here we show that sensitivity to RHPS4 is comparable in ALT-positive (U2OS; SAOS-2) and telomerase-positive (HOS) osteosarcoma cell lines, unlinking the telomere maintenance mechanism and RHPS4 responsiveness. To investigate the impact of G4 stabilization on ALT, the cardinal ALT hallmarks were analyzed. A significant induction of telomeric doublets, telomeric clusterized DNA damage, ALT-associated Promyelocytic Leukaemia-bodies (APBs), telomere sister chromatid exchanges (T-SCE) and c-circles was found exclusively in RHPS4-treated ALT cells. We surmise that RHPS4 affects ALT mechanisms through the induction of replicative stress that in turn is converted in DNA damage at telomeres, fueling recombination. In conclusion, our work indicates that RHPS4-induced telomeric DNA damage promotes overactivation of telomeric recombination in ALT cells, opening new questions on the therapeutic employment of G4 ligands in the treatment of ALT positive tumors.
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Marchesini, M., R. Matocci, L. Tasselli, V. Cambiaghi, A. Orleth, L. Furia, C. Marinelli, et al. "PML is required for telomere stability in non-neoplastic human cells." Oncogene 35, no. 14 (June 29, 2015): 1811–21. http://dx.doi.org/10.1038/onc.2015.246.
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Abstract Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that in normal human fibroblasts the PML protein associates with few telomeres, preferentially when they are damaged. Proliferation-induced telomere attrition or their damage due to alteration of the shelterin complex enhances the telomeric localization of PML, which is increased in human T-lymphocytes derived from patients genetically deficient in telomerase. In normal fibroblasts, PML depletion induces telomere damage, nuclear and chromosomal abnormalities, and senescence. Expression of the leukemia protein PML/RARα in hematopoietic progenitors displaces PML from telomeres and induces telomere shortening in the bone marrow of pre-leukemic mice. Our work provides a novel view of the physiologic function of PML, which participates in telomeres surveillance in normal cells. Our data further imply that a diminished PML function may contribute to cell senescence, genomic instability, and tumorigenesis.
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Idilli, Aurora Irene, Francesca Pagani, Emanuela Kerschbamer, Francesco Berardinelli, Manuel Bernabé, María Luisa Cayuela, Silvano Piazza, Pietro Luigi Poliani, Emilio Cusanelli, and Maria Caterina Mione. "Changes in the Expression of Pre-Replicative Complex Genes in hTERT and ALT Pediatric Brain Tumors." Cancers 12, no. 4 (April 22, 2020): 1028. http://dx.doi.org/10.3390/cancers12041028.
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Background: The up-regulation of a telomere maintenance mechanism (TMM) is a common feature of cancer cells and a hallmark of cancer. Routine methods for detecting TMMs in tumor samples are still missing, whereas telomerase targeting treatments are becoming available. In paediatric cancers, alternative lengthening of telomeres (ALT) is found in a subset of sarcomas and malignant brain tumors. ALT is a non-canonical mechanism of telomere maintenance developed by cancer cells with no-functional telomerase. Methods: To identify drivers and/or markers of ALT, we performed a differential gene expression analysis between two zebrafish models of juvenile brain tumors, that differ only for the telomere maintenance mechanism adopted by tumor cells: one is ALT while the other is telomerase-dependent. Results: Comparative analysis of gene expression identified five genes of the pre-replicative complex, ORC4, ORC6, MCM2, CDC45 and RPA3 as upregulated in ALT. We searched for a correlation between telomerase levels and expression of the pre-replicative complex genes in a cohort of paediatric brain cancers and identified a counter-correlation between telomerase expression and the genes of the pre-replicative complex. Moreover, the analysis of ALT markers in a group of 20 patients confirmed the association between ALT and increased RPA and decreased H3K9me3 localization at telomeres. Conclusions: Our study suggests that telomere maintenance mechanisms may act as a driver of telomeric DNA replication and chromatin status in brain cancers and identifies markers of ALT that could be exploited for precise prognostic and therapeutic purposes.
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Auchter, Morgan, Sandrine Medves, Laetitia Chambeau, Sophie Gazzo, Etienne Moussay, Wim Ammerlaan, Hamid Morjani, et al. "Mechanisms of Telomere Maintenance Dysfunction in B-Chronic Lymphocytic Leukemia Through CpG Island Methylation." Blood 120, no. 21 (November 16, 2012): 3489. http://dx.doi.org/10.1182/blood.v120.21.3489.3489.
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Abstract Abstract 3489 Telomeres are a repetitive DNA sequences associated with a protein complex named shelterin that protect chromosome ends. Two types of mechanisms maintain telomere in cancer cells. The first involves telomerase an enzyme able to copy the telomeric motif that consists of three principal subunits, including the telomerase reverse transcriptase hTERT. The second, named ALT (Alternative Lengthening of Telomere), corresponds to the recombination between telomeres that involves notably a complex formed by the topoisomerase III alpha (hTopoIIIa), BLM, RMI1 and RMI2. Little is known about the involvement of the ALT mechanism in B-chronic lymphocytic leukemia (B-CLL). In fact this leukemic disease shows low telomerase activity, shelterin defect and telomeric dysfunction. In an effort to characterize ALT cells from 31 B-CLL patients, we analyzed their telomere length and telomerase activity. B-CLL patients showed almost no hTERT transcript (detected in three cases), low telomerase activity (detected in 7 cases) and a telomere average size ranging from 3 to 10 kb. Moreover, a strong deregulation of genes encoding three shelterin proteins, TRF1, TRF2, Pot1, and an at least two fold downregulation of hTopoIIIa gene expression in 21 cases were observed, suggesting the presence of a telomere maintenance dysfunction affecting both mechanisms, telomerase dependent and ALT. CpG island methylation has been mapped for both promoters and if hTERT shows a disseminated methylation profile in 22 patients, for hTopoIIIα we identified nine CpG upstream the minimal promoter, being methylated in 19 of our 31 analyzed patients. We then performed luciferase experiments and we showed that methylation in this 9 CpG induced a strong inhibition of hTopoIIIa transcription. Finally we correlated telomere length and hTopoIIIa methylation status as we observed that 25.4% of the hTopoIIIa promoters were methylated in patients with shorter chromosomes and only 11.1 % were methylated in patients with longer telomeres (p<0.0025). As nearly no telomerase activity have been detected in our patients and as downregulation of hTopoIIIa could increase recombination rate between sister chromatid, methylation of hTERT and hTopoIIIa promoter CpG islands may lead to telomere dysfunction and increased genetic instability in B-CLL. Disclosures: No relevant conflicts of interest to declare.
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Tian, Xiao, Katherine Doerig, Rosa Park, Alice Can Ran Qin, Chaewon Hwang, Alexander Neary, Michael Gilbert, Andrei Seluanov, and Vera Gorbunova. "Evolution of telomere maintenance and tumour suppressor mechanisms across mammals." Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1741 (January 15, 2018): 20160443. http://dx.doi.org/10.1098/rstb.2016.0443.
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Mammalian species differ dramatically in telomere biology. Species larger than 5–10 kg repress somatic telomerase activity and have shorter telomeres, leading to replicative senescence. It has been proposed that evolution of replicative senescence in large-bodied species is an anti-tumour mechanism counteracting increased risk of cancer due to increased cell numbers. By contrast, small-bodied species express high telomerase activity and have longer telomeres. To counteract cancer risk due to longer lifespan, long-lived small-bodied species evolved additional telomere-independent tumour suppressor mechanisms. Here, we tested the connection between telomere biology and tumorigenesis by analysing the propensity of fibroblasts from 18 rodent species to form tumours. We found a negative correlation between species lifespan and anchorage-independent growth. Small-bodied species required inactivation of Rb and/or p53 and expression of oncogenic H-Ras to form tumours. Large-bodied species displayed a continuum of phenotypes requiring additional genetic ‘hits’ for malignant transformation. Based on these data we refine the model of the evolution of tumour suppressor mechanisms and telomeres. We propose that two different strategies evolved in small and large species because small-bodied species cannot tolerate small tumours that form prior to activation of the telomere barrier, and must instead use telomere-independent strategies that act earlier, at the hyperplasia stage. This article is part of the theme issue ‘Understanding diversity in telomere dynamics’.
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De Vitis, Marco, Francesco Berardinelli, Elisa Coluzzi, Jessica Marinaccio, Roderick J. O’Sullivan, and Antonella Sgura. "X-rays Activate Telomeric Homologous Recombination Mediated Repair in Primary Cells." Cells 8, no. 7 (July 12, 2019): 708. http://dx.doi.org/10.3390/cells8070708.
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Cancer cells need to acquire telomere maintenance mechanisms in order to counteract progressive telomere shortening due to multiple rounds of replication. Most human tumors maintain their telomeres expressing telomerase whereas the remaining 15%–20% utilize the alternative lengthening of telomeres (ALT) pathway. Previous studies have demonstrated that ionizing radiations (IR) are able to modulate telomere lengths and to transiently induce some of the ALT-pathway hallmarks in normal primary fibroblasts. In the present study, we investigated the telomere length modulation kinetics, telomeric DNA damage induction, and the principal hallmarks of ALT over a period of 13 days in X-ray-exposed primary cells. Our results show that X-ray-treated cells primarily display telomere shortening and telomeric damage caused by persistent IR-induced oxidative stress. After initial telomere erosion, we observed a telomere elongation that was associated to the transient activation of a homologous recombination (HR) based mechanism, sharing several features with the ALT pathway observed in cancer cells. Data indicate that telomeric damage activates telomeric HR-mediated repair in primary cells. The characterization of HR-mediated telomere repair in normal cells may contribute to the understanding of the ALT pathway and to the identification of novel strategies in the treatment of ALT-positive cancers.
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Siddiqa, Aisha, David Cavazos, Jeffery Chavez, Linda Long, and Robert A. Marciniak. "Modulation of Telomeres in Alternative Lengthening of Telomeres Type I Like Human Cells by the Expression of Werner Protein and Telomerase." Journal of Oncology 2012 (2012): 1–11. http://dx.doi.org/10.1155/2012/806382.
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The alternative lengthening of telomeres (ALT) is a recombination-based mechanism of telomere maintenance activated in 5–20% of human cancers. InSaccharomyces cerevisiae, survivors that arise after inactivation of telomerase can be classified as type I or type II ALT. In type I, telomeres have a tandem array structure, with each subunit consisting of a subtelomeric Y′ element and short telomere sequence. Telomeres in type II have only long telomere repeats and require Sgs1, theS. cerevisiaeRecQ family helicase. We previously described the first human ALT cell line, AG11395, that has a telomere structure similar to type I ALT yeast cells. This cell line lacks the activity of the Werner syndrome protein, a human RecQ helicase. The telomeres in this cell line consist of tandem repeats containing SV40 DNA, including the origin of replication, and telomere sequence. We investigated the role of the SV40 origin of replication and the effects of Werner protein and telomerase on telomere structure and maintenance in AG11395 cells. We report that the expression of Werner protein facilitates the transition in human cells of ALT type I like telomeres to type II like telomeres in some aspects. These findings have implications for the diagnosis and treatment of cancer.
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Okamoto, Keiji, and Hiroyuki Seimiya. "Revisiting Telomere Shortening in Cancer." Cells 8, no. 2 (January 31, 2019): 107. http://dx.doi.org/10.3390/cells8020107.
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Telomeres, the protective structures of chromosome ends are gradually shortened by each cell division, eventually leading to senescence or apoptosis. Cancer cells maintain the telomere length for unlimited growth by telomerase reactivation or a recombination-based mechanism. Recent genome-wide analyses have unveiled genetic and epigenetic alterations of the telomere maintenance machinery in cancer. While telomerase inhibition reveals that longer telomeres are more advantageous for cell survival, cancer cells often have paradoxically shorter telomeres compared with those found in the normal tissues. In this review, we summarize the latest knowledge about telomere length alterations in cancer and revisit its rationality. Finally, we discuss the potential utility of telomere length as a prognostic biomarker.
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Chiba, Kunitoshi, Franziska K. Lorbeer, A. Hunter Shain, David T. McSwiggen, Eva Schruf, Areum Oh, Jekwan Ryu, Xavier Darzacq, Boris C. Bastian, and Dirk Hockemeyer. "Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism." Science 357, no. 6358 (August 17, 2017): 1416–20. http://dx.doi.org/10.1126/science.aao0535.
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TERT promoter mutations (TPMs) are the most common noncoding mutations in cancer. The timing and consequences of TPMs have not been fully established. Here, we show that TPMs acquired at the transition from benign nevus to malignant melanoma do not support telomere maintenance. In vitro experiments revealed that TPMs do not prevent telomere attrition, resulting in cells with critically short and unprotected telomeres. Immortalization by TPMs requires a gradual up-regulation of telomerase, coinciding with telomere fusions. These data suggest that TPMs contribute to tumorigenesis by promoting immortalization and genomic instability in two phases. In an initial phase, TPMs do not prevent bulk telomere shortening but extend cellular life span by healing the shortest telomeres. In the second phase, the critically short telomeres lead to genome instability and telomerase is further up-regulated to sustain cell proliferation.
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Counter, CM, J. Gupta, CB Harley, B. Leber, and S. Bacchetti. "Telomerase activity in normal leukocytes and in hematologic malignancies." Blood 85, no. 9 (May 1, 1995): 2315–20. http://dx.doi.org/10.1182/blood.v85.9.2315.bloodjournal8592315.
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Telomeres are essential for function and stability of eukaryotic chromosomes. In the absence of telomerase, the enzyme that synthesizes telomeric DNA, telomeres shorten with cell division, a process thought to contribute to cell senescence and the proliferative crisis of transformed cells. We reported telomere stabilization concomitant with detection of telomerase activity in cells immortalized in vitro and in ovarian carcinoma cells, and suggested that telomerase is essential for unlimited cell proliferation. We have now examined the temporal pattern of telomerase expression in selected hematologic malignancies. We found that, unlike other somatic tissues, peripheral, cord blood, and bone marrow leukocytes from normal donors expressed low levels of telomerase activity. In leukocytes from chronic lymphocytic leukemia (CLL) patients, activity was lower than in controls in early disease, and comparable with controls in late disease. Relative to bone marrow, telomerase activity was enhanced in myelodysplastic syndrome (MDS) and more significantly so in acute myeloid leukemia (AML). Regardless of telomerase levels, telomeres shortened with progression of the diseases. Our results suggest that early CLL and MDS cells lack an efficient mechanism of telomere maintenance and that telomerase is activated late in the progression of these cancers, presumably when critical telomere loss generates selective pressure for cell immortality.
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Groff-Vindman, Cindy, Anthony J. Cesare, Shobhana Natarajan, Jack D. Griffith, and Michael J. McEachern. "Recombination at Long Mutant Telomeres Produces Tiny Single- and Double-Stranded Telomeric Circles." Molecular and Cellular Biology 25, no. 11 (June 1, 2005): 4406–12. http://dx.doi.org/10.1128/mcb.25.11.4406-4412.2005.
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ABSTRACT Recombinational telomere elongation (RTE) known as alternate lengthening of telomeres is the mechanism of telomere maintenance in up to 5 to 10% of human cancers. The telomeres of yeast mutants lacking telomerase can also be maintained by recombination. Previously, we proposed the roll-and-spread model to explain this elongation in the yeast Kluveromyces lactis. This model suggests that a very small (∼100-bp) circular molecule of telomeric DNA is copied by a rolling circle event to generate a single long telomere. The sequence of this primary elongated telomere is then spread by recombination to all remaining telomeres. Here we show by two-dimensional gel analysis and electron microscopy that small circles of single- and double-stranded telomeric DNA are commonly made by recombination in a K. lactis mutant with long telomeres. These circles were found to be especially abundant between 100 and 400 bp (or nucleotides). Interestingly, the single-stranded circles consist of only the G-rich telomeric strand sequence. To our knowledge this is the first report of single-stranded telomeric circles as a product of telomere dysfunction. We propose that the small telomeric circles form through the resolution of an intratelomeric strand invasion which resembles a t-loop. Our data reported here demonstrate that K. lactis can, in at least some circumstances, make telomeric circles of the very small sizes predicted by the roll-and-spread model. The very small circles seen here are both predicted products of telomere rapid deletion, a process observed in both human and yeast cells, and predicted templates for roll-and-spread RTE.
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Hou, Kailong, Yuyang Yu, Duda Li, Yanduo Zhang, Ke Zhang, Jinkai Tong, Kunxian Yang, and Shuting Jia. "Alternative Lengthening of Telomeres and Mediated Telomere Synthesis." Cancers 14, no. 9 (April 27, 2022): 2194. http://dx.doi.org/10.3390/cancers14092194.
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Telomeres are DNA–protein complexes that protect eukaryotic chromosome ends from being erroneously repaired by the DNA damage repair system, and the length of telomeres indicates the replicative potential of the cell. Telomeres shorten during each division of the cell, resulting in telomeric damage and replicative senescence. Tumor cells tend to ensure cell proliferation potential and genomic stability by activating telomere maintenance mechanisms (TMMs) for telomere lengthening. The alternative lengthening of telomeres (ALT) pathway is the most frequently activated TMM in tumors of mesenchymal and neuroepithelial origin, and ALT also frequently occurs during experimental cellular immortalization of mesenchymal cells. ALT is a process that relies on homologous recombination (HR) to elongate telomeres. However, some processes in the ALT mechanism remain poorly understood. Here, we review the most recent understanding of ALT mechanisms and processes, which may help us to better understand how the ALT pathway is activated in cancer cells and determine the potential therapeutic targets in ALT pathway-stabilized tumors.
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Rosso, Ilaria, and Fabrizio d’Adda di Fagagna. "Detection of Telomeric DNA:RNA Hybrids Using TeloDRIP-qPCR." International Journal of Molecular Sciences 21, no. 24 (December 21, 2020): 9774. http://dx.doi.org/10.3390/ijms21249774.
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Because of their intrinsic characteristics, telomeres are genomic loci that pose significant problems during the replication of the genome. In particular, it has been observed that telomeres that are maintained in cancer cells by the alternative mechanism of the lengthening of telomeres (ALT) harbor higher levels of replicative stress compared with telomerase-positive cancer cells. R-loops are three-stranded structures formed by a DNA:RNA hybrid and a displaced ssDNA. Emerging evidence suggests that controlling the levels of R-loops at ALT telomeres is critical for telomere maintenance. In fact, on the one hand, they favor telomere recombination, but on the other, they are a source of detrimental replicative stress. DRIP (DNA:RNA immunoprecipitation) is the main technique used for the detection of R-loops, and it is based on the use of the S9.6 antibody, which recognizes preferentially DNA:RNA hybrids in a sequence-independent manner. The detection of DNA:RNA hybrids in repetitive sequences such as telomeres requires some additional precautions as a result of their repetitive nature. Here, we share an optimized protocol for the detection of telomeric DNA:RNA hybrids, and we demonstrate its application in an ALT and in a telomerase-positive cell line. We demonstrate that ALT telomeres bear higher levels of DNA:RNA hybrids, and we propose this method as a reliable way to detect them in telomeres.
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Webb, Christopher J., and Virginia A. Zakian. "Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding." Proceedings of the National Academy of Sciences 112, no. 36 (August 24, 2015): 11312–17. http://dx.doi.org/10.1073/pnas.1503157112.
Full textAbstract:
The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.
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de la Guardia, Rafael Díaz, Carolina Elosua, Purificación Catalina, Brian A. Walker, David C. Johnson, David Gonzalez, Faith E. Davies, Gareth J. Morgan, and Paola Leone. "Expression Profile and up-Regulation of Telomere-Associated Proteins In Multiple Myeloma." Blood 116, no. 21 (November 19, 2010): 4050. http://dx.doi.org/10.1182/blood.v116.21.4050.4050.
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Abstract Abstract 4050 The role of the telomeres in the mechanisms of ageing and carcinogenesis has generated a considerable interest as a novel approach to the treatment of many cancers. Telomeres are nucleoproteins structures that protect the ends of eukaryotic chromosomes, which are particularly vulnerable due to progressive shortening in almost all dividing cells. The telomere length was observed as a critical factor in the initiation and progression of human cancers, and it is associated to chromosomal instability. Most immortal cells possess enzymatic activity of telomerase. This suggests that telomerase activity and telomere length maintenance may be required for unlimited cell proliferation, tumorigenesis, and protection, allowing the evasion of apoptosis in cancer development. The telomerase activity could also be regulated positively or negatively by post-trancriptional and/or post-translational modification of the enzyme without transcriptional up-regulation of human telomerase reverse transcriptase (hTERT) mRNA. In this work, we analyze the expression data of all genes involved in telomerase activity. Patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), multiple myeloma (MM) and plasma cell leukemia (PLC) were studied through gene expression profiling analysis (Human Genome U133 Plus 2.0 arrays, Affymetrix). We identify 21 deregulated genes, implicated directly in telomere length maintenance activity in clonal plasma cells compared with normal cells (20 up-regulated and 1 down-regulated). These genes are MYC, KRAS, HSPA9, RB1 and members of the families: Small nucleolar ribonucleoproteins (H/ACA snoRNPs), A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs), and 14-3 -3 family. In conclusion, the myeloma cells acquire the telomere maintenance capability without deregulation of the human telomerase RNA gene (hTERC) and hTERT gene expression. It is an alternative lengthening of telomeres mechanism that has effect in the regulation of the BAD activity in apoptosis. The mechanism is based on preventing the partially-denatured proteins from aggregating, telomere maintenance through the correct processing and intranuclear trafficking of hTERC, telomerase reactivation and telomere stabilization, and efficient accumulation of hTERT in the nucleus. Thus, the findings of this study may help to improve telomerase-based therapy for multiple myeloma. Disclosures: No relevant conflicts of interest to declare.
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Swiggers, Susan J. J., Marianne A. Kuijpers, Maartje J. de Cort, Berna Beverloo, and J. Mark J. M. Zijlmans. "Extensive Chromosome Instability in Acute Myeloid Leukemia Is Associated with Critical Telomere Shortening." Blood 104, no. 11 (November 16, 2004): 3376. http://dx.doi.org/10.1182/blood.v104.11.3376.3376.
Full textAbstract:
Abstract Telomeres, the ends of linear chromosomes, have a critical role in protection against chromosome end-to-end fusion. Telomeres shorten in every cell division due to the end replication problem. Telomerase is a reverse transcriptase that adds telomeric DNA repeats to the ultimate chromosome end. In vitro models of long-term fibroblast cultures have identified two sequential mortality stages, senescence (M1) and crisis (M2). Senescence can be bypassed by loss of p53 or Rb function, whereas escape from crisis can only be achieved by activating a telomere maintenance mechanism, mostly telomerase reactivation. Cells that bypass senescence (M1) did not reactivate telomerase, resulting in further telomere shortening to a critical telomere length upon reaching crisis (M2). In these models, critical telomere shortening induces extensive chromosome instability, most likely via chromosome end-to-end fusions. Dicentric chromosomes lead to anaphase breakage-fusion-bridges resulting in multiple chromosomal aberrations. To investigate whether similar mechanisms may be involved in the development of genetic instability in human cancer, we studied telomere length and expression of critical telomeric proteins (TRF2 and POT1) in acute myeloid leukemia (AML) patients. AML is a good model for these studies since distinct subgroups of AML are characterized by either exchanges along chromosome arms (translocation or inversion), or by a complex karyotype with multiple chromosome aberrations. Groups were age-matched. Telomere length was studied in metaphase arrested leukemic cells using quantitative fluorescence in situ hybridization (Q-FISH) using a telomere-specific probe. Subsequently, metaphase spreads were hybridized with a leukemia-specific probe to confirm leukemic origin of each metaphase. Telomeres were significantly shorter in AML samples with multiple chromosomal abnormalities in comparison to AML samples with a reciprocal translocation/inversion or no abnormalities (mean±SEM=16±1.7 AFU, n=12 versus 29±4.3 AFU, n=18; p=0.015). Interestingly, telomerase activity level is significantly higher in AML samples with multiple chromosomal abnormalities, compared to AML samples with a reciprocal translocation or inversion (mean±SEM=330±95, n=11 versus 70±21, n=13; p=0.02). Expression levels of telomeric proteins TRF2 and POT1 were similar in these AML groups. Our observations suggest that, consistent with previous in vitro models in fibroblasts, critical telomere shortening may have a role in the development of genetic instability in human AML. Critically short telomeres in association with high levels of telomerase activity suggest that AML cells with multiple chromosomal abnormalities have bypassed crisis (M2). The longer telomeres and low levels of telomerase activity in AML cells with a reciprocal translocation or inversion suggest that they originate from an earlier stage, preceding crisis. Consequently, telomere length modulation may have a role in cancer prevention.
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Thompson, Connor A. H., and Judy M. Y. Wong. "Non-canonical Functions of Telomerase Reverse Transcriptase: Emerging Roles and Biological Relevance." Current Topics in Medicinal Chemistry 20, no. 6 (April 13, 2020): 498–507. http://dx.doi.org/10.2174/1568026620666200131125110.
Full textAbstract:
Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere repeat synthesis activity, telomerase may have other biologically important functions. The canonical roles of telomerase are at the telomere ends where they elongate telomeres and maintain genomic stability and cellular lifespan. The catalytic protein component Telomerase Reverse Transcriptase (TERT) is preferentially expressed at high levels in cancer cells despite the existence of an alternative mechanism for telomere maintenance (alternative lengthening of telomeres or ALT). TERT is also expressed at higher levels than necessary for maintaining functional telomere length, suggesting other possible adaptive functions. Emerging non-canonical roles of TERT include regulation of non-telomeric DNA damage responses, promotion of cell growth and proliferation, acceleration of cell cycle kinetics, and control of mitochondrial integrity following oxidative stress. Non-canonical activities of TERT primarily show cellular protective effects, and nuclear TERT has been shown to protect against cell death following double-stranded DNA damage, independent of its role in telomere length maintenance. TERT has been suggested to act as a chromatin modulator and participate in the transcriptional regulation of gene expression. TERT has also been reported to regulate transcript levels through an RNA-dependent RNA Polymerase (RdRP) activity and produce siRNAs in a Dicer-dependent manner. At the mitochondria, TERT is suggested to protect against oxidative stress-induced mtDNA damage and promote mitochondrial integrity. These extra-telomeric functions of TERT may be advantageous in the context of increased proliferation and metabolic stress often found in rapidly-dividing cancer cells. Understanding the spectrum of non-canonical functions of telomerase may have important implications for the rational design of anti-cancer chemotherapeutic drugs.
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Bessler, Monica, Rachida Bouharich, Shashikant Kulkarni, Sara Freeman, Hong-Yan Du, Philip J. Mason, Arturo Londono-Vallejo, and Fred Goldman. "Accelerated Shortening of Long Telomeres and Accumulation of Short Telomeres in Dyskeratosis Congenita." Blood 106, no. 11 (November 16, 2005): 1053. http://dx.doi.org/10.1182/blood.v106.11.1053.1053.
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Abstract Dyskeratosis congenita (DC) is the first human disease whose pathogenesis has been directly linked to an impairment of telomere maintenance. Telomeres protect chromosome ends from end to end fusion and degradation. Loss of telomere function causes cell cycle arrest or cell death. Telomeres are maintained by the telomerase ribonucloprotein complex whose integral RNA component, the telomerase RNA or TERC RNA, contains the sequences that act as a template for the synthesis of telomeric repeats. Autosomal dominant DC (AD DC), a rare inherited bone marrow failure syndrome, is caused by mutations in TERC, the RNA component of telomerase. Patients with AD DC have very short telomeres. Haploinsufficiency has been proposed to be the mechanism for telomere shortening in TERC gene mutation carriers. Individuals with AD DC not only inherited the TERC gene mutation but also the shortened telomeres from the affected parent. Here we studied the telomere dynamics over 3 generations in a 32-member extended family with AD DC due to a TERC gene deletion. The investigation of telomere length within a single family has the advantage that the molecular lesion responsible for telomere shortening is uniform and that the contribution of other genetic components influencing telomere length is similar. Our analysis shows that peripheral blood cells from family members haploinsufficient for TERC have very short telomeres (6.68 kb, range 5.53–8.45, SD 1.13, normal controls: 9.15 kb rage 8.56–10.77, SD 1.22). In contrast to normal controls, whose telomere lengths shorten with age, the telomere lengths in all individuals carrying the TERC gene deletion are equally short irrespective of their age. To study the inheritance of short telomeres and the effect of TERC haploinsufficiency on specific telomere lengths in affected individuals and their relatives we carried out Q-FISH analysis using polymorphic subtelomeric probes on chromosomes 11p, 7p, and 1p, which are able to distinguish the parental origin of telomeres in this family. Our analysis showed that in children of affected parents who have inherited the gene deletion, paternal and maternal telomeres are similarly short, and similar in length to those of the affected parent. In children of affected parents who have normal TERC genes paternal and maternal telomeres are again similar in length, and similar to those of the unaffected parent. These results are consistent with a model in which telomerase preferentially acts on the shortest telomeres. When TERC is limiting this leads to the accelerated shortening of longer telomeres and the accumulation of short telomeres. The limited amount of active telomerase in TERC RNA haploinsufficiency may not be able to maintain the minimal length of the increasing number of short telomeres. Thus, the number of critically short telomeres and the degree of residual telomerase activity may determine the onset of disease in patients with DC.
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Razak, Zaineb R. Abdul, Robert J. Varkonyi, Michelle Kulp-McEliece, Corrado Caslini, Joseph R. Testa, Maureen E. Murphy, and Dominique Broccoli. "p53 Differentially Inhibits Cell Growth Depending on the Mechanism of Telomere Maintenance." Molecular and Cellular Biology 24, no. 13 (July 1, 2004): 5967–77. http://dx.doi.org/10.1128/mcb.24.13.5967-5977.2004.
Full textAbstract:
ABSTRACT Telomere stabilization is critical for tumorigenesis. A number of tumors and cell lines use a recombination-based mechanism, alternative lengthening of telomeres (ALT), to maintain telomere repeat arrays. Current data suggest that the mutation of p53 facilitates the activation of this pathway. In addition to its functions in response to DNA damage, p53 also acts to suppress recombination, independent of transactivation activity, raising the possibility that p53 might regulate the ALT mechanism via its role as a regulator of recombination. To test the role of p53 in ALT we utilized inducible alleles of human p53. We show that expression of transactivation-incompetent p53 inhibits DNA synthesis in ALT cell lines but does not affect telomerase-positive cell lines. The expression of temperature-sensitive p53 in clonal cell lines results in ALT-specific, transactivation-independent growth inhibition, due in part to the perturbation of S phase. Utilizing chromatin immunoprecipitation assays, we demonstrate that p53 is associated with the telomeric complex in ALT cells. Furthermore, the inhibition of DNA synthesis in ALT cells by p53 requires intact specific DNA binding and suppression of recombination functions. We propose that p53 causes transactivation-independent growth inhibition of ALT cells by perturbing telomeric recombination.
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Mentegari, Elisa, Federica Bertoletti, Miroslava Kissova, Elisa Zucca, Silvia Galli, Giulia Tagliavini, Anna Garbelli, et al. "A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres." International Journal of Molecular Sciences 22, no. 5 (February 27, 2021): 2365. http://dx.doi.org/10.3390/ijms22052365.
Full textAbstract:
Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.
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Criscuolo, F., S. Smith, S. Zahn, B. J. Heidinger, and M. F. Haussmann. "Experimental manipulation of telomere length: does it reveal a corner-stone role for telomerase in the natural variability of individual fitness?" Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1741 (January 15, 2018): 20160440. http://dx.doi.org/10.1098/rstb.2016.0440.
Full textAbstract:
Telomeres, the non-coding ends of linear chromosomes, are thought to be an important mechanism of individual variability in performance. Research suggests that longer telomeres are indicative of better health and increased fitness; however, many of these data are correlational and whether these effects are causal are poorly understood. Experimental tests are emerging in medical and laboratory-based studies, but these types of experiments are rare in natural populations, which precludes conclusions at an evolutionary level. At the crossroads between telomere length and fitness is telomerase, an enzyme that can lengthen telomeres. Experimental modulation of telomerase activity is a powerful tool to manipulate telomere length, and to look at the covariation of telomerase, telomeres and individual life-history traits. Here, we review studies that manipulate telomerase activity in laboratory conditions and emphasize the associated physiological and fitness consequences. We then discuss how telomerase's impact on ageing may go beyond telomere maintenance. Based on this overview, we then propose several research avenues for future studies to explore how individual variability in health, reproduction and survival may have coevolved with different patterns of telomerase activity and expression. Such knowledge is of prime importance to fully understand the role that telomere dynamics play in the evolution of animal ageing. This article is part of the theme issue ‘Understanding diversity in telomere dynamics’.
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Tsai, Yun-Luen, Shun-Fu Tseng, Shih-Husan Chang, Chuan-Chuan Lin, and Shu-Chun Teng. "Involvement of Replicative Polymerases, Tel1p, Mec1p, Cdc13p, and the Ku Complex in Telomere-Telomere Recombination." Molecular and Cellular Biology 22, no. 16 (August 15, 2002): 5679–87. http://dx.doi.org/10.1128/mcb.22.16.5679-5687.2002.
Full textAbstract:
ABSTRACT Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of the reverse transcriptase telomerase. In both tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Genetic studies have led to the identification of DNA polymerases, cell cycle checkpoint proteins, and telomere binding proteins involved in the telomerase pathway. However, how these proteins affect telomere-telomere recombination has not been identified to date. Using an assay to trace the in vivo recombinational products throughout the course of survivor development, we show here that three major replicative polymerases, α, δ, and ε, play roles in telomere-telomere recombination and that each causes different effects and phenotypes when they as well as the telomerase are defective. Polymerase δ appears to be the main activity for telomere extension, since neither type I nor type II survivors arising via telomere-telomere recombination were seen in its absence. The frequency of type I versus type II is altered in the polymerase α and ε mutants relative to the wild type. Each prefers to develop a particular type of survivor. Moreover, type II recombination is mediated by the cell cycle checkpoint proteins Tel1 and Mec1, and telomere-telomere recombination is regulated by telomere binding protein Cdc13 and the Ku complex. Together, our results suggest that coordination between DNA replication machinery, DNA damage signaling, DNA recombination machinery, and the telomere protein-DNA complex allows telomere recombination to repair telomeric ends in the absence of telomerase.
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Akter, Jesmin, and Takehiko Kamijo. "How Do Telomere Abnormalities Regulate the Biology of Neuroblastoma?" Biomolecules 11, no. 8 (July 28, 2021): 1112. http://dx.doi.org/10.3390/biom11081112.
Full textAbstract:
Telomere maintenance plays important roles in genome stability and cell proliferation. Tumor cells acquire replicative immortality by activating a telomere-maintenance mechanism (TMM), either telomerase, a reverse transcriptase, or the alternative lengthening of telomeres (ALT) mechanism. Recent advances in the genetic and molecular characterization of TMM revealed that telomerase activation and ALT define distinct neuroblastoma (NB) subgroups with adverse outcomes, and represent promising therapeutic targets in high-risk neuroblastoma (HRNB), an aggressive childhood solid tumor that accounts for 15% of all pediatric-cancer deaths. Patients with HRNB frequently present with widely metastatic disease, with tumors harboring recurrent genetic aberrations (MYCN amplification, TERT rearrangements, and ATRX mutations), which are mutually exclusive and capable of promoting TMM. This review provides recent insights into our understanding of TMM in NB tumors, and highlights emerging therapeutic strategies as potential treatments for telomerase- and ALT-positive tumors.
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Recagni, Marta, Joanna Bidzinska, Nadia Zaffaroni, and Marco Folini. "The Role of Alternative Lengthening of Telomeres Mechanism in Cancer: Translational and Therapeutic Implications." Cancers 12, no. 4 (April 11, 2020): 949. http://dx.doi.org/10.3390/cancers12040949.
Full textAbstract:
Telomere maintenance mechanisms (i.e., telomerase activity (TA) and the alternative lengthening of telomere (ALT) mechanism) contribute to tumorigenesis by providing unlimited proliferative capacity to cancer cells. Although the role of either telomere maintenance mechanisms seems to be equivalent in providing a limitless proliferative ability to tumor cells, the contribution of TA and ALT to the clinical outcome of patients may differ prominently. In addition, several strategies have been developed to interfere with TA in cancer, including Imetelstat that has been the first telomerase inhibitor tested in clinical trials. Conversely, the limited information available on the molecular underpinnings of ALT has hindered thus far the development of genuine ALT-targeting agents. Moreover, whether anti-telomerase therapies may be hampered or not by possible adaptive responses is still debatable. Nonetheless, it is plausible hypothesizing that treatment with telomerase inhibitors may exert selective pressure for the emergence of cancer cells that become resistant to treatment by activating the ALT mechanism. This notion, together with the evidence that both telomere maintenance mechanisms may coexist within the same tumor and may distinctly impinge on patients’ outcomes, suggests that ALT may exert an unexpected role in tumor biology that still needs to be fully elucidated.
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Batista, Luis, Franklin Zhong, Sharon A. Savage, and Steven Artandi. "TIN2 Mutations In Dyskeratosis Congenita Cause Telomere Shortening In Induced Pluripotent Stem Cells Through Potent Dominant Negative Inhibition Of Telomerase." Blood 122, no. 21 (November 15, 2013): 590. http://dx.doi.org/10.1182/blood.v122.21.590.590.
Full textAbstract:
Abstract Dyskeratosis congenita (DC) is a bone marrow failure syndrome characterized by widespread defects in diverse tissues and a strong predisposition to cancer. DC is caused by germline mutations in genes controlling maintenance of telomeres, nucleoprotein caps that protect chromosome ends. Mutations in components of the telomerase enzyme comprise a large share of cases, including in TERT, TERC, dyskerin, TCAB1, NOP10 and NHP2. These mutations compromise telomerase function leading to telomere shortening, which in turn impairs stem cell function. We previously created patient-derived iPS cells from patients with mutations in TERT, dyskerin or TCAB1 and analyzed these cells to understand the biochemical defects in the telomerase pathway. In each case we found a unique mechanism underlying these telomerase defects, including: reduced catalytic function (TERT mutations), impaired telomerase assembly (dyskerin mutations) and mislocalization of the enzyme to nucleoli (TCAB1 mutations). A six-member protein complex – shelterin - is essential for proper function of telomeres. Despite the critical importance of shelterin proteins in telomere regulation, only a single telomere binding protein – TIN2 – is mutated in DC. However, how these mutations compromise telomere maintenance remains poorly understood. TIN2 mutations occur in a common, autosomal dominant form of DC, presenting in early life, with particularly severe clinical manifestations and poor outcomes. Mutations in the TIN2 gene are clustered in exon 6a, which corresponds to a protein domain of unknown function. To understand how TIN2 mutations impair telomere maintenance and cause DC, we reprogrammed fibroblasts from patients with TIN2 mutations to iPS cells. We succeeded in generating pluripotent iPS cells from a patient with a frame shift mutation at position 284 of the protein. TIN2-mutant iPS cells expressed all the markers of wild-type iPS cells and human ES cells and could be differentiated to all three germ cell layers in culture. With reprogramming from fibroblasts to iPS cells, telomerase is upregulated and causes telomere elongation in wild-type cells. In analyzing telomeres from TIN2-mutant iPS cells, we found that telomere elongation was abrogated. Instead of telomere elongation, TIN2-mutant iPS cells showed telomere shortening with reprogramming and during passage in cell culture. After extended cell passage, TIN2-mutant iPS cells lost the ability to self-renew and differentiated, concomitant with the activation of the telomere surveillance checkpoint p53. To better understand how TIN2 mutant proteins interfere with telomere maintenance, we overexpressed GFP, wild-type TIN2, or TIN2 truncation mutants from DC patients into human, telomerase-positive cancer cells. Genomic DNA was collected from these cells during passage and analyzed for telomere lengths by Southern blot. Expression of GFP or wild-type TIN2 had no effect on telomere lengths, which were stably maintained during the experiment. In marked contrast, expression of the TIN2 truncation mutants from DC patients led to progressive and dramatic telomere shortening with cell passage. Together, these data in patient-derived iPS cells and in human cancer cells suggest that TIN2 mutants inhibit the action of telomerase at telomeres. These results constitute a new molecular mechanism at play in DC and yield new insight into one of the most common forms of DC. Disclosures: No relevant conflicts of interest to declare.
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Sommer, Aurore, and Nicola J. Royle. "ALT: A Multi-Faceted Phenomenon." Genes 11, no. 2 (January 27, 2020): 133. http://dx.doi.org/10.3390/genes11020133.
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One of the hallmarks of cancer cells is their indefinite replicative potential, made possible by the activation of a telomere maintenance mechanism (TMM). The majority of cancers reactivate the reverse transcriptase, telomerase, to maintain their telomere length but a minority (10% to 15%) utilize an alternative lengthening of telomeres (ALT) pathway. Here, we review the phenotypes and molecular markers specific to ALT, and investigate the significance of telomere mutations and sequence variation in ALT cell lines. We also look at the recent advancements in understanding the different mechanisms behind ALT telomere elongation and finally, the progress made in identifying potential ALT-targeted therapies, including those already in use for the treatment of both hematological and solid tumors.
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Armendáriz-Castillo, Isaac, Andrés López-Cortés, Jennyfer García-Cárdenas, Patricia Guevara-Ramírez, Paola E. Leone, Andy Pérez-Villa, Verónica Yumiceba, Ana K. Zambrano, Santiago Guerrero, and César Paz-y-Miño. "TCGA Pan-Cancer Genomic Analysis of Alternative Lengthening of Telomeres (ALT) Related Genes." Genes 11, no. 7 (July 21, 2020): 834. http://dx.doi.org/10.3390/genes11070834.
Full textAbstract:
Telomere maintenance mechanisms (TMM) are used by cancer cells to avoid apoptosis, 85–90% reactivate telomerase, while 10–15% use the alternative lengthening of telomeres (ALT). Due to anti-telomerase-based treatments, some tumors switch from a telomerase-dependent mechanism to ALT; in fact, the co-existence between both mechanisms has been observed in some cancers. Although different elements in the ALT pathway are uncovered, some molecular mechanisms are still poorly understood. Therefore, with the aim to identify potential molecular markers for the study of ALT, we combined in silico approaches in a 411 telomere maintenance gene set. As a consequence, we conducted a genomic analysis of these genes in 31 Pan-Cancer Atlas studies from The Cancer Genome Atlas and found 325,936 genomic alterations; from which, we identified 20 genes highly mutated in the cancer studies. Finally, we made a protein-protein interaction network and enrichment analysis to observe the main pathways of these genes and discuss their role in ALT-related processes, like homologous recombination and homology directed repair. Overall, due to the lack of understanding of the molecular mechanisms of ALT cancers, we proposed a group of genes, which after ex vivo validations, could represent new potential therapeutic markers in the study of ALT.
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Winkler, Thomas, Jake E. Decker, So Gun Hong, Chuanfeng Wu, Mary J. Morgan, Cynthia E. Dunbar, Neal S. Young, and Rodrigo T. Calado. "Telomere Dynamics in Pluripotent Stem Cells Derived From Patients with Telomere Diseases." Blood 118, no. 21 (November 18, 2011): 51. http://dx.doi.org/10.1182/blood.v118.21.51.51.
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Abstract Abstract 51 Telomeres are ribonucleoprotein structures located at the end of linear chromosomes that serve to maintain genomic integrity and cellular proliferative capacity. In highly proliferative cells, the enzyme complex telomerase is responsible for the maintenance and elongation of telomeres, as the process of DNA replication inherently results in loss of terminal nucleotides. Critically short telomeres and deficiency in telomerase activity are etiologic contributors in the bone marrow failure syndromes, idiopathic pulmonary fibrosis, and liver cirrhosis. Although dysfunctional telomere machinery clearly is pathogenic in humans, important clinical features such as highly variable penetrance of phenotype and organ specificity are not well understood. A major obstacle in the investigation of these diseases is the lack of primary tissue, especially early in the clinical course. Direct reprogramming of somatic cells to a pluripotent phenotype (induced pluripotent stem cells, iPSc) by forced expression of a set of defined transcription factors may allow investigation of these phenotypes and specificity in a patient-specific manner. One hallmark of all pluripotent cells, embryonic stem cells and iPSc, is the maintenance of telomere length, most likely due to upregulation of telomerase. Thus, these cells are good candidates through which to directly investigate the effect of loss-of-function mutations within the telomerase complex. Using forced expression of the reprogramming factors Oct4, Sox2, Klf4, and c-myc via retroviral (single transgenes) or lentiviral (polycistronic) gene transfer, we derived multiple iPSc lines from dermal fibroblasts of patients harboring heterozygous loss-of-function mutations in the telomerase genes TERT([R1084P], [R889X]) and TERC([-58C>G]), as well as from healthy subjects. These mutations were shown in vitro to reduce telomerase activity of the mutant allele. Generated iPSc lines morphologically resembled human ES cells, expressed endogenous pluripotency markers (such as TRA 1–60, TRA 1–81, SSEA4, NANOG, OCT4), and showed a similar mRNA expression profile as compared to embryonic stem cells in microarray analysis. Over multiple passages (currently up to 40), iPSc retained their self-renewal capacity and formed teratomas in immune-compromised NSG mice. We randomly chose three iPSc lines from each patient and healthy controls to study telomere dynamics. Telomerase-mutant iPS cells elongated their telomeres during the first 10 passages compared to parental (telomerase negative) fibroblasts, as determined by quantitative real-time PCR and Southern blot. However, telomere elongation was significantly less than in iPSc derived from healthy individuals (p=0.003). Moreover, the pattern and extent of elongation varied among different iPSc lines harboring the same mutation. Telomerase mRNA expression was lower in telomerase-mutant iPS cells than in healthy controls. Additionally, telomerase activity, measured by standard TRAP assay, in early telomerase-mutant iPS cells was reduced relative to control iPSc, but later passage cells tended to have similar activity, suggesting a passage-effect on telomerase activity levels. In conclusion, iPSc can be derived from human telomerase-deficient cells. These cells elongate telomeres to a lesser extent than iPSc from healthy controls, indicating that functional telomerase is the main mechanism of telomere elongation in iPSc. Therefore these cells could be valuable tools in the study of human telomerase deficiencies. Unlike previous studies (Agarwal et al., Nature 2010) investigating iPSc from patients with X-linked dyskeratosis congenita (in which a loss-of-function mutation within DKC1 results in short telomeres), we did not observe significant upregulation of TERC as a compensating mechanism during reprogramming in our telomerease-mutant iPSc. Furthermore, in contrast to the DKC1-mutant iPSc described by Batista et al. (Nature 2011), TERT/TERC mutant iPSc did not show signs of impaired proliferation or self-renewal capacity in long-term culture, likely reflecting clinical differences among patients with TERT/TERC and DKC1 mutations. However, elongation patterns and telomerase activity levels are heterogeneous among clones and passages, indicating the importance of kinetic studies and sample size when studying telomere dynamics in iPSc. Disclosures: Dunbar: ASH: Honoraria.
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Kim, S., T. Chowdhury, H. Yu, S. Choi, K. Kim, H. Kang, J. Lee, et al. "P02.01.B The telomere maintenance mechanism spectrum and its dynamics in gliomas." Neuro-Oncology 24, Supplement_2 (September 1, 2022): ii29. http://dx.doi.org/10.1093/neuonc/noac174.094.
Full textAbstract:
Abstract The activation of the telomere maintenance mechanism (TMM) is one of the critical drivers of cancer cell immortality. In gliomas, TERT expression and TERT promoter mutation are considered to reliably indicate telomerase activation, while ATRX mutation indicates alternative lengthening of telomeres (ALT). However, these relationships have not been extensively validated in tumor tissues. Here, we show through the direct measurement of telomerase activity and ALT in a large set of glioma samples that the TMM in glioma cannot be defined in the dichotomy of telomerase activity and ALT, regardless of TERT expression, TERT promoter mutation and ATRX mutation. Moreover, we observed that a considerable proportion of gliomas lack both telomerase activity and ALT (Neither group). And this Neither group exhibited evidence of slow growth potential. From a set of longitudinal samples from a separate cohort of glioma patients, we discovered that the TMM is not fixed but changes with glioma progression. Collectively, these results suggest that the TMM is a dynamic entity and that reflects the plasticity of the oncogenic biological status of tumor cells and that the TMM should be defined by the direct measurement of telomerase enzyme activity and evidence of ALT.
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Sun, Hong, Zhenfang Wu, Yuanze Zhou, Yanjia Lu, Huaisheng Lu, Hongwen Chen, Shaohua Shi, Zhixiong Zeng, Jian Wu, and Ming Lei. "Structural insights into Pot1-ssDNA, Pot1-Tpz1 and Tpz1-Ccq1 Interactions within fission yeast shelterin complex." PLOS Genetics 18, no. 7 (July 18, 2022): e1010308. http://dx.doi.org/10.1371/journal.pgen.1010308.
Full textAbstract:
The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1OB1-GGTTAC and Pot1OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1DBD-single-stranded-DNA, Pot1372-555-Tpz1185-212 and Tpz1425-470-Ccq1123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3’ ss telomeric overhang.
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Sung, Ji-Yong, Hee-Woong Lim, Je-Gun Joung, and Woong-Yang Park. "Pan-Cancer Analysis of Alternative Lengthening of Telomere Activity." Cancers 12, no. 8 (August 7, 2020): 2207. http://dx.doi.org/10.3390/cancers12082207.
Full textAbstract:
Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism that extends telomeres in cancer cells. It influences tumorigenesis and patient survival. Despite the clinical significance of ALT in tumors, the manner in which ALT is activated and influences prognostic outcomes in distinct cancer types is unclear. In this work, we profiled distinct telomere maintenance mechanisms (TMMs) using 8953 transcriptomes of 31 different cancer types from The Cancer Genome Atlas (TCGA). Our results demonstrated that approximately 29% of cancer types display high ALT activity with low telomerase activity in the telomere-lengthening group. Among the distinct ALT mechanisms, homologous recombination was frequently observed in sarcoma, adrenocortical carcinoma, and kidney chromophobe. Five cancer types showed a significant difference in survival in the presence of high ALT activity. Sarcoma patients with elevated ALT had unfavorable risks (p < 0.038) coupled with a high expression of TOP2A, suggesting this as a potential drug target. On the contrary, glioblastoma patients had favorable risks (p < 0.02), and showed low levels of antigen-presenting cells. Together, our analyses highlight cancer type-dependent TMM activities and ALT-associated genes as potential therapeutic targets.
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Schrumpfová, Petra Procházková, and Jiří Fajkus. "Composition and Function of Telomerase—A Polymerase Associated with the Origin of Eukaryotes." Biomolecules 10, no. 10 (October 8, 2020): 1425. http://dx.doi.org/10.3390/biom10101425.
Full textAbstract:
The canonical DNA polymerases involved in the replication of the genome are unable to fully replicate the physical ends of linear chromosomes, called telomeres. Chromosomal termini thus become shortened in each cell cycle. The maintenance of telomeres requires telomerase—a specific RNA-dependent DNA polymerase enzyme complex that carries its own RNA template and adds telomeric repeats to the ends of chromosomes using a reverse transcription mechanism. Both core subunits of telomerase—its catalytic telomerase reverse transcriptase (TERT) subunit and telomerase RNA (TR) component—were identified in quick succession in Tetrahymena more than 30 years ago. Since then, both telomerase subunits have been described in various organisms including yeasts, mammals, birds, reptiles and fish. Despite the fact that telomerase activity in plants was described 25 years ago and the TERT subunit four years later, a genuine plant TR has only recently been identified by our group. In this review, we focus on the structure, composition and function of telomerases. In addition, we discuss the origin and phylogenetic divergence of this unique RNA-dependent DNA polymerase as a witness of early eukaryotic evolution. Specifically, we discuss the latest information regarding the recently discovered TR component in plants, its conservation and its structural features.
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Pan, Xiaolei, William C. Drosopoulos, Louisa Sethi, Advaitha Madireddy, Carl L. Schildkraut, and Dong Zhang. "FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres." Proceedings of the National Academy of Sciences 114, no. 29 (July 3, 2017): E5940—E5949. http://dx.doi.org/10.1073/pnas.1708065114.
Full textAbstract:
In the mammalian genome, certain genomic loci/regions pose greater challenges to the DNA replication machinery (i.e., the replisome) than others. Such known genomic loci/regions include centromeres, common fragile sites, subtelomeres, and telomeres. However, the detailed mechanism of how mammalian cells cope with the replication stress at these loci/regions is largely unknown. Here we show that depletion of FANCM, or of one of its obligatory binding partners, FAAP24, MHF1, and MHF2, induces replication stress primarily at the telomeres of cells that use the alternative lengthening of telomeres (ALT) pathway as their telomere maintenance mechanism. Using the telomere-specific single-molecule analysis of replicated DNA technique, we found that depletion of FANCM dramatically reduces the replication efficiency at ALT telomeres. We further show that FANCM, BRCA1, and BLM are actively recruited to the ALT telomeres that are experiencing replication stress and that the recruitment of BRCA1 and BLM to these damaged telomeres is interdependent and is regulated by both ATR and Chk1. Mechanistically, we demonstrated that, in FANCM-depleted ALT cells, BRCA1 and BLM help to resolve the telomeric replication stress by stimulating DNA end resection and homologous recombination (HR). Consistent with their roles in resolving the replication stress induced by FANCM deficiency, simultaneous depletion of BLM and FANCM, or of BRCA1 and FANCM, leads to increased micronuclei formation and synthetic lethality in ALT cells. We propose that these synthetic lethal interactions can be explored for targeting the ALT cancers.
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Lai, Tsung-Po, Woodring E. Wright, and Jerry W. Shay. "Comparison of telomere length measurement methods." Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1741 (January 15, 2018): 20160451. http://dx.doi.org/10.1098/rstb.2016.0451.
Full textAbstract:
The strengths and limitations of the major methods developed to measure telomere lengths (TLs) in cells and tissues are presented in this review. These include Q-PCR ( Q uantitative P olymerase C hain R eaction), TRF ( T erminal R estriction F ragment) analysis, a variety of Q-FISH ( Q uantitative F luorescence I n S itu H ybridization) methods, STELA ( S ingle TE lomere L ength A nalysis) and TeSLA ( Te lomere S hortest L ength A ssay). For each method, we will cover information about validation studies, including reproducibility in independent laboratories, accuracy, reliability and sensitivity for measuring not only the average but also the shortest telomeres. There is substantial evidence that it is the shortest telomeres that trigger DNA damage responses leading to replicative senescence in mammals. However, the most commonly used TL measurement methods generally provide information on average or relative TL, but it is the shortest telomeres that leads to telomere dysfunction (identified by TIF, T elomere dysfunction I nduced F oci) and limit cell proliferation in the absence of a telomere maintenance mechanism, such as telomerase. As the length of the shortest telomeres is a key biomarker determining cell fate and the onset of senescence, a new technique (TeSLA) that provides quantitative information about all the shortest telomeres will be highlighted. This article is part of the theme issue ‘Understanding diversity in telomere dynamics’.
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Grobelny, J. V., A. K. Godwin, and D. Broccoli. "ALT-associated PML bodies are present in viable cells and are enriched in cells in the G(2)/M phase of the cell cycle." Journal of Cell Science 113, no. 24 (December 15, 2000): 4577–85. http://dx.doi.org/10.1242/jcs.113.24.4577.
Full textAbstract:
Telomere maintenance is essential for the unlimited proliferative potential of human cells, and hence immortalization. However, a number of tumors, tumor-derived cell lines and in vitro immortalized cell lines have been described that do not express detectable telomerase activity. These lines utilize a mechanism, termed Alternative Lengthening of Telomeres (ALT), to provide telomere maintenance. A subset of the cells in each ALT cell line contain a novel form of the promyelocytic leukemia nuclear body (PML NB) in which telomeric DNA and the telomere binding proteins TRF1 and TRF2 co-localize with the PML protein, termed ALT-associated PML bodies (AA-PBs). In contrast, in non-ALT, telomerase-positive cell lines these telomeric proteins and the PML NB occupy distinct and separate subnuclear domains. PML NBs have been implicated in terminal differentiation, growth suppression and apoptosis. The role, if any, of AA-PBs in telomere maintenance or culture viability in telomerase negative cell lines is unclear, but it has been suggested that cells containing these structures are no longer viable and are marked for eventual death. We utilized a series of human ovarian surface epithelium (HOSE) cell lines that use ALT for telomere maintenance to determine if AA-PBs are indeed markers of cells in these cultures that are no longer cycling. We show that AA-PB positive cells incorporate BrdU and thus are able to carry out DNA replication. In addition, AA-PBs are present in mitotic cells and the frequency of cells containing these structures is increased when cultures are enriched for cells in the G(2)/M phase of the cell cycle suggesting that the formation of AA-PBs is coordinately regulated with the cell cycle. Finally, we demonstrate that the majority of the AA-PB positive cells in the culture are not destined for immediate apoptosis. Taken together the data argue against AA-PBs marking cells destined for death and, instead, raise the possibility that these structures may be actively involved in telomere maintenance via the ALT pathway.
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Pal, Jagannath, Jason Wong, Puru Nanjappa, Saem Lee, Masood Shammas, and Nikhil C. Munshi. "Inhibition of Homologous Recombination Pathway Promotes Telomere Shortening and Cell Survival without Affecting Telomerase Activity In Myeloma." Blood 116, no. 21 (November 19, 2010): 786. http://dx.doi.org/10.1182/blood.v116.21.786.786.
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Abstract Abstract 786 Recombinase (RAD51) expression and homologous recombination (HR) activity are low in normal human cells including plasma cells. It is significantly induced following exposure of normal human cells to carcinogen, and is constitutively elevated in cancer cells including multiple myeloma (MM) cells. Besides its effect on genomic stability, elevated or dysregulated HR has also been implicated in telomere maintenance in tumor and immortalized cells. These cells usually lack telomerase activity and maintain telomere length by ALT mechanism (alternate lengthening of telomeres). Inhibitors of homologous recombination, therefore, have potential not only to prevent/reduce genomic instability, but also inhibit telomere maintenance, and cancer survival. We have here investigated the effect of inhibitor of HR on telomere maintenance mechanism in MM. We have evaluated effect of Nilotinib, a tyrosine kinase inhibitor and RAD51 shRNA on HR in MM. First we observed that nilotinib inhibits and RAD51 phosphorylation in MM. Nilotinib at both 5 and 10 mM concentration also led to dose-dependent inhibition of recombinase expression in MM cells. Importantly, Nilotinib also inhibited HR activity in MM cells as well as other cancer cell lines, as measured by a plasmid based assay in which leuciferase activity is generated following homologous recombination. We next evaluated effect of nilotinib on telomere maintenance alone as well as in combination with agents inhibiting telomere maintenance. The MM cells were treated for 48 hrs, either with nilotinib, telomerase inhibitor, or both nilotinib and telomerase inhibitor and evaluated for telomerase activity as well as effect on telomere length. As expected, the treatment of myeloma cells with telomerase inhibitor at 1 mM led to 88% inhibition of telomerase activity relative to control cells. Nilotinib, either alone or in the presence of telomerase inhibitor, did not have any major effect on telomerase activity in these cells. The cells were cultured in the presence of these agents for 2 weeks and evaluated for telomere length, using telomere specific real time PCR. Cells in presence of Telomerase inhibitor at 1 mM in fact had slightly increased telomere length (9%), probably due to presence or activation of ALT mechanism, following loss of telomerase activity. Importantly, nilotinib alone at 10 mM led to 20% reduction in telomere length and when combined with telomerease inhibitor at 1 mM concentrations led to reduction in the telomere length in MM cells by 52%. Moreover we have observed that transduction of MM cells with shRNA targeting RAD51 combined with telomerase inhibitor induced greater and quicker MM cell kill compared to either of these treatments alone. These data indicate that elevated HR pathway contributes to telomere maintenance in MM and combining inhibitors of HR with telomerase would expedite telomere shortening and cell death providing more effective therapeutic strategy. Disclosures: Munshi: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.
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Blasco, Maria A. "Therapeutic Strategies for the Treatment of Telomere-Associated Diseases." Blood 126, no. 23 (December 3, 2015): SCI—50—SCI—50. http://dx.doi.org/10.1182/blood.v126.23.sci-50.sci-50.
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Abstract Aplastic anemia is a rare but life-threatening disorder often accompanied by the presence of short telomeres in the hematopoietic compartment in both the acquired and the inherited form. We recently generated a mouse model that faithfully recapitulates the bone marrow phenotype observed in aplastic anemia patients. In this model, incomplete conditional Trf1 gene deletion specifically in the hematopoietic system causes acute telomere uncapping and persistent activation of a DNA damage response at telomeres, leading to a fast elimination of those HSCs lacking Trf1 and a compensatory hyperproliferation along with rapid telomere shortening in those which retain intact Trf1. Thus, this model recapitulates the high turnover and hyperproliferation observed in aplastic anemia patients of autoimmune origin, as well as presence of very short telomeres owing to mutations in telomere maintenance genes. We are currently employing this model to test two different therapeutic strategies for the reactivation of telomerase in the hematopoietic system, therefore counteracting telomere attrition and alleviating the aplastic anemia phenotype owed to the presence of short telomeres. First, we explored the possibility that the recently described regulation of telomerase activity by sex hormones may be the responsible mechanism for the frequently observed induction of remission in aplastic anemia patients following hormone therapy. We show that androgen therapy results in telomerase up-regulation, improved blood counts, and a significant extension of life-span in our mouse model. Importantly, longitudinal follow-up studies revealed longer telomeres in peripheral blood in mice subjected to hormone treatment. In addition to this chemical approach for telomerase activation we are also testing the efficacy of increasing telomerase gene dosage by virtue of AAV9 gene therapy. Our preliminary data indicate that AAV9-Tert treatment leads to a significantly reduces aplastic anemia related morbidity which coincides with telomere elongation in blood and bone marrow cell compared to empty vector treated mice. In summary, we used a mouse model of aplastic anemia produced by short telomeres and demonstrate two different strategies of telomerase activation which are effective in treating the disease. Thus this work may pave the way for the development of novel treatment options for aplastic anemia in humans. Disclosures No relevant conflicts of interest to declare.
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Brault, Marie Eve, and Chantal Autexier. "Telomeric recombination induced by dysfunctional telomeres." Molecular Biology of the Cell 22, no. 2 (January 15, 2011): 179–88. http://dx.doi.org/10.1091/mbc.e10-02-0173.
Full textAbstract:
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.
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Buttarelli, Francesca Romana, Simone Minasi, Maria Luisa Garrè, Maura Massimino, Veronica Biassoni, Tobias Goschzik, Torsten Pietsch, and Felice Giangaspero. "MEDB-68. Analysis of telomeres length and Alternative Lengthening of Telomeres (ALT) in molecular subgroups of infant medulloblastoma." Neuro-Oncology 24, Supplement_1 (June 1, 2022): i122. http://dx.doi.org/10.1093/neuonc/noac079.442.
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Abstract We investigated the association between the molecular profile and telomere length in a infant medulloblastoma (iMB) cohort, retrospectively studied. Activation of telomeres maintenance mechanisms was analyzed to determine whether the senescence escape triggered by telomere-elongation mechanisms could explain the aggressivity of some iMB belonging to the same molecular subgroup. Interestingly, several telomerase- and ALT-targeted therapies have recently been tested on pediatric cancers and might represent a promising strategy for the future treatment of aggressive telomerase- or ALT-positive iMB. We analyzed a cohort of 50 FFPE tissues from young MB patients (age ≤ 3); IHC, FISH, and an Illumina 850K methylation profile were used to identify molecular subgroups. Telomere length was measured using Telo-quantitative FISH, and image analysis was performed using TFL-Telo software. Three distinct telomere intensity categories (low (L), medium (M), and high (H)) were identified by comparing neoplastic- to endothelial-cell signals in each sample. ATRX loss and TERTp mutation/methylation were investigated using IHC and Sanger sequencing/methylation-specific PCR. SHH-MBs accounted for 59% of our cohort, while Group3/4-MBs accounted for 41%; no WNT-MBs were detected. ALT was found to be activated in 10% of iMBs and was not exclusive to any molecular subgroup, implying that it could be a potential mechanism associated with aggressive behaviour in a subset of iMBs. Promising results have been found in the telomere length distribution among the iMB molecular subgroups: SHH iMBs had a higher frequency of High (H) telomeres length (85%) than NON-SHH/NON-WNT iMBs (p=0.046), which were more frequently associated with Medium (M) telomeres length. CONCLUSIONS: ALT activation in infant MBs (10%) could be a novel target for risk-stratification and personalized therapy. It may be useful to examine ALT as a potential predictor of aggressive behaviour and as a promising novel therapeutic approach for a subset of these tumors in the diagnostic workup.
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Toubiana, Shir, Aya Tzur-Gilat, and Sara Selig. "Epigenetic Characteristics of Human Subtelomeres Vary in Cells Utilizing the Alternative Lengthening of Telomeres (ALT) Pathway." Life 11, no. 4 (March 26, 2021): 278. http://dx.doi.org/10.3390/life11040278.
Full textAbstract:
Most human cancers circumvent senescence by activating a telomere length maintenance mechanism, most commonly involving telomerase activation. A minority of cancers utilize the recombination-based alternative lengthening of telomeres (ALT) pathway. The exact requirements for unleashing normally repressed recombination at telomeres are yet unclear. Epigenetic modifications at telomeric regions were suggested to be pivotal for activating ALT; however, conflicting data exist regarding their exact nature and necessity. To uncover common ALT-positive epigenetic characteristics, we performed a comprehensive analysis of subtelomeric DNA methylation, histone modifications, and TERRA expression in several ALT-positive and ALT-negative cell lines. We found that subtelomeric DNA methylation does not differentiate between the ALT-positive and ALT-negative groups, and most of the analyzed subtelomeres within each group do not share common DNA methylation patterns. Additionally, similar TERRA levels were measured in the ALT-positive and ALT-negative groups, and TERRA levels varied significantly among the members of the ALT-positive group. Subtelomeric H3K4 and H3K9 trimethylation also differed significantly between samples in the ALT-positive group. Our findings do not support a common route by which epigenetic modifications activate telomeric recombination in ALT-positive cells, and thus, different therapeutic approaches will be necessary to overcome ALT-dependent cellular immortalization.
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Sloand, Elaine M., Rodrigo Calado, Simant Shah, Loretta Pfannes, Jan Blancato, and Neal Young. "Telomere Shortening and Genomic Instability: Primary Cells from Patients with Telomere Repair Complex Mutations Are Susceptible to End-to-End Chromosome Fusion and Aneuploidy." Blood 108, no. 11 (November 1, 2006): 2079. http://dx.doi.org/10.1182/blood.v108.11.2079.2079.
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Abstract Telomeres that cap the ends of chromosomes, protect them from recombination, end-to-end fusion, and recognition as damaged DNA. Telomere shorteningmay result in cell cycle arrest and senescence or in genomic instability that precedes malignancy. Maintenance of the integrity of telomeres requires the telomerase ribonucleoprotein complex, mainly the telomerase reverse transcriptase (TERT) and its integral RNA template (TERC). Mutations in TERC and TERT cause progressive telomere shortening; short telomeres are found in constitutional marrow failure syndromes and also in apparently acquired aplastic anemia (Fogarty PF et al, Lancet362:1628, 2003;. Yamaguchi H et al, NEJM352:1413, 2005). Myelodysplasia and acute leukemia are often present in these kindreds and associated with pathogenic mutations. The mechanism resulting in genomic instability in TERT and TERC-deficient individuals is unclear. End-to-end chromosome fusion in TERT-”knock-out” embryonic mouse stem cells are seen after multiple passages in culture and are associated with profound telomere shortening and the development of aneuploidy (Lee HW et al. Nature 9:569–74, 1998). End-to-end fusion of sister chromatids presumably results in disruption of chromosome segregation during metaphase. In the present study, we examined blood and marrow cells from 7 patients with TERT, and TERC gene mutations; 10 healthy young controls; and 2 individuals older than age 60 years of age, as well as 6 umbilical cord bloods. Leukocytes with TERT or TERC mutations had short telomeres as previously measured by flow-fluorescent in situ hybridization (FISH) and confirmed by Southern hybridization (mean telomere shortening in comparison to age-matched controls, 3.4 ± 1.7 kb) and low telomerase enzymatic activity (144±13 vs. 270±56 respectively; P<0.02)compared to normal controls, using the telomeric repeat amplification protocol assay. Bone marrow mononuclear cells were cultured for several days, synchronized with colcemid, and metaphase spreads were prepared for FISH using a telomeric probe. Samples from 4 patients who had significant numbers of metaphases demonstrated end-to-end fusions of chromosomes in 2- -20 % of metaphases. One elderly healthy donor demonstrated small numbers of metaphases with end to end fusions; no fusions were identified in any cord blood samples or in the 5 samples from younger donors. To measure aneuploidy, bone marrow cells from 5 patients were cultured for two weeks with 400ng/mL of G-CSF. Three patients with TERT and two patients with TERC mutant cells showed trisomy 8 (4–24% of interphases, mean=12%), with fewer samples (N=2) showing monosomy 7 (2–40%, mean 14%) and trisomy 9 by FISH; none of the ten samples from young healthy controls showed any anueploidy after culture (p=0.007). We have established links between telomere shortening, end-to-end chromosome fusion, and genomic instability, predicted from murine models, in human cells. Telomere shortening-secondary to genetic lesions in crucial genes of the repair complex and perhaps also due to normal aging (and, in theory, marrow “stress”) may underlie the genomic instability that leads to aneuploidy, providing a mechanism for chromosome aberrations observed in patients with dyskeratosis congenita, in “clonal evolution” of aplastic anemia, and in the most frequent form of bone marrow failure in the elderly, myelodysplasia.
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Gu, Baiwei, Jun He, Monica Bessler, and Philip J. Mason. "Unbalanced X Chromosome Inactivation Independent of Telomere Shortening in Mice Heterozygous for a Mutant Dyskerin Allele." Blood 108, no. 11 (November 16, 2006): 184. http://dx.doi.org/10.1182/blood.v108.11.184.184.
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Abstract X-linked Dyskeratosis Congenita (DC) is a rare recessive disorder caused by mutations in the DKC1 gene that encodes dyskerin. Dyskerin is part of ribonucleoprotein complexes that participate in two different pathways: ribosome biogenesis and telomere maintenance. It is the subject of intense debate whether disease manifestations in DC are due to dysfunctional telomere maintenances or are caused by a defect in ribosome biogenesis. Pathogenic mutations in dyskerin cause telomere shortening and patients with X-linked DC have critically short telomeres, However, whether there is an additional defect in ribosome biogenesis is difficult to investigate. To dissect the impact of a pathogenic dyskerin mutation on telomeres from the possible additional impact on ribosome biogenesis in an in vivo model, we generated mice expressing a mutant dyskerin protein. Because laboratory mice have very long telomeres a short telomere phenotype requires several generations of inbreeding, whereas a phenotype seen in the first generation is likely to be caused by the defect in ribosome biogenesis. To delete the last 21 amino acids of dyskerin (Del15) we used homologous recombination followed by conditional gene deletion in murine embryonic stem (ES) cells and in mice. Six independent ES cell clones with the deleted Dkc1 gene were obtained. In vitro analysis of the ES cells showed that the Del15 mutation led to dramatically decreased expression of a truncated dyskerin protein with decreased accumulation of the telomerase RNA. In addition, both reduction in telomerase activity and significant telomere shortening after 65 passages were observed. These findings indicate that the Del15 mutation impairs the telomerase maintenance pathway. After testing the accumulation of a series of mouse H/ACA snoRNA in Del15 ES cells, we found a decrease of the mU68 and mE1 snoRNAs suggesting the mutation may also confer effects which are outside the telomerase pathway. We therefore went on to produce a line of mice expressing the truncated Dkc1 protein and were able to obtain male mice hemizygous for the mutant Dkc1 gene as well as female heterozgotes. The male mice express the truncated dyskerin protein and show no gross abnormality up to 6 months of age. Interestingly, heterozygous female mice were healthy as well but the truncated dyskerin protein was dramatically decreased in expression compared to the wild type dyskerin in spleen, thymus, and bone marrow, but not in liver and brain. This result must derive from preferential proliferation of cells expressing wild type dyskerin after random X-inactivation in early embryogenesis. Our analysis indicates that the mutant dyskerin impairs the proliferation in hematopoietic tissues while it does not affect cells which are not rapidly proliferating such as those in liver and brain. Because of the early appearance of the skewed X-inactivation phenotype we conclude that skewing in these mice is caused by a telomere independent mechanism. Interestingly, the lack of overt DC-like abnormalities in the male hemizygous mice indicates that this proliferative disadvantage is insufficient to cause bone marrow failure but in combination with impaired telomere maintenance may accelerate the onset and severity of disease and thus explain the earlier and more severe manifestation in X-linked DC compared to autosomal dominant DC which only affects the telomerase pathway.
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Choo, Seunga, Franziska K. Lorbeer, Samuel G. Regalado, Sarah B. Short, Shannon Wu, Gabrielle Rieser, Alison A. Bertuch, and Dirk Hockemeyer. "Genetic Dissection of Dyskeratosis Congenita-Causing Mutations in TINF2 Using Human Pluripotent and Hematopoietic Stem Cell Models." Blood 138, Supplement 1 (November 5, 2021): 2178. http://dx.doi.org/10.1182/blood-2021-149798.
Full textAbstract:
Abstract The length of telomeres, which cap the ends of linear chromosomes and provide genomic stability, is tightly regulated in adult stem cells. The telomere reserve in the stem cell population sets the replicative potential of its differentiated progeny. For this reason, abnormally short telomeres in stem cells restrict the number of cell divisions that their differentiated progenies can undergo, eventually resulting in stem cell depletion and tissue failure syndromes. Telomere biology disorders (TBDs) display a broad range of clinical features, age of onset, and severity, which are all correlated with the extent of abnormal telomere shortening. One such early-onset TBD is dyskeratosis congenita (DC), which is a bone marrow predisposition syndrome characterized by a mucocutaneous triad (oral leukoplakia, nail dystrophy, and abnormal skin pigmentation) as well as other conditions driven by premature tissue aging. The leading cause of death in DC patients is bone marrow failure and hematopoietic stem cell transplantation is the only definite intervention to restore hematopoiesis. TINF2, which encodes the TIN2 protein, is mutated in 12% of patients and thereby the second most frequently altered gene in DC cases. TIN2 is a member of the shelterin protein complex bridging the double-strand binding shelterin proteins TRF1/TRF2, and the TPP1/POT1 heterodimer. Such interactions implicate a complex role of TIN2 in telomere length regulation: First, TIN2 stabilizes TRF1, which is a negative regulator of telomere length. Secondly, TPP1, which recruits telomerase, strictly requires TIN2 for telomere elongation and maintenance. TINF2-DC mutations are uniformly heterozygous and localize to a 30 amino acid coding stretch in exon 6 called the 'DC cluster'. TINF2-DC mutations usually arise de novo and result in an earlier disease onset, shorter telomeres, and a more severe manifestation compared to other heterozygous DC-causing mutations in genes such as TR and TERT, which encode the components of the telomerase enzyme. How TINF2-DC mutations cause telomere shortening is unknown. Specifically, whether telomere shortening is caused by reduced telomerase action at telomeres or by degradation of telomeric DNA remains unresolved as studies using different model systems report contrasting results. The discrepancy could be attributed to differences in the model systems used in the studies, highlighting the need for a genetically trackable, primary preclinical human model system. Here, we report the development of two novel endogenous, isogenic model systems to study TINF2-DC mutations. First, we generated human embryonic stem cells (hESCs) engineered to express the TINF2-DC T284R mutation from the endogenous locus, which recapitulated the short telomere phenotype observed in DC patients. Using this model, we identified a gene editing strategy that elongates telomeres in the mutant stem cells and eventually restores replicative potential of the differentiated cells. Next, we used a xenotransplantation model of donor-derived human hematopoietic stem cells (hHSCs) to test the effects of target gene modifications on telomere length and proliferative capacity in vivo. We demonstrate that our models robustly complement each other and offer direct insights into the disease mechanism as well as avenues to potential therapeutic approaches. Figure 1 Figure 1. Disclosures Bertuch: Elixirgen Therapeutics: Consultancy; ImmunityBio: Current equity holder in publicly-traded company; NIH/NCI,: Research Funding; DOD: Research Funding; Hyundai Hope on Wheels: Research Funding.