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1
Tommerup, H., A. Dousmanis y T. de Lange. "Unusual chromatin in human telomeres". Molecular and Cellular Biology 14, n.º 9 (septiembre de 1994): 5777–85. http://dx.doi.org/10.1128/mcb.14.9.5777-5785.1994.
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We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere.
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Tommerup, H., A. Dousmanis y T. de Lange. "Unusual chromatin in human telomeres." Molecular and Cellular Biology 14, n.º 9 (septiembre de 1994): 5777–85. http://dx.doi.org/10.1128/mcb.14.9.5777.
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We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere.
3
Cook, Brandoch D., Jasmin N. Dynek, William Chang, Grigoriy Shostak y Susan Smith. "Role for the Related Poly(ADP-Ribose) Polymerases Tankyrase 1 and 2 at Human Telomeres". Molecular and Cellular Biology 22, n.º 1 (1 de enero de 2002): 332–42. http://dx.doi.org/10.1128/mcb.22.1.332-342.2002.
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ABSTRACT Telomere maintenance is essential for the continuous growth of tumor cells. In most human tumors telomeres are maintained by telomerase, a specialized reverse transcriptase. Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase (PARP), positively regulates telomere length through its interaction with TRF1, a telomeric DNA-binding protein. Tankyrase 1 ADP-ribosylates TRF1, inhibiting its binding to telomeric DNA. Overexpression of tankyrase 1 in the nucleus promotes telomere elongation, suggesting that tankyrase 1 regulates access of telomerase to the telomeric complex. The recent identification of a closely related homolog of tankyrase 1, tankyrase 2, opens the possibility for a second PARP at telomeres. We therefore sought to establish the role of tankyrase 1 at telomeres and to determine if tankyrase 2 might have a telomeric function. We show that endogenous tankyrase 1 is a component of the human telomeric complex. We demonstrate that telomere elongation by tankyrase 1 requires the catalytic activity of the PARP domain and does not occur in telomerase-negative primary human cells. To investigate a potential role for tankyrase 2 at telomeres, recombinant tankyrase 2 was subjected to an in vitro PARP assay. Tankyrase 2 poly(ADP-ribosyl)ated itself and TRF1. Overexpression of tankyrase 2 in the nucleus released endogenous TRF1 from telomeres. These findings establish tankyrase 2 as a bona fide PARP, with itself and TRF1 as acceptors of ADP-ribosylation, and suggest the possibility of a role for tankyrase 2 at telomeres.
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Mattern, Karin A., Susan J. J. Swiggers, Alex L. Nigg, Bob Löwenberg, Adriaan B. Houtsmuller y J. Mark J. M. Zijlmans. "Dynamics of Protein Binding to Telomeres in Living Cells: Implications for Telomere Structure and Function". Molecular and Cellular Biology 24, n.º 12 (15 de junio de 2004): 5587–94. http://dx.doi.org/10.1128/mcb.24.12.5587-5594.2004.
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ABSTRACT Telomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is largely unknown. By using green fluorescent protein tagging and photobleaching, we investigated in vivo interactions of human telomeric DNA-binding proteins with telomeric DNA. Our results show that telomeric proteins interact with telomeres in a complex dynamic fashion: TRF2, which has a dual role in chromosome end protection and telomere length homeostasis, resides at telomeres in two distinct pools. One fraction (∼73%) has binding dynamics similar to TRF1 (residence time of ∼44 s). Interestingly, the other fraction of TRF2 binds with similar dynamics as the putative end-protecting factor hPOT1 (residence time of ∼11 min). Our data support a dynamic model of telomeres in which chromosome end-protection and telomere length homeostasis are governed by differential binding of telomeric proteins to telomeric DNA.
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Brault, Marie Eve y Chantal Autexier. "Telomeric recombination induced by dysfunctional telomeres". Molecular Biology of the Cell 22, n.º 2 (15 de enero de 2011): 179–88. http://dx.doi.org/10.1091/mbc.e10-02-0173.
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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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Fernandes, Stina George, Rebecca Dsouza, Gouri Pandya, Anuradha Kirtonia, Vinay Tergaonkar, Sook Y. Lee, Manoj Garg y Ekta Khattar. "Role of Telomeres and Telomeric Proteins in Human Malignancies and Their Therapeutic Potential". Cancers 12, n.º 7 (14 de julio de 2020): 1901. http://dx.doi.org/10.3390/cancers12071901.
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Telomeres are the ends of linear chromosomes comprised of repetitive nucleotide sequences in humans. Telomeres preserve chromosomal stability and genomic integrity. Telomere length shortens with every cell division in somatic cells, eventually resulting in replicative senescence once telomere length becomes critically short. Telomere shortening can be overcome by telomerase enzyme activity that is undetectable in somatic cells, while being active in germline cells, stem cells, and immune cells. Telomeres are bound by a shelterin complex that regulates telomere lengthening as well as protects them from being identified as DNA damage sites. Telomeres are transcribed by RNA polymerase II, and generate a long noncoding RNA called telomeric repeat-containing RNA (TERRA), which plays a key role in regulating subtelomeric gene expression. Replicative immortality and genome instability are hallmarks of cancer and to attain them cancer cells exploit telomere maintenance and telomere protection mechanisms. Thus, understanding the role of telomeres and their associated proteins in cancer initiation, progression and treatment is very important. The present review highlights the critical role of various telomeric components with recently established functions in cancer. Further, current strategies to target various telomeric components including human telomerase reverse transcriptase (hTERT) as a therapeutic approach in human malignancies are discussed.
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Lin, Chih-Yi Gabriela, Anna Christina Näger, Thomas Lunardi, Aleksandra Vančevska, Gérald Lossaint y Joachim Lingner. "The human telomeric proteome during telomere replication". Nucleic Acids Research 49, n.º 21 (8 de noviembre de 2021): 12119–35. http://dx.doi.org/10.1093/nar/gkab1015.
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Abstract Telomere shortening can cause detrimental diseases and contribute to aging. It occurs due to the end replication problem in cells lacking telomerase. Furthermore, recent studies revealed that telomere shortening can be attributed to difficulties of the semi-conservative DNA replication machinery to replicate the bulk of telomeric DNA repeats. To investigate telomere replication in a comprehensive manner, we develop QTIP-iPOND - Quantitative Telomeric chromatin Isolation Protocol followed by isolation of Proteins On Nascent DNA - which enables purification of proteins that associate with telomeres specifically during replication. In addition to the core replisome, we identify a large number of proteins that specifically associate with telomere replication forks. Depletion of several of these proteins induces telomere fragility validating their importance for telomere replication. We also find that at telomere replication forks the single strand telomere binding protein POT1 is depleted, whereas histone H1 is enriched. Our work reveals the dynamic changes of the telomeric proteome during replication, providing a valuable resource of telomere replication proteins. To our knowledge, this is the first study that examines the replisome at a specific region of the genome.
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Boccardi, Virginia, Luigi Cari, Giuseppe Nocentini, Carlo Riccardi, Roberta Cecchetti, Carmelinda Ruggiero, Beatrice Arosio, Giuseppe Paolisso, Utz Herbig y Patrizia Mecocci. "Telomeres Increasingly Develop Aberrant Structures in Aging Humans". Journals of Gerontology: Series A 75, n.º 2 (2 de noviembre de 2018): 230–35. http://dx.doi.org/10.1093/gerona/gly257.
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Abstract Telomeres progressively shorten with age, and it has been proposed that critically short and dysfunctional telomeres contribute to aging and aging-associated diseases in humans. For many years it was thought that telomere erosion was strictly a consequence of the “end replication problem,” or the inability of replicative polymerases to completely duplicate linear DNA ends. It is becoming increasingly evident, however, that telomere shortening of cultured human cells is also caused because of other replication defects in telomeric repeats, those that cause fragile telomeres and other aberrant telomeric structures that can be detected on metaphase chromosomes. Whether these replication defects contribute to telomere erosion also in human tissues is currently unknown. By analyzing peripheral blood mononuclear cells from a total of 35 healthy subjects ranging in age from 23 to 101 years, we demonstrated that telomeres increasingly display aberrant structures with advancing donor age. Although the percentages of fragile telomeres increased only until adulthood, the percentages of chromosomes displaying sister telomere loss and sister telomere chromatid fusions increased consistently throughout the entire human life span. Our data, therefore, suggest that telomeric replication defects other than the end replication problem contribute to aging-associated telomere erosion in humans.
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Groff-Vindman, Cindy, Anthony J. Cesare, Shobhana Natarajan, Jack D. Griffith y Michael J. McEachern. "Recombination at Long Mutant Telomeres Produces Tiny Single- and Double-Stranded Telomeric Circles". Molecular and Cellular Biology 25, n.º 11 (1 de junio de 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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Calado, Rodrigo T., Solomon A. Graf y Neal S. Young. "Telomeric Recombination in Lymphocytes Implicates ALT, an Alternative Mechanism for Telomere Length Maintenance, in Normal Human Hematopoietic Cells." Blood 110, n.º 11 (16 de noviembre de 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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Hsieh, Yi-Ching, Pei-Jung Tu, Ying-Yuan Lee, Chun-Chen Kuo, Yi-Chien Lin, Chi-Fang Wu y Jing-Jer Lin. "The U3 small nucleolar ribonucleoprotein component Imp4p is a telomeric DNA-binding protein". Biochemical Journal 408, n.º 3 (28 de noviembre de 2007): 387–93. http://dx.doi.org/10.1042/bj20070968.
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Imp4p is a component of U3 snoRNP (small nucleolar ribonucleoprotein) involved in the maturation of 18S rRNA. We have shown that Imp4p interacts with Cdc13p, a single-stranded telomere-binding protein involved in telomere maintenance. To understand the role of Imp4p in telomeres, we purified recombinant Imp4p protein and tested its binding activity towards telomeric DNA using electrophoretic mobility-shift assays. Our results showed that Imp4p bound specifically to single-stranded telomeric DNA in vitro. The interaction of Imp4p to telomeres in vivo was also demonstrated by chromatin immunoprecipitation experiments. Significantly, the binding of Imp4p to telomeres was not limited to yeast proteins, since the hImp4 (human Imp4) also bound to vertebrate single-stranded telomeric DNA. Thus we conclude that Imp4p is a novel telomeric DNA-binding protein that, in addition to its role in rRNA processing, might participate in telomere function.
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Li, Bibo y Titia de Lange. "Rap1 Affects the Length and Heterogeneity of Human Telomeres". Molecular Biology of the Cell 14, n.º 12 (diciembre de 2003): 5060–68. http://dx.doi.org/10.1091/mbc.e03-06-0403.
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Telomere length is controlled in part by cis-acting negative regulators that limit telomere extension by telomerase. In budding yeast, the major telomere length regulator scRap1 binds to telomeric DNA and acts to inhibit telomere elongation in cis. Because the human Rap1 ortholog hRap1 does not bind to telomeric DNA directly but is recruited to telomeres by TRF2, we examined its role in telomere length control. The data are consistent with hRap1 being a negative regulator of telomere length, indicating functional conservation. Deletion mapping confirmed that hRap1 is tethered to telomeres through interaction of its C terminus with TRF2. The telomere length phenotypes of hRap1 deletion mutants implicated both the BRCT and Myb domain as protein interaction domains involved in telomere length regulation. By contrast, scRap1 binds to telomeres with its Myb domains and uses its C terminus to recruit the telomere length regulators Rif1 and Rif2. Together, our data show that although the role of Rap1 at telomeres has been largely conserved, the domains of Rap1 have undergone extensive functional changes during eukaryotic evolution. Surprisingly, hRap1 alleles lacking the BRCT domain diminished the heterogeneity of human telomeres, indicating that hRap1 also plays a role in the regulation of telomere length distribution.
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Caslini, Corrado, James A. Connelly, Amparo Serna, Dominique Broccoli y Jay L. Hess. "MLL Associates with Telomeres and Regulates Telomeric Repeat-Containing RNA Transcription". Molecular and Cellular Biology 29, n.º 16 (15 de junio de 2009): 4519–26. http://dx.doi.org/10.1128/mcb.00195-09.
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ABSTRACT Mammalian telomeres consist of TTAGGG repeats organized in nucleosomes and associated with a six-protein complex known as shelterin, which preserves telomere structure and protects chromosome ends from the cellular DNA damage response. Recent studies have found that telomeres are transcribed into telomeric UUAGGG repeat-containing RNA (TERRA) starting from subtelomeric regions. TERRA binding at telomeres appears to be involved in cis-based mechanisms of telomeric chromatin organization and maintenance. A number of histone methyltransferases (HMTs) are known to influence telomeric chromatin status; however, the regulatory mechanisms of telomere transcription are poorly understood. Here, we show that the histone 3/lysine 4 (H3/K4) HMT and the transcriptional regulator MLL associate with telomeres and contribute to their H3/K4 methylation and transcription in a telomere length-dependent manner. In human diploid fibroblasts, RNA interference-mediated MLL depletion affects telomere chromatin modification and transcription and induces the telomere damage response. Telomere uncapping through either TRF2 shelterin protein knockdown or exposure to telomere G-strand DNA oligonucleotides significantly increases the transcription of TERRA, an effect mediated by the functional cooperation between MLL and the tumor suppressor p53. In total, our findings identify a previously unrecognized role of MLL in modifying telomeric chromatin and provide evidence for the functional interaction between MLL, p53, and the shelterin complex in the regulation of telomeric transcription and stability.
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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, n.º 14 (29 de junio de 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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Esnault, Germain, Stefano Majocchi, Danielle Martinet, Nathalie Besuchet-Schmutz, Jacques S. Beckmann y Nicolas Mermod. "Transcription Factor CTF1 Acts as a Chromatin Domain Boundary That Shields Human Telomeric Genes from Silencing". Molecular and Cellular Biology 29, n.º 9 (9 de marzo de 2009): 2409–18. http://dx.doi.org/10.1128/mcb.00779-08.
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ABSTRACT Telomeres are associated with chromatin-mediated silencing of genes in their vicinity. However, how epigenetic markers mediate mammalian telomeric silencing and whether specific proteins may counteract this effect are not known. We evaluated the ability of CTF1, a DNA- and histone-binding transcription factor, to prevent transgene silencing at human telomeres. CTF1 was found to protect a gene from silencing when its DNA-binding sites were interposed between the gene and the telomeric extremity, while it did not affect a gene adjacent to the telomere. Protein fusions containing the CTF1 histone-binding domain displayed similar activities, while mutants impaired in their ability to interact with the histone did not. Chromatin immunoprecipitation indicated the propagation of a hypoacetylated histone structure to various extents depending on the telomere. The CTF1 fusion protein was found to recruit the H2A.Z histone variant at the telomeric locus and to restore high histone acetylation levels to the insulated telomeric transgene. Histone lysine trimethylations were also increased on the insulated transgene, indicating that these modifications may mediate expression rather than silencing at human telomeres. Overall, these results indicate that transcription factors can act to delimit chromatin domain boundaries at mammalian telomeres, thereby blocking the propagation of a silent chromatin structure.
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Yang, Qin, Yun-Ling Zheng y Curtis C. Harris. "POT1 and TRF2 Cooperate To Maintain Telomeric Integrity". Molecular and Cellular Biology 25, n.º 3 (1 de febrero de 2005): 1070–80. http://dx.doi.org/10.1128/mcb.25.3.1070-1080.2005.
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ABSTRACT Mammalian telomeric DNA contains duplex TTAGGG repeats and single-stranded overhangs. POT1 (protection of telomeres 1) is a telomere-specific single-stranded DNA-binding protein, highly conserved in eukaryotes. The biological function of human POT1 is not well understood. In the present study, we demonstrate that POT1 plays a key role in telomeric end protection. The reduction of POT1 by RNA interference led to the loss of telomeric single-stranded overhangs and induced apoptosis, chromosomal instability, and senescence in cells. POT1 and TRF2 interacted with each other to form a complex with telomeric DNA. A dominant negative TRF2, TRF2ΔBΔM, bound to POT1 and prevented it from binding to telomeres. POT1 overexpression protected against TRF2ΔBΔM-induced loss of telomeric single-stranded overhangs, chromosomal instability, and senescence. These results demonstrate that POT1 and TRF2 share in part in the same pathway for telomere capping and suggest that POT1 binds to the telomeric single-stranded DNA in the D-loop and cooperates with TRF2 in t-loop maintenance.
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Wu, Guanhui, Luying Chen, Wenting Liu y Danzhou Yang. "Molecular Recognition of the Hybrid-Type G-Quadruplexes in Human Telomeres". Molecules 24, n.º 8 (22 de abril de 2019): 1578. http://dx.doi.org/10.3390/molecules24081578.
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G-quadruplex (G4) DNA secondary structures formed in human telomeres have been shown to inhibit cancer-specific telomerase and alternative lengthening of telomere (ALT) pathways. Thus, human telomeric G-quadruplexes are considered attractive targets for anticancer drugs. Human telomeric G-quadruplexes are structurally polymorphic and predominantly form two hybrid-type G-quadruplexes, namely hybrid-1 and hybrid-2, under physiologically relevant solution conditions. To date, only a handful solution structures are available for drug complexes of human telomeric G-quadruplexes. In this review, we will describe two recent solution structural studies from our labs. We use NMR spectroscopy to elucidate the solution structure of a 1:1 complex between a small molecule epiberberine and the hybrid-2 telomeric G-quadruplex, and the structures of 1:1 and 4:2 complexes between a small molecule Pt-tripod and the hybrid-1 telomeric G-quadruplex. Structural information of small molecule complexes can provide important information for understanding small molecule recognition of human telomeric G-quadruplexes and for structure-based rational drug design targeting human telomeric G-quadruplexes.
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Vinayagamurthy, Soujanya, Akansha Ganguly y Shantanu Chowdhury. "Extra-telomeric impact of telomeres: Emerging molecular connections in pluripotency or stemness". Journal of Biological Chemistry 295, n.º 30 (22 de mayo de 2020): 10245–54. http://dx.doi.org/10.1074/jbc.rev119.009710.
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Telomeres comprise specialized nucleic acid–protein complexes that help protect chromosome ends from DNA damage. Moreover, telomeres associate with subtelomeric regions through looping. This results in altered expression of subtelomeric genes. Recent observations further reveal telomere length–dependent gene regulation and epigenetic modifications at sites spread across the genome and distant from telomeres. This regulation is mediated through the telomere-binding protein telomeric repeat–binding factor 2 (TRF2). These observations suggest a role of telomeres in extra-telomeric functions. Most notably, telomeres have a broad impact on pluripotency and differentiation. For example, cardiomyocytes differentiate with higher efficacy from induced pluripotent stem cells having long telomeres, and differentiated cells obtained from human embryonic stem cells with relatively long telomeres have a longer lifespan. Here, we first highlight reports on these two seemingly distinct research areas: the extra-telomeric role of telomere-binding factors and the role of telomeres in pluripotency/stemness. On the basis of the observations reported in these studies, we draw attention to potential molecular connections between extra-telomeric biology and pluripotency. Finally, in the context of the nonlocal influence of telomeres on pluripotency and stemness, we discuss major opportunities for progress in molecular understanding of aging-related disorders and neurodegenerative diseases.
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Smogorzewska, Agata, Bas van Steensel, Alessandro Bianchi, Stefan Oelmann, Matthias R. Schaefer, Gisela Schnapp y Titia de Lange. "Control of Human Telomere Length by TRF1 and TRF2". Molecular and Cellular Biology 20, n.º 5 (1 de marzo de 2000): 1659–68. http://dx.doi.org/10.1128/mcb.20.5.1659-1668.2000.
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ABSTRACT Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3′ telomere terminus in TRF1- and TRF2-induced telomeric loops.
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Higa, Mitsunori, Yukihiro Matsuda, Jumpei Fujii, Nozomi Sugimoto, Kazumasa Yoshida y Masatoshi Fujita. "TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress". Nucleic Acids Research 49, n.º 21 (11 de noviembre de 2021): 12234–51. http://dx.doi.org/10.1093/nar/gkab1004.
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Abstract Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2–ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2–ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244–511), which competitively inhibited the TRF2–ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.
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Britt-Compton, B. y D. M. Baird. "Intra-allelic mutation at human telomeres". Biochemical Society Transactions 34, n.º 4 (21 de julio de 2006): 581–82. http://dx.doi.org/10.1042/bst0340581.
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The maintenance of telomere length is important in upholding the integrity of the genome. However, it is clear from detailed observations of both telomere length and internal repeat structure that human telomeres are extremely dynamic structures and are subjected to multiple processes that create considerable heterogeneity. Genetic evidence suggests that meiotic recombination within telomeres is rare. However, there are various lines of evidence that implicate the involvement of intra-allelic processes in human telomere dynamics. In this paper, we briefly review some of this evidence and the putative mechanisms of intra-allelic telomeric mutation.
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Gineitis, Arunas A., Irina A. Zalenskaya, Peter M. Yau, E. Morton Bradbury y Andrei O. Zalensky. "Human Sperm Telomere–Binding Complex Involves Histone H2b and Secures Telomere Membrane Attachment". Journal of Cell Biology 151, n.º 7 (25 de diciembre de 2000): 1591–98. http://dx.doi.org/10.1083/jcb.151.7.1591.
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Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization. A telomere-binding protein complex from human sperm (hSTBP) has been isolated by detergent treatment and was partially purified. hSTBP specifically binds double-stranded telomeric DNA and does not contain known somatic telomere proteins TRF1, TRF2, and Ku. Surprisingly, the essential component of this complex has been identified as a specific variant of histone H2B. Indirect immunofluorescence shows punctate localization of H2B in sperm nuclei, which in part coincides with telomeric DNA localization established by fluorescent in situ hybridization. Anti–H2B antibodies block interactions of hSTBP with telomere DNA, and spH2B forms specific complex with this DNA in vitro, indicating that this protein plays a role in telomere DNA recognition. We propose that hSTBP participates in the membrane attachment of telomeres that may be important for ordered chromosome withdrawal after fertilization.
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Ancelin, Katia, Michele Brunori, Serge Bauwens, Catherine-Elaine Koering, Christine Brun, Michelle Ricoul, Jean-Patrick Pommier, Laure Sabatier y Eric Gilson. "Targeting Assay To Study the cis Functions of Human Telomeric Proteins: Evidence for Inhibition of Telomerase by TRF1 and for Activation of Telomere Degradation by TRF2". Molecular and Cellular Biology 22, n.º 10 (15 de mayo de 2002): 3474–87. http://dx.doi.org/10.1128/mcb.22.10.3474-3487.2002.
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ABSTRACT We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.
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Henderson, S., R. Allsopp, D. Spector, S. S. Wang y C. Harley. "In situ analysis of changes in telomere size during replicative aging and cell transformation." Journal of Cell Biology 134, n.º 1 (1 de julio de 1996): 1–12. http://dx.doi.org/10.1083/jcb.134.1.1.
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Telomeres have been shown to gradually shorten during replicative aging in human somatic cells by Southern analysis. This study examines telomere shortening at the single cell level by fluorescence in situ hybridization (FISH). FISH and confocal microscopy of interphase human diploid fibroblasts (HDFs) demonstrate that telomeres are distributed throughout the nucleus with an interchromosomal heterogeneity in size. Analysis of HDFs at increasing population doubling levels shows a gradual increase in spot size, intensity, and detectability of telomeric signal. FISH of metaphase chromosomes prepared from young and old HDFs shows a heterogeneity in detection frequency for telomeres on chromosomes 1, 9, 15, and Y. The interchromosomal distribution of detection frequencies was similar for cells at early and late passage. The telomeric detection frequency for metaphase chromosomes also decreased with age. These observations suggest that telomeres shorten at similar rates in normal human somatic cels. T-antigen transformed HDFs near crisis contained telomere signals that were low compared to nontransformed HDFs. A large intracellular heterogeneity in telomere lengths was detected in two telomerase-negative cell lines compared to normal somatic cells and the telomerase-positive 293 cell line. Many telomerase-negative immortal cells had telomeric signals stronger than those in young HDFs, suggesting a different mechanism for telomere length regulation in telomerase-negative immortal cells. These studies provide an in situ demonstration of interchromosomal heterogeneity in telomere lengths. Furthermore, FISH is a reliable and sensitive method for detecting changes in telomere size at the single cell level.
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Kelleher, Colleen, Isabel Kurth y Joachim Lingner. "Human Protection of Telomeres 1 (POT1) Is a Negative Regulator of Telomerase Activity In Vitro". Molecular and Cellular Biology 25, n.º 2 (15 de enero de 2005): 808–18. http://dx.doi.org/10.1128/mcb.25.2.808-818.2005.
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ABSTRACT The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans. Human POT1 appears to interact with telomeres both through direct binding to the 3′ overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex. The influence of POT1 on telomerase activity has not been studied at the molecular level. We show here that POT1 negatively effects telomerase activity in vitro. We find that the DNA binding activity of POT1 is required for telomerase inhibition. Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase.
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Majerska, Jana, Marianna Feretzaki, Galina Glousker y Joachim Lingner. "Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1". Life Science Alliance 1, n.º 4 (17 de julio de 2018): e201800121. http://dx.doi.org/10.26508/lsa.201800121.
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Telomeres play crucial roles during tumorigenesis, inducing cellular senescence upon telomere shortening and extensive chromosome instability during telomere crisis. However, it has not been investigated if and how cellular transformation and oncogenic stress alter telomeric chromatin composition and function. Here, we transform human fibroblasts by consecutive transduction with vectors expressing hTERT, the SV40 early region, and activated H-RasV12. Pairwise comparisons of the telomeric proteome during different stages of transformation reveal up-regulation of proteins involved in chromatin remodeling, DNA repair, and replication at chromosome ends. Depletion of several of these proteins induces telomere fragility, indicating their roles in replication of telomeric DNA. Depletion of SAMHD1, which has reported roles in DNA resection and homology-directed repair, leads to telomere breakage events in cells deprived of the shelterin component TRF1. Thus, our analysis identifies factors, which accumulate at telomeres during cellular transformation to promote telomere replication and repair, resisting oncogene-borne telomere replication stress.
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Bechard, Laura H., Bilge D. Butuner, George J. Peterson, Will McRae, Zeki Topcu y Michael J. McEachern. "Mutant Telomeric Repeats in Yeast Can Disrupt the Negative Regulation of Recombination-Mediated Telomere Maintenance and Create an Alternative Lengthening of Telomeres-Like Phenotype". Molecular and Cellular Biology 29, n.º 3 (24 de noviembre de 2008): 626–39. http://dx.doi.org/10.1128/mcb.00423-08.
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ABSTRACT Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent of human ALT cells. In this study, we generated telomeres composed of either of two types of mutant telomeric repeats, Acc and SnaB, that each alter the binding site for the telomeric protein Rap1. We show here that arrays of both types of mutant repeats present basally on a telomere were defective in negatively regulating telomere length in the presence of telomerase. Similarly, when each type of mutant repeat was spread to all chromosome ends in cells lacking telomerase, they led to the formation of telomeres produced by RTE that were much longer than those seen in cells with only wild-type telomeric repeats. The Acc repeats produced the more severe defect in both types of telomere maintenance, consistent with their more severe Rap1 binding defect. Curiously, although telomerase deletion mutants with telomeres composed of Acc repeats invariably showed extreme telomere elongation, they often also initially showed persistent very short telomeres with few or no Acc repeats. We suggest that these result from futile cycles of recombinational elongation and truncation of the Acc repeats from the telomeres. The presence of extensive 3′ overhangs at mutant telomeres suggests that Rap1 may normally be involved in controlling 5′ end degradation.
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Izumi, Hiroto y Keiko Funa. "Telomere Function and the G-Quadruplex Formation are Regulated by hnRNP U". Cells 8, n.º 5 (28 de abril de 2019): 390. http://dx.doi.org/10.3390/cells8050390.
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We examine the role of the heterogenous ribonucleoprotein U (hnRNP U) as a G-quadruplex binding protein in human cell lines. Hypothesizing that hnRNP U is associated with telomeres, we investigate what other telomere-related functions it may have. Telomeric G-quadruplexes have been fully characterized in vitro, but until now no clear evidence of their function or in vivo interactions with proteins has been revealed in mammalian cells. Techniques used were immunoprecipitation, DNA pull-down, binding assay, and Western blots. We identified hnRNP U as a G-quadruplex binding protein. Immunoprecipitations disclosed that endogenous hnRNP U associates with telomeres, and DNA pull-downs showed that the hnRNP U C-terminus specifically binds telomeric G-quadruplexes. We have compared the effect of telomere repeat containing RNA (TERRA) on binding between hnRNP U and telomeric (Tel) or single- stranded Tel (ssTel) oligonucleotides and found that ssTel binds stronger to TERRA than to Tel. We also show that hnRNP U prevents replication protein A (RPA) accumulation at telomeres, and the recognition of telomeric ends by hnRNP suggests that a G-quadruplex promoting protein regulates its accessibility. Thus, hnRNP U-mediated formation has important functions for telomere biology.
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De Vitis, Marco, Francesco Berardinelli, Elisa Coluzzi, Jessica Marinaccio, Roderick J. O’Sullivan y Antonella Sgura. "X-rays Activate Telomeric Homologous Recombination Mediated Repair in Primary Cells". Cells 8, n.º 7 (12 de julio de 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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Coluzzi, Elisa, Stefano Leone y Antonella Sgura. "Oxidative Stress Induces Telomere Dysfunction and Senescence by Replication Fork Arrest". Cells 8, n.º 1 (3 de enero de 2019): 19. http://dx.doi.org/10.3390/cells8010019.
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Oxidative DNA damage, particularly 8-oxoguanine, represents the most frequent DNA damage in human cells, especially at the telomeric level. The presence of oxidative lesions in the DNA can hinder the replication fork and is able to activate the DNA damage response. In this study, we wanted to understand the mechanisms by which oxidative damage causes telomere dysfunction and senescence in human primary fibroblasts. After acute oxidative stress at telomeres, our data demonstrated a reduction in TRF1 and TRF2, which are involved in proper telomere replication and T-loop formation, respectively. Furthermore, we observed a higher level of γH2AX with respect to 53BP1 at telomeres, suggesting a telomeric replication fork stall rather than double-strand breaks. To confirm this finding, we studied the replication of telomeres by Chromosome Orientation-FISH (CO-FISH). The data obtained show an increase in unreplicated telomeres after hydrogen peroxide treatment, corroborating the idea that the presence of 8-oxoG can induce replication fork arrest at telomeres. Lastly, we analyzed the H3K9me3 histone mark after oxidative stress at telomeres, and our results showed an increase of this marker, most likely inducing the heterochromatinization of telomeres. These results suggest that 8-oxoG is fundamental in oxidative stress-induced telomeric damage, principally causing replication fork arrest.
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Caslini, Corrado y Jay L. Hess. "MLL Modulates Telomere Length in Mammalian Cells." Blood 108, n.º 11 (1 de noviembre de 2006): 2209. http://dx.doi.org/10.1182/blood.v108.11.2209.2209.
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Abstract In mammalian cells, the telomeric complex at the end of chromosomes consists of several thousand copies of the exanucleotide TTAGGG and associated proteins attached to the nuclear matrix. Chromatin modifying enzymes involved in histone H3/lysine 9 and histone H4/lysine 20 trimethylation, and DNA methylation are known to preserve the telomere heterochromatic structure, length and capping function. Loss of these heterochromatic marks leads to telomere lengthening, most likely through the negative regulation of telomerase or alternative lengthening of telomeres (ALT) mechanisms. The MLL protein is a chromatin modifying enzyme with histone H3/lysine 4 methyltransferase activity, which maintains active transcriptional state of target genes in a large multiprotein complex. Analogously, the yeast’s MLL homologous protein Set1 is part of a multiprotein complex required for maintenance of target genes expression. In addition, Set1 deletion mutants show disruption of telomeric silencing along with telomere shortening or lengthening, respectively in budding and fission yeast. This raised the question of whether MLL, like Set1, plays a role in epigenetic maintenance of telomeric heterochromatin. Here, using chromatin immunoprecipitation (ChIP) analysis, we show that MLL associates with the heterochromatic complex at telomeres of primary and transformed human cell lines. ChIP analysis of cell lines conditionally expressing Flag-tagged MLL chimeric proteins and deletion mutants shows the amino terminus of MLL, which confers association to the nuclear matrix, is responsible for targeting to the telomeric complex. MLL associates with the telomeres of telomerase and ALT positive cell lines in amount that is proportional to the telomere length, as revealed by Southern blot terminal restriction fragment analysis. Moreover, immunoprecipitation analysis evidenced the association of MLL with the terminal-repeat binding factor TRF2, a protein known to play a key role in telomere capping, and indirect immunofluorescence analysis showed MLL and TRF2 colocalization at ALT-associated PML nuclear bodies. In search for possible biological functions of MLL at the telomeric complex, we found abnormally longer telomeres in Mll-null mouse embryonic stem (ES) cells and fibroblasts (MEFs) than in wild-type control cells. In Mll-null MEFs, a significant telomere shortening was obtained by stable reexpression of an MLL allele. In addition, we found that in aging human cells the MLL binding to the telomeric complex is abrogated by the progressive telomere shortening due to telomere attrition, suggesting a possible involvement of MLL in signaling for replicative senescence.
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Stock, Amanda, Kun Wang, Chengyu Liu, Ross McDevitt, Chongkui Sun, Yi Gong y Yie Liu. "Age-Related Phenotypes Linked to Aberrant Expression and Localization of a Telomeric Protein". Innovation in Aging 5, Supplement_1 (1 de diciembre de 2021): 661–62. http://dx.doi.org/10.1093/geroni/igab046.2498.
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Abstract Telomere attrition is associated with telomere biology disorders and age-related diseases. In telomere biology disorders, telomere uncapping induces a DNA damage response that evokes cell death or senescence. However, a causal mechanism for telomere attrition in age-related diseases remains elusive. Telomere capping and integrity are maintained by shelterin, a six-protein complex. Rap1 is the only shelterin member that is not required for telomere capping and is expressed at non-telomeric genomic and cytosolic regions. The objective of this study was to determine aberrant phenotypes attributed to non-telomeric Rap1. To test this, we generated a Rap1 mutant knock-in (KI) mouse model using CRISPR/Cas9 editing, in which Rap1 at telomeres is prevented, leaving only non-telomeric Rap1. Cell fractionation/western blotting of primary fibroblasts from Rap1 KI mice demonstrated decreased Rap1 expression and Rap1 re-localization off telomeres, with an altered cellular distribution. This same difference in Rap1 is also observed in human cells with telomere erosion, indicating that aberrant Rap1 in our model may recapitulate Rap1 in aging and human telomere biology disorders. Compared to wild-type control mice, Rap1 KI mice exhibited increased body weight, altered cytokine levels, behavioral deficits, and decreased lifespan. In conclusion, our results reveal a novel mechanism by which telomere shortening may contribute to age-related pathologies by disrupting Rap1 expression and cell localization.
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Stock, Amanda, Kun Wang, Chongkui Sun, Chengyu Liu, Yi Gong y Yie Liu. "A DNA Damage Response-Independent Mechanism for Telomere Shortening-Elicited Age-Related Pathologies". Innovation in Aging 4, Supplement_1 (1 de diciembre de 2020): 885. http://dx.doi.org/10.1093/geroni/igaa057.3268.
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Abstract Telomere attrition is associated with telomeropathies and age-related pathologies. In telomeropathies, telomere uncapping induces a DNA damage response (DDR) that drives apoptosis or senescence. However, a defined mechanism by which telomere attrition contributes to other age-related pathologies has not been determined. Telomere integrity is maintained by shelterin, a six-protein complex. Rap1 is the only shelterin member that is not essential for telomere capping but engages non-telomeric DNA and regulates gene transcription. We hypothesized that non-telomeric Rap1 accumulation could contribute to age-related pathologies in a DDR-independent manner. To test this, we used CRISPR/Cas9 editing to generate a Rap1 mutant mouse model in which Rap1 at telomeres is prevented, leaving only non-telomeric Rap1. Indirect immunostaining showed no differences in telomere dysfunction-induced DDR foci in Rap1 mutant compared to wild-type primary fibroblasts. Cell fractionation/western blotting of fibroblasts from Rap1 mutants demonstrated decreased Rap1 expression and Rap1 re-localization off telomeres, which mimics the same alteration of Rap1 in human cells with telomere attrition. Rap1 mutant mice exhibited increased body weight and altered metabolic and immune-response transcripts in various tissues, indicating that altered transcription could account for some of the observed phenotypes related to telomere attrition. In conclusion, telomere shortening may facilitate non-telomeric Rap1, which alters gene transcription and drives metabolic and immune dysfunction in a DDR-independent manner.
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Amato, Roberta, Martina Valenzuela, Francesco Berardinelli, Erica Salvati, Carmen Maresca, Stefano Leone, Antonio Antoccia y Antonella Sgura. "G-quadruplex Stabilization Fuels the ALT Pathway in ALT-positive Osteosarcoma Cells". Genes 11, n.º 3 (13 de marzo de 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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Bassham, Susan, Aaron Beam y Janis Shampay. "Telomere Variation in Xenopus laevis". Molecular and Cellular Biology 18, n.º 1 (1 de enero de 1998): 269–75. http://dx.doi.org/10.1128/mcb.18.1.269.
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ABSTRACT Eukaryotic telomeres are variable at several levels, from the length of the simple sequence telomeric repeat tract in different cell types to the presence or number of telomere-adjacent DNA sequence elements in different strains or individuals. We have investigated the sequence organization of Xenopus laevis telomeres by use of the vertebrate telomeric repeat (TTAGGG) n and blot hybridization analysis. The (TTAGGG) n -hybridizing fragments, which ranged from less than 10 to over 50 kb with frequently cutting enzymes, defined a pattern that was polymorphic between individuals. BAL 31 exonuclease treatment confirmed that these fragments were telomeric. The polymorphic fragments analyzed did not hybridize to 5S RNA sequences, which are telomeric according to in situ hybridization. When telomeric fragments from offspring (whole embryos) were compared to those from the spleens of the parents, the inheritance pattern of some bands was found to be unusual. Furthermore, in one cross, the telomeres of the embryo were shorter than the telomeres of the parents’ spleen, and in another, the male’s testis telomeres were shorter than those of the male’s spleen. Our data are consistent with a model for chromosome behavior that involves a significant amount of DNA rearrangement at telomeres and suggest that length regulation ofXenopus telomeres is different from that observed forMus spretus and human telomeres.
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Douglas, Max E. y John F. X. Diffley. "Budding yeast Rap1, but not telomeric DNA, is inhibitory for multiple stages of DNA replication in vitro". Nucleic Acids Research 49, n.º 10 (28 de mayo de 2021): 5671–83. http://dx.doi.org/10.1093/nar/gkab416.
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Abstract Telomeres are copied and reassembled each cell division cycle through a multistep process called telomere replication. Most telomeric DNA is duplicated semiconservatively during this process, but replication forks frequently pause or stall at telomeres in yeast, mouse and human cells, potentially causing chronic telomere shortening or loss in a single cell cycle. We have investigated the cause of this effect by examining the replication of telomeric templates in vitro. Using a reconstituted assay for eukaryotic DNA replication in which a complete eukaryotic replisome is assembled and activated with purified proteins, we show that budding yeast telomeric DNA is efficiently duplicated in vitro unless the telomere binding protein Rap1 is present. Rap1 acts as a roadblock that prevents replisome progression and leading strand synthesis, but also potently inhibits lagging strand telomere replication behind the fork. Both defects can be mitigated by the Pif1 helicase. Our results suggest that GC-rich sequences do not inhibit DNA replication per se, and that in the absence of accessory factors, telomere binding proteins can inhibit multiple, distinct steps in the replication process.
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Stroik, Susanna, Kevin Kurtz y Eric A. Hendrickson. "CtIP is essential for telomere replication". Nucleic Acids Research 47, n.º 17 (5 de agosto de 2019): 8927–40. http://dx.doi.org/10.1093/nar/gkz652.
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Abstract The maintenance of telomere length is critical to longevity and survival. Specifically, the failure to properly replicate, resect, and/or form appropriate telomeric structures drives telomere shortening and, in turn, genomic instability. The endonuclease CtIP is a DNA repair protein that is well-known to promote genome stability through the resection of endogenous DNA double-stranded breaks. Here, we describe a novel role for CtIP. We show that in the absence of CtIP, human telomeres shorten rapidly to non-viable lengths. This telomere dysfunction results in an accumulation of fusions, breaks, and frank telomere loss. Additionally, CtIP suppresses the generation of circular, extrachromosomal telomeric DNA. These latter structures appear to arise from arrested DNA replication forks that accumulate in the absence of CtIP. Hence, CtIP is required for faithful replication through telomeres via its roles at stalled replication tracts. Our findings demonstrate a new role for CtIP as a protector of human telomere integrity.
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Xu, Lifeng y Elizabeth H. Blackburn. "Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules". Journal of Cell Biology 167, n.º 5 (6 de diciembre de 2004): 819–30. http://dx.doi.org/10.1083/jcb.200408181.
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We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.
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Bryan, Christopher, Cory Rice, Michael Harkisheimer, David Schultz y Emmanuel Skordalakes. "Structure of the Human Telomeric Stn1-Ten1 Complex". Acta Crystallographica Section A Foundations and Advances 70, a1 (5 de agosto de 2014): C1587. http://dx.doi.org/10.1107/s2053273314084125.
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The telomeric CST complex plays a central role in chromosome end capping and replication in budding yeast, and homologues of CST were identified recently in higher eukaryotes. The human CST (Ctc1, hStn1, hTen1) has been shown to play a role in telomere maintenance, but the extent of conservation across species has been in question because of low sequence identity (below 10% for Ctc1, the core subunit of the CST complex) and data suggesting subtle differences in function between complexes. We solved the high-resolution crystal structure of the human Stn1-Ten1 complex, which revealed striking structural similarity between the yeast and human CST complexes. We also showed using southern blots and fluorescence in situ hybridization experiments that disruption of the hStn1-Ten1 binding interface in vivo produces elongated telomeres and telomere defects in accordance with what has been previously observed for the yeast CST complex. Our results support structural and functional conservation of telomeric CST across species.
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Gupta, Aditi, Bor-Jang Hwang, Daniel Benyamien-Roufaeil, Sara Jain, Sophie Liu, Rex Gonzales, Robert A. Brown, Michal Zalzman y A.-Lien Lu. "Mammalian MutY Homolog (MYH or MUTYH) is Critical for Telomere Integrity under Oxidative Stress". OBM Geriatrics 6, n.º 2 (22 de julio de 2021): 1. http://dx.doi.org/10.21926/obm.geriatr.2202196.
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Telomeres consist of special features and proteins to protect the ends of each chromosome from deterioration and fusion. The telomeric DNA repeats are highly susceptible to oxidative damage that can accelerate telomere shortening and affect telomere integrity. Several DNA repair factors including MYH/MUTYH DNA glycosylase, its interacting partners Rad9/Rad1/Hus1 checkpoint clamp, and SIRT6 aging regulator, are associated with the telomeres. MYH prevents C:G to A:T mutation by removing adenine mispaired with a frequent oxidative DNA lesion, 8-oxoguanine. Here, we show that hMYH knockout (KO) human HEK-293T cells are more sensitive to H2O2 treatment, have higher levels of DNA strand breaks and shorter telomeres than the control hMYH+/+ cells. SIRT6 foci increase at both the global genome and at telomeric regions in H2O2-treated hMYH+/+ cells. However, in untreated hMYH KO HEK-293T cells, SIRT6 foci only increase at the global genome, but not at the telomeric regions. In addition, the hMYH KO HEK-293T cells have increased extra-chromosomal and intra-chromosomal telomeres compared to the control cells, even in the absence of H2O2 treatment. After H2O2 treatment, the frequency of extra-chromosomal telomeres increased in control HEK-293T cells. Remarkably, in H2O2-treated hMYH KO cells, the frequencies of extra-chromosomal telomeres, intra-chromosomal telomeres, and telomere fusions are further increased. We further found that the sensitivity to H2O2 and shortened telomeres of hMYH KO cells, are restored by expressing wild-type hMYH, and partially rescued by expressing hMYHQ324H mutant (defective in Hus1 interaction only), but not by expressing hMYHV315A mutant (defective in both SIRT6 and Hus1 interactions). Thus, MYH interactions with SIRT6 and Hus1 are critical for maintaining cell viability and telomeric stability. Therefore, the failure to coordinate 8-oxoG repair is detrimental to telomere integrity.
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Gu, Wei, Zihan Lin, Shengchao Zhao, Guanzhen Wang, Ziyi Shen, Wei Liu, Yi Cai, Kaibo Wang, Chunpeng Craig Wan y Tingdong Yan. "Research Progress on G-Quadruplexes in Human Telomeres and Human Telomerase Reverse Transcriptase (hTERT) Promoter". Oxidative Medicine and Cellular Longevity 2022 (6 de junio de 2022): 1–11. http://dx.doi.org/10.1155/2022/2905663.
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The upregulation telomerase activity is observed in over 85-90% of human cancers and provides an attractive target for cancer therapies. The high guanine content in the telomere DNA sequences and the hTERT promoter can form G-quadruplexes (G4s). Small molecules targeting G4s in telomeres and hTERT promoter could stabilize the G4s and inhibit hTERT expression and telomere extension. Several G4 ligands have shown inhibitory effects in cancer cells and xenograft mouse models, indicating these ligands have a potential for cancer therapies. The current review article describes the concept of the telomere, telomerase, and G4s. Moreover, the regulation of telomerase and G4s in telomeres and hTERT promoter is discussed as well. The summary of the small molecules targeting G4s in telomeric DNA sequences and the hTERT promoter will also be shown.
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Calado, Rodrigo T., Joshua A. Regal, Sachiko Kajigaya y Neal S. Young. "Erosion of Telomeric Single-Stranded Overhang in Patients with Aplastic Anemia Carrying Telomerase Mutations." Blood 114, n.º 22 (20 de noviembre de 2009): 1079. http://dx.doi.org/10.1182/blood.v114.22.1079.1079.
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Abstract Abstract 1079 Poster Board I-101 Loss-of-function mutations in telomerase complex genes reduce telomerase activity and shorten overall telomere length in leukocytes, and they can clinically manifest as bone marrow failure (aplastic anemia and dyskeratosis congenita), familial pulmonary fibrosis, and hepatic cirrhosis. The double-stranded tandem telomeric TTAGGG repeats are followed by a 3' G-rich single-stranded overhang, a crucial structural component responsible for protective t-loop formation. We investigated the length of telomeric overhangs in 25 healthy individuals from 0 to 76 years, 16 patients with aplastic anemia, and 13 of their immediate relatives using a non-denaturing in-gel method and the telomere-oligonucleotide ligation assay (T-OLA). Among healthy controls, overall telomeric length of leukocytes shortened as a function of age. It was longest in umbilical cord blood samples and eroded as a function of a third-order polynomial until 76 years (R2=0.9517; Spearman=0.90; P<0.0001). However, the lengths of single-stranded overhangs were constant with age, as determined by both non-denaturing and the T-OLA methods. In contrast, most patients with marrow failure carrying a telomerase gene mutation showed marked erosion of telomeric overhangs associated with critically short telomeres (P<0.05). Seven telomerase complex-mutant aplastic patients had overhang lengths below the 5% percentile established for healthy volunteers. In aplastic patients with normal genotypes, normal overall telomere lengths, and who effectively responded to immunosuppressive therapy, telomeric overhangs were maintained and were all within the range established for control individuals. In conclusion, telomeric overhang erosion does not participate in physiological aging but eroded telomeric overhangs and abnormal telomere structure appear in pathologic shortening of telomeres, especially caused by loss-of-function telomerase mutations. Disrupted telomere structure caused by short telomeric overhangs may contribute to the mechanisms of abnormal hematopoietic compartment senescence and chromosomal instability in human bone marrow failure. Disclosures: No relevant conflicts of interest to declare.
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Pedram, Mehrdad, Carl N. Sprung, Qing Gao, Anthony W. I. Lo, Gloria E. Reynolds y John P. Murnane. "Telomere Position Effect and Silencing of Transgenes near Telomeres in the Mouse". Molecular and Cellular Biology 26, n.º 5 (1 de marzo de 2006): 1865–78. http://dx.doi.org/10.1128/mcb.26.5.1865-1878.2006.
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ABSTRACT Reversible transcriptional silencing of genes located near telomeres, termed the telomere position effect (TPE), is well characterized in Saccharomyces cerevisiae. TPE has also been observed in human tumor cell lines, but its function remains unknown. To investigate TPE in normal mammalian cells, we developed clones of mouse embryonic stem (ES) cells that contain single-copy marker genes integrated adjacent to different telomeres. Analysis of these telomeric transgenes demonstrated that they were expressed at very low levels compared to the same transgenes integrated at interstitial sites. Similar to the situation in yeast, but in contrast to studies with human tumor cell lines, TPE in mouse ES cells was not reversed with trichostatin A. Prolonged culturing without selection resulted in extensive DNA methylation and complete silencing of telomeric transgenes, which could be reversed by treatment with 5-azacytidine. Thus, complete silencing of the telomeric transgenes appears to involve a two-step process in which the initial repression is reinforced by DNA methylation. Extensive methylation of the telomeric transgenes was also observed in various tissues and embryonic fibroblasts isolated from transgenic mice. In contrast, telomeric transgenes were not silenced in ES cell lines isolated from 3-day-old preimplantation embryos, consistent with the hypothesis that TPE plays a role in the development of the embryo.
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Baumann, Peter, Elaine Podell y Thomas R. Cech. "Human Pot1 (Protection of Telomeres) Protein: Cytolocalization, Gene Structure, and Alternative Splicing". Molecular and Cellular Biology 22, n.º 22 (15 de noviembre de 2002): 8079–87. http://dx.doi.org/10.1128/mcb.22.22.8079-8087.2002.
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ABSTRACT Fission yeast Pot1 (protection of telomeres) is a single-stranded telomeric DNA binding protein with a critical role in ensuring chromosome stability. A putative human homolog (hPot1) was previously identified, based on moderate sequence similarity with fission yeast Pot1 and telomere end-binding proteins from ciliated protozoa. Using indirect immunofluorescence, we show here that epitope-tagged hPot1 localizes to telomeres in interphase nuclei of human cells, consistent with a direct role in telomere end protection. The hPOT1 gene contains 22 exons, most of which are present in all cDNAs examined. However, four exons are subject to exon skipping in some transcripts, giving rise to five splice variants. Four of these are ubiquitously expressed, whereas the fifth appears to be specific to leukocytes. The resultant proteins vary significantly in their ability to form complexes with single-stranded telomeric DNA as judged by electrophoretic mobility shift assays. In addition to these splice variants, the Pot1 family is expanded by the identification of six more genes from diverse species. Pot1-like proteins have now been found in plants, animals, yeasts, and microsporidia.
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Klingelhutz, A. J., S. A. Barber, P. P. Smith, K. Dyer y J. K. McDougall. "Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells". Molecular and Cellular Biology 14, n.º 2 (febrero de 1994): 961–69. http://dx.doi.org/10.1128/mcb.14.2.961-969.1994.
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Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.
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Klingelhutz, A. J., S. A. Barber, P. P. Smith, K. Dyer y J. K. McDougall. "Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells." Molecular and Cellular Biology 14, n.º 2 (febrero de 1994): 961–69. http://dx.doi.org/10.1128/mcb.14.2.961.
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Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.
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Smith, S. y T. de Lange. "Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes". Journal of Cell Science 112, n.º 21 (1 de noviembre de 1999): 3649–56. http://dx.doi.org/10.1242/jcs.112.21.3649.
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Tankyrase is a human poly(ADP-ribose) polymerase that was initially identified through its interaction with the telomeric protein TRF1, a negative regulator of telomere length. In vitro poly(ADP-ribosyl)ation by tankyrase inhibits TRF1 binding to telomeric DNA suggesting a role for tankyrase in telomere function. We previously demonstrated that tankyrase co-localizes with TRF1 at the ends of human chromosomes in metaphase. Here we show that tankyrase localizes to additional subcellular sites in a cell cycle dependent manner. In interphase, tankyrase co-localized with TRF1 to telomeres, but in addition was found to reside at nuclear pore complexes, as evidenced by indirect immunofluorescence, subcellular fractionation and immunoelectron microscopy. At mitosis, concomitant with nuclear envelope breakdown and nuclear pore complex disassembly, tankyrase was found to relocate around the pericentriolar matrix of mitotic centrosomes. This complex staining pattern along with the observation that tankyrase did not contain a nuclear localization signal suggested that its telomeric localization might be regulated, perhaps by TRF1. Indeed, localization of exogenously-expressed tankyrase to telomeres was dependent upon co-transfection with TRF1. These data indicate that the subcellular localization of tankyrase can be regulated by both the cell cycle and TRF1.
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Soman, Aghil, Chong Wai Liew, Hsiang Ling Teo, Nikolay V. Berezhnoy, Vincent Olieric, Nikolay Korolev, Daniela Rhodes y Lars Nordenskiöld. "The human telomeric nucleosome displays distinct structural and dynamic properties". Nucleic Acids Research 48, n.º 10 (6 de mayo de 2020): 5383–96. http://dx.doi.org/10.1093/nar/gkaa289.
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Abstract Telomeres protect the ends of our chromosomes and are key to maintaining genomic integrity during cell division and differentiation. However, our knowledge of telomeric chromatin and nucleosome structure at the molecular level is limited. Here, we aimed to define the structure, dynamics as well as properties in solution of the human telomeric nucleosome. We first determined the 2.2 Å crystal structure of a human telomeric nucleosome core particle (NCP) containing 145 bp DNA, which revealed the same helical path for the DNA as well as symmetric stretching in both halves of the NCP as that of the 145 bp ‘601’ NCP. In solution, the telomeric nucleosome exhibited a less stable and a markedly more dynamic structure compared to NCPs containing DNA positioning sequences. These observations provide molecular insights into how telomeric DNA forms nucleosomes and chromatin and advance our understanding of the unique biological role of telomeres.
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Cesare, Anthony J. y Jack D. Griffith. "Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops". Molecular and Cellular Biology 24, n.º 22 (15 de noviembre de 2004): 9948–57. http://dx.doi.org/10.1128/mcb.24.22.9948-9957.2004.
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ABSTRACT A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.
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Cong, Yu-Sheng, Woodring E. Wright y Jerry W. Shay. "Human Telomerase and Its Regulation". Microbiology and Molecular Biology Reviews 66, n.º 3 (septiembre de 2002): 407–25. http://dx.doi.org/10.1128/mmbr.66.3.407-425.2002.
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SUMMARY The telomere is a special functional complex at the end of linear eukaryotic chromosomes, consisting of tandem repeat DNA sequences and associated proteins. It is essential for maintaining the integrity and stability of linear eukaryotic genomes. Telomere length regulation and maintenance contribute to normal human cellular aging and human diseases. The synthesis of telomeres is mainly achieved by the cellular reverse transcriptase telomerase, an RNA-dependent DNA polymerase that adds telomeric DNA to telomeres. Expression of telomerase is usually required for cell immortalization and long-term tumor growth. In humans, telomerase activity is tightly regulated during development and oncogenesis. The modulation of telomerase activity may therefore have important implications in antiaging and anticancer therapy. This review describes the currently known components of the telomerase complex and attempts to provide an update on the molecular mechanisms of human telomerase regulation.