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      Replication Timing of Human Telomeres Is Chromosome Arm–Specific, Influenced by Subtelomeric Structures and Connected to Nuclear Localization

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          Abstract

          The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of β-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4–associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm–specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.

          Author Summary

          Functional telomeres are essential for genome stability. While replication of telomeres has been extensively studied in model organisms such as the baker's yeast, little is known about the mechanisms that govern the replication of human telomeres. In this study, we have determined the timing of replication of telomeres of individual human chromosomes and its association with potential modulating factors such as particular subtelomeric sequences, the presence of heterochromatic regions, and nuclear localization. We have found that native telomeres associated with D4Z4 sequences—a macrosatellite naturally located in the subtelomeric regions of 4q, 10q, and acrocentric chromosome extremities—replicate later than others. We also present descriptive and experimental evidence indicating that nuclear localization influences the timing of telomere replication. These results contribute to our understanding of telomere metabolism in humans.

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          Most cited references58

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          Extension of life-span by introduction of telomerase into normal human cells.

          Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.
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            A telomerase component is defective in the human disease dyskeratosis congenita.

            The X-linked form of the human disease dyskeratosis congenita (DKC) is caused by mutations in the gene encoding dyskerin. Sufferers have defects in highly regenerative tissues such as skin and bone marrow, chromosome instability and a predisposition to develop certain types of malignancy. Dyskerin is a putative pseudouridine synthase, and it has been suggested that DKC may be caused by a defect in ribosomal RNA processing. Here we show that dyskerin is associated not only with H/ACA small nucleolar RNAs, but also with human telomerase RNA, which contains an H/ACA RNA motif. Telomerase adds simple sequence repeats to chromosome ends using an internal region of its RNA as a template, and is required for the indefinite proliferation of primary human cells. We find that primary fibroblasts and lymphoblasts from DKC-affected males are not detectably deficient in conventional H/ACA small nucleolar RNA accumulation or function; however, DKC cells have a lower level of telomerase RNA, produce lower levels of telomerase activity and have shorter telomeres than matched normal cells. The pathology of DKC is consistent with compromised telomerase function leading to a defect in telomere maintenance, which may limit the proliferative capacity of human somatic cells in epithelia and blood.
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              Telomere end-replication problem and cell aging.

              Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                April 2010
                April 2010
                22 April 2010
                : 6
                : 4
                : e1000920
                Affiliations
                [1 ]Telomeres and Cancer Laboratory, Institut Curie, CNRS, UPMC University Paris 06, Paris, France
                [2 ]Epigenetics and Telomere Regulation, CNRS ENS UCBL IFR128, Ecole Normale Supérieure de Lyon, Lyon, France
                [3 ]Functional Organization and Plasticity of Mammalian Genomes, Institut Curie, UPMC University Paris 06, Paris, France
                [4 ]Lawrence Berkeley Laboratory, Berkeley, California, United States of America
                [5 ]Buck Institute for Age Research, Novato, California, United States of America
                Brandeis University, United States of America
                Author notes
                [¤]

                Current address: Vattikuti Urology Institute, Henry Ford Health System, Detroit, Michigan, United States of America

                Conceived and designed the experiments: ALV. Performed the experiments: NA CSB ID RBB AB. Analyzed the data: NA CSB EG ALV. Contributed reagents/materials/analysis tools: AL JC ShK AO FM EG ALV. Wrote the paper: ALV.

                Article
                09-PLGE-RA-1677R4
                10.1371/journal.pgen.1000920
                2858680
                20421929
                e4bdb9d7-39b1-4f5c-b148-fa7bb39abc1f
                Arnoult et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 21 September 2009
                : 22 March 2010
                Page count
                Pages: 15
                Categories
                Research Article
                Cell Biology/Nuclear Structure and Function
                Genetics and Genomics/Functional Genomics
                Genetics and Genomics/Nuclear Structure and Function
                Molecular Biology/Chromosome Structure

                Genetics
                Genetics

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