Internal genomic regions mobilized for telomere maintenance in C. elegans.
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ABSTRACT:
SUBMITTER: Kim C
PROVIDER: S-EPMC4805358 | biostudies-other | 2016 Jan-Mar
REPOSITORIES: biostudies-other
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Project description:Cells surviving crisis are often tumorigenic and their telomeres are commonly maintained through the reactivation of telomerase. However, surviving cells occasionally activate a recombination-based mechanism called alternative lengthening of telomeres (ALT). Here we establish stably maintained survivors in telomerase-deleted Caenorhabditis elegans that escape from sterility by activating ALT. ALT survivors trans-duplicate an internal genomic region, which is already cis-duplicated to chromosome ends, across the telomeres of all chromosomes. These 'Template for ALT' (TALT) regions consist of a block of genomic DNA flanked by telomere-like sequences, and are different between two genetic background. We establish a model that an ancestral duplication of a donor TALT region to a proximal telomere region forms a genomic reservoir ready to be incorporated into telomeres on ALT activation.
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Project description:Cancers require telomere maintenance mechanisms for unlimited replicative potential. They achieve this through TERT activation or alternative telomere lengthening associated with ATRX or DAXX loss. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we dissect whole-genome sequencing data of over 2500 matched tumor-control samples from 36 different tumor types aggregated within the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium to characterize the genomic footprints of these mechanisms. While the telomere content of tumors with ATRX or DAXX mutations (ATRX/DAXXtrunc) is increased, tumors with TERT modifications show a moderate decrease of telomere content. One quarter of all tumor samples contain somatic integrations of telomeric sequences into non-telomeric DNA. This fraction is increased to 80% prevalence in ATRX/DAXXtrunc tumors, which carry an aberrant telomere variant repeat (TVR) distribution as another genomic marker. The latter feature includes enrichment or depletion of the previously undescribed singleton TVRs TTCGGG and TTTGGG, respectively. Our systematic analysis provides new insight into the recurrent genomic alterations associated with telomere maintenance mechanisms in cancer.
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Project description:Telomeres are composed of simple tandem DNA repeats that protect the ends of linear chromosomes from replicative erosion or inappropriate DNA damage response mechanisms. The mammalian Protection Of Telomeres (POT1) protein interacts with single-stranded telomeric DNA and can exert positive and negative effects on telomere length. Of four distinct POT1 homologs in the roundworm Caenorhabditis elegans, deficiency for POT-1 or POT-2 resulted in progressive telomere elongation that occurred because both proteins negatively regulate telomerase. We created a POT-1::mCherry fusion protein that forms discrete foci at C. elegans telomeres, independent of POT-2, allowing for live analysis of telomere dynamics. Transgenic pot-1::mCherry repressed telomerase in pot-1 mutants. Animals deficient for pot-1, but not pot-2, displayed mildly enhanced telomere erosion rates in the absence of the telomerase reverse transcriptase, trt-1. However, trt-1; pot-1 double mutants exhibited delayed senescence in comparison to trt-1 animals, and senescence was further delayed in trt-1; pot-2; pot-1 triple mutants, some of which survived robustly in the absence of telomerase. Our results indicate that POT-1 and POT-2 play independent roles in suppressing a telomerase-independent telomere maintenance pathway but may function together to repress telomerase.
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Project description:Homology-directed DNA repair is essential for genome maintenance through templated DNA synthesis. Alternative lengthening of telomeres (ALT) necessitates homology-directed DNA repair to maintain telomeres in about 10-15% of human cancers. How DNA damage induces assembly and execution of a DNA replication complex (break-induced replisome) at telomeres or elsewhere in the mammalian genome is poorly understood. Here we define break-induced telomere synthesis and demonstrate that it utilizes a specialized replisome, which underlies ALT telomere maintenance. DNA double-strand breaks enact nascent telomere synthesis by long-tract unidirectional replication. Proliferating cell nuclear antigen (PCNA) loading by replication factor C (RFC) acts as the initial sensor of telomere damage to establish predominance of DNA polymerase δ (Pol δ) through its POLD3 subunit. Break-induced telomere synthesis requires the RFC-PCNA-Pol δ axis, but is independent of other canonical replisome components, ATM and ATR, or the homologous recombination protein Rad51. Thus, the inception of telomere damage recognition by the break-induced replisome orchestrates homology-directed telomere maintenance.
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Project description:BackgroundTelomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis.Methodology/principal findingsWe used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana.Conclusion/significanceAblation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.
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Project description:Tumour cells can adopt telomere maintenance mechanisms (TMMs) to avoid telomere shortening, an inevitable process due to successive cell divisions. In most tumour cells, telomere length (TL) is maintained by reactivation of telomerase, while a small part acquires immortality through the telomerase-independent alternative lengthening of telomeres (ALT) mechanism. In the last years, a great amount of data was generated, and different TMMs were reported and explained in detail, benefiting from genome-scale studies of major importance. In this review, we address seven different TMMs in tumour cells: mutations of the TERT promoter (TERTp), amplification of the genes TERT and TERC, polymorphic variants of the TERT gene and of its promoter, rearrangements of the TERT gene, epigenetic changes, ALT, and non-defined TMM (NDTMM). We gathered information from over fifty thousand patients reported in 288 papers in the last years. This wide data collection enabled us to portray, by organ/system and histotypes, the prevalence of TERTp mutations, TERT and TERC amplifications, and ALT in human tumours. Based on this information, we discuss the putative future clinical impact of the aforementioned mechanisms on the malignant transformation process in different setups, and provide insights for screening, prognosis, and patient management stratification.
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Project description:A variety of human tumors employ alternative and recombination-mediated lengthening for telomere maintenance (ALT). Human RecQ helicases, such as BLM and WRN, can efficiently unwind alternate/secondary structures during telomere replication and/or recombination. Here, we report a novel role for RECQL1, the most abundant human RecQ helicase but functionally least studied, in telomere maintenance. RECQL1 associates with telomeres in ALT cells and actively resolves telomeric D-loops and Holliday junction substrates. RECQL1 physically and functionally interacts with telomere repeat-binding factor 2 that in turn regulates its helicase activity on telomeric substrates. The telomeric single-stranded binding protein, protection of telomeres 1 efficiently stimulates RECQL1 on telomeric substrates containing thymine glycol, a replicative blocking lesion. Loss of RECQL1 results in dysfunctional telomeres, telomere loss and telomere shortening, elevation of telomere sister-chromatid exchanges and increased aphidicolin-induced telomere fragility, indicating a role for RECQL1 in telomere maintenance. Further, our results indicate that RECQL1 may participate in the same pathway as WRN, probably in telomere replication.
| S-EPMC4027191 | biostudies-literature
Project description:Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in Barrett's esophageal adenocarcinoma (BAC). The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for ?-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those that resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contributes to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase renders telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore demonstrate that a therapy targeting HR and telomerase has the potential to prevent both tumor growth and genomic evolution in BAC.
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Project description:BackgroundInternal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns.Methodology/principal findingsIn this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level.ConclusionsIdentification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.
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Project description:Telomeres are responsible for the protection of the chromosome ends and shortened telomere length has been associated with risk of multiple cancers. Genetic variation in telomere-related genes may alter cancer risk associated with telomere length. Using lung cancer cases (n=120) and population-based controls (n=110) from Xuanwei, China, we analyzed telomere length separately and in conjunction with single nucleotide polymorphisms in the telomere maintenance genes POT1, TERT, and TERF2, which we have previously reported were associated with risk of lung cancer in this study. POT1 rs10244817, TERT rs2075786, and TERF2 rs251796 were significantly associated with lung cancer (p(trend)< or =0.05). The shortest tertile of telomere length was not significantly associated with risk of lung cancer (OR=1.58; 95% CI=0.79-3.18) when compared to the longest tertile of telomere length. When stratified by genotype, there was a suggestion of a dose-response relationship between tertiles of telomere length and risk of lung cancer among the POT1 rs10244817 common variant carriers (OR (95% CI)=1.33 (0.47-3.75), 3.30 (1.14-9.56), respectively) but not among variant genotype carriers (p(interaction)=0.05). Our findings provide evidence that telomere length and genetic variation in telomere maintenance genes may be associated with risk of lung cancer susceptibility and warrant replication in larger studies.
| S-EPMC2783462 | biostudies-literature