Telomeric Repeat Research Articles

Single-Molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA.

Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single-stranded guanine-rich (G-rich) DNA invades at a complementary duplex telomere repeat sequence, forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single-stranded DNA. Single-stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQs) in vitro and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here, we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single-stranded DNA of a TDL fold into a GQ. This study provides new insight into telomere structure and establishes a framework for the development of novel therapeutics designed to target GQs at telomeres in cancer cells.

Structural insights into Pot1-ssDNA, Pot1-Tpz1 and Tpz1-Ccq1 Interactions within fission yeast shelterin complex.

The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1OB1-GGTTAC and Pot1OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1DBD-single-stranded-DNA, Pot1372-555-Tpz1185-212 and Tpz1425-470-Ccq1123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3' ss telomeric overhang.

Telomere Attrition With Concomitant hTERT Overexpression Involved in the Progression of Gastric Cancer May Have Prognostic and Clinical Implications in High-Risk Population Group From North India.

Genetic instabilities exacerbated by the dysfunction of telomeres can lead to the development of cancer. Nearly 90% of all human malignancies are linked with telomere dysregulation and overexpression of telomerase, an enzyme that catalyzes the synthesis of telomeric DNA repeats at the ends of chromosomes. The burden of gastric cancer continues to inflict a deterring impact on the global health scenario, accounting for over one million new cases in 2020. The disease is asymptomatic in its early stages of progression, which is attributed to the poor prognosis and overall surge in mortality rate worldwide. Exploiting telomere physiology can provide extensive mechanistic insight into telomere-associated gastric cancer progression and its use as a target in a variety of therapeutic interventions. In this study, we aimed to evaluate the clinical implications of c-Myc, human telomerase reverse transcriptase (hTERT) expression, and telomere length in patients with gastric cancer. A total of 57 gastric cancer cases and adjacent controls were included in the study. RT-PCR and immunohistochemistry were used to assess the expression levels of c-Myc and hTERT. The relative telomere length was measured by MMQPCR using the Cawthon method. Our results indicated that the shorter telomere and increased hTERT expression were associated with gastric cancer progression. The study also highlighted the role of short telomeres and increased expression of hTERT in gastric cancer progression and its association with various etiological risk factors, transcriptional activators, and overall survival among the ethnic Kashmiri population of North India.

Environment-Sensitive Nucleoside Probe Unravels the Complex Structural Dynamics of i-Motif DNAs.

Although evidence for the existence and biological role of i-motif (iM) DNA structures in cells is emerging, probing their structural polymorphism and identifying physiologically active conformations using currently available tools remain a major challenge. Here, we describe the development of an innovative device to investigate the conformation equilibrium of different iMs formed by C-rich telomeric repeat and oncogenic B-raf promoter sequences using a new conformation-sensitive dual-purpose nucleoside probe. The nucleoside is composed of a trifluoromethyl-benzofuran-2-yl moiety at the C5 position of 2'-deoxyuridine, which functions as a responsive fluorescent and 19F NMR probe. While the fluorescent component is useful in monitoring and estimating the folding process, the 19F label provides spectral signatures for various iMs, thereby enabling a systematic analysis of their complex population equilibrium under different conditions (e.g., pH, temperature, metal ions, and cell lysate). Distinct 19F signals exhibited by the iMs formed by the human telomeric repeat helped in calculating their relative population. A battery of fluorescence and 19F NMR studies using native and mutated B-raf oligonucleotides gave valuable insights into the iM structure landscape and its dependence on environmental conditions and also helped in predicting the structure of the major iM conformation. Overall, our findings indicate that the probe is highly suitable for studying complex nucleic acid systems.

Anticancer activity of G4-targeting phenoxazine derivatives in vitro

G4-stabilizing ligands are now being considered as anticancer, antiviral and antibacterial agents. Phenoxazine is a promising scaffold for the development of G4 ligands. Here, we profiled two known phenoxazine-based nucleoside analogs and five new nucleoside and non-nucleoside derivatives against G4 targets from telomere repeats and the KIT promoter region. Leading new derivatives exhibited remarkably high G4-stabilizing effects (comparable or superior to the effects of the commonly used selective G4 ligands PDS and NMM) and selectivity toward G4s over duplex (superior to BRACO-19). All phenoxazine-based ligands inhibited cellular metabolic activity. The phenoxazine derivatives were particularly toxic for lung adenocarcinoma cells A549' and human liver cancer cells HepG2 (CC50 of the nucleoside analogues in the nanomolar range), but also affected breast cancer cells MCF7, as well as immortalized fibroblasts VA13 and embryonic kidney cells HEK293t (CC50 in the micromolar range). Importantly, the CC50 values varied mostly in accordance with G4-binding affinities and G4-stabilizing effects, and the phenoxazine derivatives localized in the cell nuclei, which corroborates G4-mediated mechanisms of action.

How Structural Features Define Biogenesis and Function of Human Telomerase RNA Primary Transcript.

Telomerase RNA has been uncovered as a component of the telomerase enzyme, which acts as a reverse transcriptase and maintains the length of telomeres in proliferated eukaryotic cells. Telomerase RNA is considered to have major functions as a template for telomeric repeat synthesis and as a structural scaffold for telomerase. However, investigations of its biogenesis and turnover, as well as structural data, have provided evidence of functions of telomerase RNA that are not associated with telomerase activity. The primary transcript produced from the human telomerase RNA gene encodes for the hTERP protein, which presents regulatory functions related to autophagy, cellular proliferation, and metabolism. This review focuses on the specific features relating to the biogenesis and structure of human telomerase RNA that support the existence of an isoform suitable for functioning as an mRNA. We believe that further investigation into human telomerase RNA biogenesis mechanisms will provide more levels for manipulating cellular homeostasis, survival, and transformation mechanisms, and may contribute to a deeper understanding of the mechanisms of aging.

Effects of Transcription-Dependent Physical Perturbations on the Chromosome Dynamics in Living Cells.

Recent studies with single-particle tracking in live cells have revealed that chromatin dynamics are directly affected by transcription. However, how transcription alters the chromatin movements followed by changes in the physical properties of chromatin has not been elucidated. Here, we measured diffusion characteristics of chromatin by targeting telomeric DNA repeats with CRISPR-labeling. We found that transcription inhibitors that directly block transcription factors globally increased the movements of chromatin, while the other inhibitor that blocks transcription by DNA intercalating showed an opposite effect. We hypothesized that the increased mobility of chromatin by transcription inhibition and the decreased chromatin movement by a DNA intercalating inhibitor is due to alterations in chromatin rigidity. We also tested how volume confinement of nuclear space affects chromatin movements. We observed decreased chromatin movements under osmotic pressure and with overexpressed chromatin architectural proteins that compact chromatin.

Telomere Length as a Marker of Suicidal Risk in Schizophrenia.

Schizophrenia and suicidal behavior are associated with shortening in the length of telomeres. The aim of the study was to compare the content (pg/mcg) of telomeric repeat in DNA isolated from peripheral blood cells in three groups of subjects: patients with schizophrenia and a history of suicide attempts, patients with schizophrenia without suicidal tendencies, and healthy control volunteers. Relapses according to gender and age were examined in 47 patients with schizophrenia with suicidal behavior, 47 patients without self-destructive conditions, and 47 volunteers with healthy control and maintenance for the content of telomeric and the number of copies of mitochondrial DNA (mtDNA) in peripheral blood leukocytes. Analysis of determining the content of telomeric repeat (TR) in the DNA of massive weight gain in the series: patients with schizophrenia and suicidal attempts - patients with schizophrenia without suicidal observations - healthy controls (225±28.4 (227 [190; 250]) vs. 243±21 (245 [228; 260]) vs. 255±17.9 (255 [242; 266]), p <0.005. The same trend is observed for the number of mtDNA copies (257±101.5 (250 [194; 297])) vs. 262.3±59.3 (254 [217; 312]) vs. 272±79.9 (274 [213; 304]); p=0.012), but no significant differences were recorded. For the first time, the phenomenon of telomere shortening was discovered in schizophrenics with suicidal risk. The length of the telomere corresponds to the parameter of a biological marker - an objectively measured indicator of normal or pathological processes, but gaining an idea of its reliability is still necessary for verification with an assessment of its sensitivity, specificity, and positive and negative predictive value. The telomere may be considered a putative predictive indicator of suicidal risk.

Telomere Length Regulation.

The number of (TTAGGG)n repeats at the ends of chromosomes is highly variable between individual chromosomes, between different cells and between species. Progressive loss of telomere repeats limits the proliferation of pre-malignant human cells but also contributes to aging by inducing apoptosis and senescence in normal cells. Despite enormous progress in understanding distinct pathways that result in loss and gain of telomeric DNA in different cell types, many questions remain. Further studies are needed to delineate the role of damage to telomeric DNA, replication errors, chromatin structure, liquid-liquid phase transition, telomeric transcripts (TERRA) and secondary DNA structures such as guanine quadruplex structures, R-loops and T-loops in inducing gains and losses of telomere repeats in different cell types. Limitations of current telomere length measurements techniques and differences in telomere biology between species and different cell types complicate generalizations about the role of telomeres in aging and cancer. Here some of the factors regulating the telomere length in embryonic and adult cells in mammals are discussed from a mechanistic and evolutionary perspective.

Cytogenetic Analysis of the Members of the Snake Genera Cylindrophis, Eryx, Python, and Tropidophis.

The recent discovery of two independently evolved XX/XY sex determination systems in the snake genera Python and Boa sparked a new drive to study the evolution of sex chromosomes in poorly studied lineages of snakes, where female heterogamety was previously assumed. Therefore, we examined seven species from the genera Eryx, Cylindrophis, Python, and Tropidophis by conventional and molecular cytogenetic methods. Despite the fact that these species have similar karyotypes in terms of chromosome number and morphology, we detected variability in the distribution of heterochromatin, telomeric repeats, and rDNA loci. Heterochromatic blocks were mainly detected in the centromeric regions in all species, although accumulations were detected in pericentromeric and telomeric regions in a few macrochromosomes in several of the studied species. All species show the expected topology of telomeric repeats at the edge of all chromosomes, with the exception of Eryx muelleri, where additional accumulations were detected in the centromeres of three pairs of macrochromosomes. The rDNA loci accumulate in one pair of microchromosomes in all Eryx species and in Cylindrophis ruffus, in one macrochromosome pair in Tropidophis melanurus and in two pairs of microchromosomes in Python regius. Sex-specific differences were not detected, suggesting that these species likely have homomorphic, poorly differentiated sex chromosomes.

Shortening of leucocyte telomere length is independently correlated with high body mass index and subcutaneous obesity (predominantly truncal), in Asian Indian women with abnormal fasting glycemia

IntroductionLeucocyte telomere length (LTL) is linked to accelerate aging and premature mortality. In this research, we aimed to explore the relations between biochemical and anthropometry markers and LTL in Asian...

DNA-RNA hybrid G-quadruplex tends to form near the 3′ end of telomere overhang

Telomeric repeat-containing RNA (TERRA) has been suggested to participate in telomere maintenance. TERRA consisting of UUAGGG repeats is capable of forming an intermolecular G-quadruplex (GQ) with single-stranded TTAGGG-repeat DNA in the telomere 3′ overhang. To explore the structural features and potential functions of this DNA-RNA hybrid GQ (HGQ), we used single-molecule FRET to study the folding patterns of DNA with four to seven telomeric tandem repeats annealed with a short RNA consisting of two or five telomeric repeats. Our data highlight that RNA prefers to form DNA-RNA HGQ near the 3′ end of telomeric DNA. Furthermore, the unfolding of secondary structures by a complementary C-rich sequence was observed for DNA GQ but not for DNA-RNA HGQ, which demonstrated the enhanced stability of the telomere 3′ end via hybridization with RNA. These conformational and physical properties of telomeric DNA-RNA HGQ suggest that TERRA might limit access to the 3′ end of the telomeric DNA overhang, which is known to be critical for the interaction with telomerase and other telomere-associated proteins.

G-quadruplex structure of the C. elegans telomeric repeat: a two tetrads basket type conformation stabilized by a non-canonical C-T base-pair.

The Caenorhabditis elegans model has greatly contributed to the understanding of the role of G-quadruplexes in genomic instability. The GGCTTA repeats of the C. elegans telomeres resemble the GGGTTA repeats of the human telomeres. However, the comparison of telomeric sequences (Homo sapiens, Tetrahymena, Oxytricha, Bombyx mori and Giardia) revealed that small changes in these repeats can drastically change the topology of the folded G-quadruplex. In the present work we determined the structure adopted by the C. elegans telomeric sequence d[GG(CTTAGG)3]. The investigated C. elegans telomeric sequence is shown to fold into an intramolecular two G-tetrads basket type G-quadruplex structure that includes a C–T base pair in the diagonal loop. This work sheds light on the telomeric structure of the widely used C. elegans animal model.

Mitochondrial dysfunction caused by targeted deletion of Mfn1 does not result in telomere shortening in oocytes.

Telomere shortening during oocyte growth and development is related to reproductive ageing and infertility. The main mechanism involved in the maintenance of telomeres is based on telomerase activity, a specialized enzyme complex, which is capable of adding TTAGGG repeats at the ends of the chromosomes. Mitochondrial dysfunction may cause progressive shortening of telomeres by promoting the generation of reactive oxygen species. Mitofusin-1 is a protein required for mitochondrial fusion. Mice with the mitofusin-1 (Mfn1) deletion in the oocyte are characterized by accelerated follicular depletion and infertility, associated with defective oocyte maturation and follicular development. We hypothesized whether mitochondrial dysfunction in oocytes with targeted deletion of Mfn1 causes telomere shortening. We analyzed telomere length in oocyte and somatic cells in 3-, 6- and 9-month-old Mfn1-/- and wild-type mice. Immunofluorescence in oocyte mice of TRF1 and H2A.X was assessed to evaluate the interplay between the end-protection functions and the response to DNA damage occurring inside the telomeric repeats. Mitochondrial dysfunction due to the deletion of Mfn1 does not seem to affect telomere length in mouse oocytes.

Sensitive naked-eye detection of telomerase activity based on exponential amplification reaction and lateral flow assay.

Telomerase is a promising diagnostic and prognostic biomarker for cancers. Sensitive, simple, and reliable telomerase activity detection is vital for cancer diagnosis. Herein, we developed an ultrasensitive visualized assay for telomerase activity that combined the exponential amplification reaction (EXPAR) and lateral flow assay for easy and quick signal readout, which we termed as a lateral flow readout-EXPAR (LFR-EXPAR) assay. In the LFR-EXPAR assay, telomerase elongation products initiate the exponential amplification reaction, the generated trigger hybridizes with the reporter to form the recognition site of the nicking enzyme, and the nicking enzyme cuts the reporter strand. The degradation of the reporter can be detected with a universal lateral flow dipstick and read out with the naked eye. After conducting a series of proof-of-concept investigations, the LFR-EXPAR assay was found to achieve a sensitivity comparable to that of a TRAP (telomere repeat amplification protocol) assay. The LFR-EXPAR assay can be used to realize ultrasensitive and point-of-care detection of telomerase without requiring specialized instruments, holding great promise for early cancer diagnosis.

Abstract 2169: New telomeres and chromosomal arm fusions in cancer genomes revealed by long-read genome sequencing

Abstract Chromosomal rearrangements are widespread features of cancer genomes, and often lead to the generation of both larger and smaller chromosomes that are readily observed by cytogenetics analysis. The generation of these chromosomal alterations likely involves alterations in the structure and location of telomeres at chromosome ends, though these events have been difficult to assess due to the long and highly repetitive nature of telomeres. Here, we developed an analytic method (TeloFuse) to identify new telomeres and chromosomal arm fusions which are characterized by telomeric repeats at intra-chromosomal locations. We applied TeloFuse to identify telomere-containing fusions as well as novel sites of telomere addition using short-read genome sequencing data from 326 cancer cell lines followed by long-read genome sequencing and cytogenetic analysis of selected cell lines. We observed new telomeres at sites of terminal deletion in long read data (telomere length = 2-9 kb), and in 76/326 (23%) of cell line genomes with short read data. Telomere sequence-containing fusions at intra-chromosomal sites were detected in 75/326 (23%) of cancer cell lines and confirmed as chromosomal arm fusion events by long-read genome sequencing. Long-read genome sequencing further suggests that new telomeres have similar telomere length as telomeres found on normal chromosomal arms (median = 5kb). Conversely, telomeric repeats at sites of chromosomal arm fusions were found to be critically short (~200-500bp long). Extending our analysis to 97 lung adenocarcinoma patient samples from The Cancer Genome Atlas, we further found new telomeres in 17/97 (18%) and chromosomal arm fusions in 28/97 (29%) patients with short-read data. Notably, new telomeres and chromosomal arm fusion events were also found to disrupt protein-coding genes, suggesting that these events represent a poorly appreciated mode for gene inactivation in cancer. Our results suggest that the formation of new telomeres and chromosomal arm fusions are prevalent events during cancer genome evolution and are likely critical for the stabilization of new chromosomes in human cancers. More broadly, our findings also highlight the utility of long-read genome sequencing in delineating chromosomal-scale alterations that have been widely observed by cytogenetics analysis. Citation Format: Kar-Tong Tan, Michael K. Slevin, Max Garrity-Janger, Heng Li, Matthew Meyerson. New telomeres and chromosomal arm fusions in cancer genomes revealed by long-read genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2169.

Genetic, parental and lifestyle factors influence telomere length

The average length of telomere repeats (TL) declines with age and is considered to be a marker of biological ageing. Here, we measured TL in six blood cell types from 1046 individuals using the clinically validated Flow-FISH method. We identified remarkable cell-type-specific variations in TL. Host genetics, environmental, parental and intrinsic factors such as sex, parental age, and smoking are associated to variations in TL. By analysing the genome-wide methylation patterns, we identified that the association of maternal, but not paternal, age to TL is mediated by epigenetics. Single-cell RNA-sequencing data for 62 participants revealed differential gene expression in T-cells. Genes negatively associated with TL were enriched for pathways related to translation and nonsense-mediated decay. Altogether, this study addresses cell-type-specific differences in telomere biology and its relation to cell-type-specific gene expression and highlights how perinatal factors play a role in determining TL, on top of genetics and lifestyle.

Partners in crime: Tbf1 and Vid22 promote expansions of long human telomeric repeats at an interstitial chromosome position in yeast.

In humans, telomeric repeats (TTAGGG)n are known to be present at internal chromosomal sites. These interstitial telomeric sequences (ITSs) are an important source of genomic instability, including repeat length polymorphism, but the molecular mechanisms responsible for this instability remain to be understood. Here, we studied the mechanisms responsible for expansions of human telomeric (Htel) repeats that were artificially inserted inside a yeast chromosome. We found that Htel repeats in an interstitial chromosome position are prone to expansions. The propensity of Htel repeats to expand depends on the presence of a complex of two yeast proteins: Tbf1 and Vid22. These two proteins are physically bound to an interstitial Htel repeat, and together they slow replication fork progression through it. We propose that slow progression of the replication fork through the protein complex formed by the Tbf1 and Vid22 partners at the Htel repeat cause DNA strand slippage, ultimately resulting in repeat expansions.

Investigating the role of G-quadruplexes at Saccharomyces cerevisiae telomeres.

The G-quadruplex consensus motif G≥3NxG≥3NxG≥3NxG≥3 is found at telomeres of many species, ranging from yeast to plants to humans, but the biological significance of this fact remains largely unknown. In this study, we examine the in vivo relevance of telomeric G-quadruplexes in the budding yeast Saccharomyces cerevisiae by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant [(TG)0–4TGG]xATTTGG telomeric repeats instead of wild-type (TG)0-6TGGGTGTG(G)0-1 repeats to the distal ends of telomeres. The tlc1-tm telomere sequences lack the GGG motif present in every wild-type repeat and, therefore, are expected to be impaired in the formation of G-quadruplexes. Circular dichroism analysis of oligonucleotides consisting of tlc1-tm telomeric sequence is consistent with this hypothesis. We have previously shown that tlc1-tm cells grow similarly to wild-type cells, suggesting that the ability to form telomeric G-quadruplexes is not essential for telomere capping in S. cerevisiae cells.

Investigation of the genome sizes and ploidy within the genus Monocercomonoides.

Monocercomonoides is a genus of anaerobic flagellates found mainly in the gut of insects and vertebrates. We explored the ploidy of six strains of Monocercomonoides using fluorescence in situ hybridization (FISH) with probes against the SufDSU gene known to be in a single copy in M. exilis. Our results show that all investigated strains are haploid, with a single clear signal displayed in most of the analyzed nuclei. Staining of the telomeric repeats TTAGGG using FISH revealed that all investigated strains, except for strains of M.merkovicensis, exhibit a similar number of telomeric signals to those of M.exilis. DNA content of the nuclei in seven strains was assessed using flow cytometry. With the knowledge of the ploidy, their haploid genome sizes were estimated to vary from 60 to 161 Mbp. The genome size variation observed in Monocercomonoides is much larger than the variation within other genera of metamonads such as Trichomonas, Tritrichomonas, or Giardia, but similar to the variations observed within genera of algae or plants.