Caenorhabditis elegans telomere-binding proteins TEBP-1 and TEBP-2 adapt the Myb module to dimerize and bind telomeric DNA.

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.

A POT1 Founder Variant Associated with Early Onset Recurrent Melanoma and Various Solid Malignancies.

POT1 (Protection of Telomeres 1) is a key component of the six-membered shelterin complex that plays a critical role in telomere protection and length regulation. Germline variants in the POT1 gene have been implicated in predisposition to cancer, primarily to melanoma and chronic lymphocytic leukemia (CLL). We report the identification of POT1 p.(I78T), previously ranked with conflicting interpretations of pathogenicity, as a founder pathogenic variant among Ashkenazi Jews (AJs) and describe its unique clinical landscape. A directed database search was conducted for individuals referred for genetic counselling from 2018 to 2023. Demographic, clinical, genetic, and pathological data were collected and analyzed. Eleven carriers, 25 to 67 years old, from ten apparently unrelated families were identified. Carriers had a total of 30 primary malignancies (range 1-6); nine carriers (82%) had recurrent melanoma between the ages of 25 and 63 years, three carriers (27%) had desmoid tumors, three (27%) had papillary thyroid cancer (PTC), and five women (63% of female carriers) had breast cancer between the ages of 44 and 67 years. Additional tumors included CLL; sarcomas; endocrine tumors; prostate, urinary, and colorectal cancers; and colonic polyps. A review of a local exome database yielded an allelic frequency of the variant of 0.06% among all ethnicities and of 0.25% in AJs. A shared haplotype was found in all carriers tested. POT1 p.(I78T) is a founder disease-causing variant associated with early-onset melanoma and additional various solid malignancies with a high tumor burden. We advocate testing for this variant in high-risk patients of AJ descent. The inclusion of POT1 in germline panels for various types of cancer is warranted.

Decreased telomere length in a subgroup of young individuals with bipolar disorders: replication in the FACE-BD cohort and association with the shelterin component POT1.

Bipolar disorder (BD) has been associated with premature cellular aging with shortened telomere length (TL) as compared to the general population. We recently identified a subgroup of young individuals with prematurely shortened TL. The aims of the present study were to replicate this observation in a larger sample and analyze the expression levels of genes associated with age or TL in a subsample of these individuals. TL was measured on peripheral blood DNA using quantitative polymerase chain reaction in a sample of 542 individuals with BD and clustering analyses were performed. Gene expression level of 29 genes, associated with aging or with telomere maintenance, was analyzed in RNA samples from a subsample of 129 individuals. Clustering analyses identified a group of young individuals (mean age 29.64 years), with shorter TL. None of the tested clinical variables were significantly associated with this subgroup. Gene expression level analyses showed significant downregulation of MYC, POT1, and CD27 in the prematurely aged young individuals compared to the young individuals with longer TL. After adjustment only POT1 remained significantly differentially expressed between the two groups of young individuals. This study confirms the existence of a subgroup of young individuals with BD with shortened TL. The observed decrease of POT1 expression level suggests a newly described cellular mechanism in individuals with BD, that may contribute to telomere shortening.

Ccq1 restrains Mre11-mediated degradation to distinguish short telomeres from double-strand breaks.

Telomeres protect chromosome ends and are distinguished from DNA double-strand breaks (DSBs) by means of a specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. We investigated the role of telomere-associated proteins in establishing end-protection by studying viable mutants lacking these proteins. Mutants were studied using a Schizosaccharomyces pombe model system that induces cutting of a 'proto-telomere' bearing telomere repeats to rapidly form a new stable chromosomal end, in contrast to the rapid degradation of a control DSB. Cells lacking the telomere-associated proteins Taz1, Rap1, Poz1 or Rif1 formed a chromosome end that was stable. Surprisingly, cells lacking Ccq1, or impaired for recruiting Ccq1 to the telomere, converted the cleaved proto-telomere to a rapidly degraded DSB. Ccq1 recruits telomerase, establishes heterochromatin and affects DNA damage checkpoint activation; however, these functions were separable from protection of the new telomere by Ccq1. In cells lacking Ccq1, telomere degradation was greatly reduced by eliminating the nuclease activity of Mre11 (part of the Mre11-Rad50-Nbs1/Xrs2 DSB processing complex), and higher amounts of nuclease-deficient Mre11 associated with the new telomere. These results demonstrate a novel function for S. pombe Ccq1 to effect end-protection by restraining Mre11-dependent degradation of the DNA end.

Exploring TRF2-Dependent DNA Distortion Through Single-DNA Manipulation Studies.

TRF2 is a component of shelterin, a telomere-specific protein complex that protects the ends of mammalian chromosomes from DNA damage signaling and improper repair. TRF2 functions as a homodimer and its interaction with telomeric DNA has been studied, but its full-length DNA-binding properties are unknown. This study examines TRF2's interaction with single-DNA strands and focuses on the conformation of the TRF2-DNA complex and TRF2's preference for DNA chirality. The results show that TRF2-DNA can switch between extended and compact conformations, indicating multiple DNA-binding modes, and TRF2's binding does not have a strong preference for DNA supercoiling chirality when DNA is under low tension. Instead, TRF2 induces DNA bending under tension. Furthermore, both the N-terminal domain of TRF2 and the Myb domain enhance its affinity for the telomere sequence, highlighting the crucial role of multivalent DNA binding in enhancing its affinity and specificity for telomere sequence. These discoveries offer unique insights into TRF2's interaction with telomeric DNA.

Characterization of novel mutations in the TEL-patch domain of the telomeric factor TPP1 associated with telomere biology disorders.

Telomeres are nucleoprotein structures that protect the chromosome ends from degradation and fusion. Telomerase is a ribonucleoprotein complex essential to maintain the length of telomeres. Germline defects that lead to short and/or dysfunctional telomeres cause telomere biology disorders (TBDs), a group of rare and heterogeneous Mendelian diseases including pulmonary fibrosis, dyskeratosis congenita, and Høyeraal-Hreidarsson syndrome. TPP1, a telomeric factor encoded by the gene ACD, recruits telomerase at telomere and stimulates its activity via its TEL-patch domain that directly interacts with TERT, the catalytic subunit of telomerase. TBDs due to TPP1 deficiency have been reported only in 11 individuals. We here report four unrelated individuals with a wide spectrum of TBD manifestations carrying either heterozygous or homozygous ACD variants consisting in the recurrent and previously described in-frame deletion of K170 (K170∆) and three novel missense mutations G179D, L184R, and E215V. Structural and functional analyses demonstrated that the four variants affect the TEL-patch domain of TPP1 and impair telomerase activity. In addition, we identified in the ACD gene several motifs associated with small deletion hotspots that could explain the recurrence of the K170∆ mutation. Finally, we detected in a subset of blood cells from one patient, a somatic TERT promoter-activating mutation that likely provides a selective advantage over non-modified cells, a phenomenon known as indirect somatic genetic rescue. Together, our results broaden the genetic and clinical spectrum of TPP1 deficiency and specify new residues in the TEL-patch domain that are crucial for length maintenance and stability of human telomeres in vivo.

A Nested PCR Telomere Fusion Assay Highlights the Widespread End-Capping Protection of Arabidopsis CTC1.

Telomeres protect the ends of linear eukaryotic chromosomes from being recognized as DNA double-strand breaks. Two major protein complexes are involved in the protection of telomeres: shelterin and CST. The dysfunction of these complexes can challenge the function of telomeres and lead to telomere fusions, breakage-fusion-bridge cycles, and cell death. Therefore, monitoring telomere fusions helps to understand telomeres biology. Telomere fusions are often analyzed by Fluorescent In Situ Hybridization (FISH) or PCR. Usually, both methods involve hybridization with a telomeric probe, which allows the detection of fusions containing telomeric sequences, but not of those lacking them. With the aim of detecting both types of fusion events, we have developed a nested PCR method to analyze telomere fusions in Arabidopsis thaliana. This method is simple, accurate, and does not require hybridization. We have used it to analyze telomere fusions in wild-type and mutant plants altered in CTC1, one of the three components of the Arabidopsis CST telomere capping complex. Our results show that null ctc1-2 mutant plants display fusions between all telomeric regions present in Arabidopsis chromosomes 1, 3 and 5, thus highlighting the widespread end-capping protection achieved by CTC1.

Germline POT1 Variants: A Critical Perspective on POT1 Tumor Predisposition Syndrome.

The Protection of Telomere 1 (POT1) gene was identified as a melanoma predisposition candidate nearly 10 years ago. Thereafter, various cancers have been proposed as associated with germline POT1 variants in the context of the so-called POT1 Predisposition Tumor Syndrome (POT1-TPD). While the key role, and related risks, of the alterations in POT1 in melanoma are established, the correlation between germline POT1 variants and the susceptibility to other cancers partially lacks evidence, due also to the rarity of POT1-TPD. Issues range from the absence of functional or segregation studies to biased datasets or the need for a revised classification of variants. Furthermore, a proposal of a surveillance protocol related to the cancers associated with POT1 pathogenic variants requires reliable data to avoid an excessive, possibly unjustified, burden for POT1 variant carriers. We propose a critical perspective regarding data published over the last 10 years that correlate POT1 variants to various types of cancer, other than cutaneous melanoma, to offer food for thought for the specialists who manage cancer predisposition syndromes and to stimulate a debate on the grey areas that have been exposed.

The shelterin component TRF2 mediates columnar stacking of human telomeric chromatin.

Telomere repeat binding factor 2 (TRF2) is an essential component of the telomeres and also plays an important role in a number of other non-telomeric processes. Detailed knowledge of the binding and interaction of TRF2 with telomeric nucleosomes is limited. Here, we study the binding of TRF2 to in vitro-reconstituted kilobasepair-long human telomeric chromatin fibres using electron microscopy, single-molecule force spectroscopy and analytical ultracentrifugation sedimentation velocity. Our electron microscopy results revealed that full-length and N-terminally truncated TRF2 promote the formation of a columnar structure of the fibres with an average width and compaction larger than that induced by the addition of Mg2+, in agreement with the in vivo observations. Single-molecule force spectroscopy showed that TRF2 increases the mechanical and thermodynamic stability of the telomeric fibres when stretched with magnetic tweezers. This was in contrast to the result for fibres reconstituted on the 'Widom 601' high-affinity nucleosome positioning sequence, where minor effects on fibre stability were observed. Overall, TRF2 binding induces and stabilises columnar fibres, which may play an important role in telomere maintenance.

The Altered Functions of Shelterin Components in ALT Cells.

Telomeres are nucleoprotein complexes that cap the ends of eukaryotic linear chromosomes. Telomeric DNA is bound by shelterin protein complex to prevent telomeric chromosome ends from being recognized as damaged sites for abnormal repair. To overcome the end replication problem, cancer cells mostly preserve their telomeres by reactivating telomerase, but a minority (10-15%) of cancer cells use a homologous recombination-based pathway called alternative lengthening of telomeres (ALT). Recent studies have found that shelterin components play an important role in the ALT mechanism. The binding of TRF1, TRF2, and RAP1 to telomeres attenuates ALT activation, while the maintenance of ALT telomere requires TRF1 and TRF2. POT1 and TPP1 can also influence the occurrence of ALT. The elucidation of how shelterin regulates the initiation of ALT remains elusive. This review presents a comprehensive overview of the current findings on the regulation of ALT by shelterin components, aiming to enhance the insight into the altered functions of shelterin components in ALT cells and to identify potential targets for the treatment of ALT tumor cells.

ZNF524 directly interacts with telomeric DNA and supports telomere integrity.

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing nonhomologous end joining (NHEJ) through its subunit TRF2. Here, we describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity, and reveal base-specific sequence recognition by cocrystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

Pot1 promotes telomere DNA replication via the Stn1-Ten1 complex in fission yeast.

Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.

Canine sperm motility is associated with telomere shortening and changes in expression of shelterin genes.

BACKGROUND: Motion quality is a critical property for essential functions. Several endogenous and exogenous factors are involved in sperm motility. Here, we measured the relative telomere length and evaluated the gene expression of its binding-proteins, shelterin complex (TRF1, TRF2, RAP1, POT1, TIN2, and TPP1) in sperm of dogs using relative quantitative real-time PCR. We compared them between two sperm subpopulations with poor and good motion qualities (separated by swim-up method). Telomere shortening and alterations of shelterin gene expression result from ROS, genotoxic insults, and genetic predisposition. RESULTS: Sperm kinematic parameters were measured in two subpopulations and then telomeric index of each parameter was calculated. Telomeric index for linearity, VSL, VCL, STR, BCF, and ALH were significantly higher in sperms with good motion quality than in sperms with poor quality. We demonstrated that poor motion quality is associated with shorter telomere, higher expression of TRF2, POT1, and TIN2 genes, and lower expression of the RAP1 gene in dog sperm. The levels of TRF1 and TPP1 gene expression remained consistent despite variations in sperm quality and telomere length. CONCLUSION: Data provided evidence that there are considerable changes in gene expression of many shelterin components (TRF2, TIN2, POT1and RAP1) associated with shortening telomere in the spermatozoa with poor motion quality. Possibly, the poor motion quality is the result of defects in the shelterin complex and telomere length. Our data suggests a new approach in the semen assessment and etiologic investigations of subfertility or infertility in male animals.

Condensin positioning at telomeres by shelterin proteins drives sister-telomere disjunction in anaphase.

The localization of condensin along chromosomes is crucial for their accurate segregation in anaphase. Condensin is enriched at telomeres but how and for what purpose had remained elusive. Here, we show that fission yeast condensin accumulates at telomere repeats through the balancing acts of Taz1, a core component of the shelterin complex that ensures telomeric functions, and Mit1, a nucleosome remodeler associated with shelterin. We further show that condensin takes part in sister-telomere separation in anaphase, and that this event can be uncoupled from the prior separation of chromosome arms, implying a telomere-specific separation mechanism. Consistent with a cis-acting process, increasing or decreasing condensin occupancy specifically at telomeres modifies accordingly the efficiency of their separation in anaphase. Genetic evidence suggests that condensin promotes sister-telomere separation by counteracting cohesin. Thus, our results reveal a shelterin-based mechanism that enriches condensin at telomeres to drive in cis their separation during mitosis.

Effects of nanomaterial exposure on telomere dysfunction, hallmarks of mammalian and zebrafish cell senescence, and zebrafish mortality.

Environmental and occupational exposure to hazardous substances accelerates biological aging. However, the toxic effects of nanomaterials on telomere and cellular senescence (major hallmarks of the biological aging) remained controversial. This study was to synthesize all published evidence to explore the effects of nanomaterial exposure on the telomere change, cellular senescence and mortality of model animals. Thirty-five studies were included by searching electronic databases (PubMed, Embase and Web of Science). The pooled analysis by Stata 15.0 software showed that compared with the control, nanomaterial exposure could significantly shorten the telomere length [measured as kbp: standardized mean difference (SMD) = -1.88; 95% confidence interval (CI) = -3.13 - - 0.64; % of control: SMD = -1.26; 95%CI = -2.11- - 0.42; < 3 kbp %: SMD = 5.76; 95%CI = 2.92 - 8.60), increase the telomerase activity (SMD = -1.00; 95%CI = -1.74 to -0.26), senescence-associated ß-galactosidase levels in cells (SMD = 8.20; 95%CI = 6.05 - 10.34) and zebrafish embryos (SMD = 7.32; 95%CI = 4.70 - 9.94) as well as the mortality of zebrafish (SMD = 3.83; 95%CI = 2.94 - 4.72)]. The expression levels of telomerase TERT, shelterin components (TRF1, TRF2 and POT1) and senescence biomarkers (p21, p16) were respectively identified to be decreased or increased in subgroup analyses. In conclusion, this meta-analysis demonstrates that nanomaterial exposure is associated with telomere attrition, cell senescence and organismal death.

POT1 involved in telomeric DNA damage repair and genomic stability of cervical cancer cells in response to radiation.

Though telomeres play a crucial role in maintaining genomic stability in cancer cells and have emerged as attractive therapeutic targets in anticancer therapy, the relationship between telomere dysfunction and genomic instability induced by irradiation is still unclear. In this study, we identified that protection of telomeres 1 (POT1), a single-stranded DNA (ssDNA)-binding protein, was upregulated in γ-irradiated HeLa cells and in cancer patients who exhibit radiation tolerance. Knockdown of POT1 delayed the repair of radiation-induced telomeric DNA damage which was associated with enhanced H3K9 trimethylation and enhanced the radiosensitivity of HeLa cells. The depletion of POT1 also resulted in significant genomic instability, by showing a significant increase in end-to-end chromosomal fusions, and the formation of anaphase bridges and micronuclei. Furthermore, knockdown of POT1 disturbed telomerase recruitment to telomere, and POT1 could interact with phosphorylated ATM (p-ATM) and POT1 depletion decreased the levels of p-ATM induced by irradiation, suggesting that POT1 could regulate the telomerase recruitment to telomeres to repair irradiation-induced telomeric DNA damage of HeLa cells through interactions with p-ATM. The enhancement of radiosensitivity in cancer cells can be achieved through the combination of POT1 and telomerase inhibitors, presenting a potential approach for radiotherapy in cancer treatment.

Models for human telomere C-strand fill-in by CST-Polα-primase.

Telomere maintenance is essential for the genome integrity of eukaryotes, and this function is underpinned by the two-step telomeric DNA synthesis process: telomere G-overhang extension by telomerase and complementary strand fill-in by DNA polymerase alpha-primase (polα-primase). Compared to the telomerase step, the telomere C-strand fill-in mechanism is less understood. Recent studies have provided new insights into how telomeric single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) and polα-primase coordinate to synthesize the telomeric C-strand for telomere overhang fill-in. Cryogenic electron microscopy (cryo-EM) structures of CST-polα-primase complexes have provided additional insights into how they assemble at telomeric templates and de novo synthesize the telomere C-strand. In this review, we discuss how these latest findings coalesce with existing understanding to develop a human telomere C-strand fill-in mechanism model.

The CST complex facilitates cell survival under oxidative genotoxic stress.

Genomic DNA is constantly exposed to a variety of genotoxic stresses, and it is crucial for organisms to be equipped with mechanisms for repairing the damaged genome. Previously, it was demonstrated that the mammalian CST (CTC1-STN1-TEN1) complex, which was originally identified as a single-stranded DNA-binding trimeric protein complex essential for telomere maintenance, is required for survival in response to hydroxyurea (HU), which induces DNA replication fork stalling. It is still unclear, however, how the CST complex is involved in the repair of diverse types of DNA damage induced by oxidizing agents such as H2O2. STN1 knockdown (KD) sensitized HeLa cells to high doses of H2O2. While H2O2 induced DNA strand breaks throughout the cell cycle, STN1 KD cells were as resistant as control cells to H2O2 treatment when challenged in the G1 phase of the cell cycle, but they were sensitive when exposed to H2O2 in S/G2/M phase. STN1 KD cells showed a failure of DNA synthesis and RAD51 foci formation upon H2O2 treatment. Chemical inhibition of RAD51 in shSTN1 cells did not exacerbate the sensitivity to H2O2, implying that the CST complex and RAD51 act in the same pathway. Collectively, our results suggest that the CST complex is required for maintaining genomic stability in response to oxidative DNA damage, possibly through RAD51-dependent DNA repair/protection mechanisms.

Human POT1 protects the telomeric ds-ss DNA junction by capping the 5' end of the chromosome.

Protection of telomeres 1 (POT1) is the 3' single-stranded overhang-binding telomeric protein that prevents an ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) at chromosome ends. What precludes the DDR machinery from accessing the telomeric double-stranded-single-stranded junction is unknown. We demonstrate that human POT1 binds this junction by recognizing the phosphorylated 5' end of the chromosome. High-resolution crystallographic structures reveal that the junction is capped by POT1 through a "POT-hole" surface, the mutation of which compromises junction protection in vitro and telomeric 5'-end definition and DDR suppression in human cells. Whereas both mouse POT1 paralogs bind the single-stranded overhang, POT1a, not POT1b, contains a POT-hole and binds the junction, which explains POT1a's sufficiency for end protection. Our study shifts the paradigm for DDR suppression at telomeres by highlighting the importance of protecting the double-stranded-single-stranded junction.

Pot1b -/- tumors activate G-quadruplex-induced DNA damage to promote telomere hyper-elongation.

Malignant cancers must activate telomere maintenance mechanisms to achieve replicative immortality. Mutations in the human Protection of Telomeres 1 (POT1) gene are frequently detected in cancers with abnormally long telomeres, suggesting that the loss of POT1 function disrupts the regulation of telomere length homeostasis to promote telomere elongation. However, our understanding of the mechanisms leading to elongated telomeres remains incomplete. The mouse genome encodes two POT1 proteins, POT1a and POT1b possessing separation of hPOT1 functions. We performed serial transplantation of Pot1b-/- sarcomas to better understand the role of POT1b in regulating telomere length maintenance. While early-generation Pot1b-/- sarcomas initially possessed shortened telomeres, late-generation Pot1b-/- cells display markedly hyper-elongated telomeres that were recognized as damaged DNA by the Replication Protein A (RPA) complex. The RPA-ATR-dependent DNA damage response at telomeres promotes telomerase recruitment to facilitate telomere hyper-elongation. POT1b, but not POT1a, was able to unfold G-quadruplex present in hyper-elongated telomeres to repress the DNA damage response. Our findings demonstrate that the repression of the RPA-ATR DDR is conserved between POT1b and human POT1, suggesting that similar mechanisms may underly the phenotypes observed in human cancers harboring human POT1 mutations.