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The heat shock protein 90 (HSP90) family members are not only widely involved in animal cellular immune response and signal transduction pathway regulation, but also play an important role in plant development and environmental stress response. Here,we identified a HSP90 family member in Ginkgo biloba, designated as GbHSP90, which performs a dual functional role to regulate telomere stability. GbHSP90 was screened by a yeast one-hybrid library using the Ginkgo biloba telomeric DNA (TTTAGGG) 5 . Fluorescence polarization, surface plasmon resonance(SPR) and EMSA technologyies revealed a specific interaction between GbHSP90 and the double-stranded telomeric DNA via its N-CR region, with no affinity for the single-stranded telomeric DNA or human double-stranded telomeric DNA. Furthermore, yeast two-hybrid system and Split-LUC assay demonstrated that GbHSP90 can interacts with two telomere end-binding proteins:the ginkgo telomerase reverse transcriptase (GbTERT) and the ginkgo Structural Maintenance of Chromosomes protein 1 (GbSMC1). Overexpression of GbHSP90 in human 293 T and HeLa cells increased cell growth rate, the content of telomerase reverse transcriptase (TERT), and promote cell division and inhibit cell apoptosis. Our results indicated GbHSP90 have dually functions: as a telomere-binding protein that binds specifically to double-stranded telomeric DNA and as a molecular chaperone that modulates cell differentiation and apoptosis by binding to telomere protein complexes in Ginkgo biloba. This study contributes to a significantly understanding of the unique telomere complex structure and regulatory mechanisms in Ginkgo biloba, a long-lived tree species., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction . Adherence is a major virulence trait in Candida glabrata that, in many strains, depends on the EPA (epithelial adhesin) genes, which confer the ability to adhere to epithelial and endothelial cells of the host. The EPA genes are generally found at subtelomeric regions, which makes them subject to subtelomeric silencing. In C. glabrata , subtelomeric silencing depends on different protein complexes, such as silent information regulator and yKu complexes, and other proteins, such as Repressor/activator protein 1 (Rap1) and Abf1. At the EPA1 locus, which encodes the main adhesin Epa1, we previously found at least two cis -acting elements, the protosilencer Sil2126 and the negative element, that contribute to the propagation of silencing from the telomere to the subtelomeric region. Hypothesis . Abf1 binds to the regulatory regions of EPA1 and other regions at the telomere E-R, thereby negatively regulating EPA1 transcription. Aim . To determine whether Abf1 and Rap1 silencing proteins bind to previously identified cis- acting elements on the right telomere of chromosome E (E-R subtelomeric region), resulting in negative regulation of EPA1 transcription and infer Abf1 and Rap1 recognition sites in C. glabrata . Methodology . We used chromatin immunoprecipitation (ChIP) followed by quantitative PCR to determine the binding sites for Abf1 and Rap1 in the intergenic regions between EPA1 and EPA2 and HYR1 and EPA1 , and mutants were used to determine the silencing level of the EPA1 promoter region. Results . We found that Abf1 predominantly binds to the EPA1 promoter region, leading to negative regulation of EPA1 expression. Furthermore, the mutant abf1-43 , which lacks the last 43 amino acids at its C-terminal end and is defective for subtelomeric silencing, exhibits hyperadherence to epithelial cells in vitro compared to the parental strain, suggesting that EPA1 is derepressed. We also determined the motif-binding sequences for Abf1 and Rap1 in C. glabrata using data from the ChIP assays. Conclusion . Together these data indicate that Abf1 negatively regulates EPA1 expression, leading to decreased adhesion of C. glabrata to epithelial cells.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome, caused by genetic mutations that principally affect telomere biology. Approximately 35% of cases remain uncharacterised at the genetic level. To explore the genetic landscape, we conducted genetic studies on a large collection of clinically diagnosed cases of DC as well as cases exhibiting features resembling DC, referred to as 'DC-like' (DCL). This led us to identify several novel pathogenic variants within known genetic loci and in the novel X-linked gene, POLA1. In addition, we have also identified several novel variants in POT1 and ZCCHC8 in multiple cases from different families expanding the allelic series of DC and DCL phenotypes. Functional characterisation of novel POLA1 and POT1 variants, revealed pathogenic effects on protein-protein interactions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunit complexes, that are critical for telomere maintenance. ZCCHC8 variants demonstrated ZCCHC8 deficiency and signs of pervasive transcription, triggering inflammation in patients' blood. In conclusion, our studies expand the current genetic architecture and broaden our understanding of disease mechanisms underlying DC and DCL disorders., (© 2024. The Author(s).)

Retinal vascular leakage is a major event in several retinal diseases, including diabetic retinopathy (DR). In a previous study, we demonstrated that the aqueous humor concentration of Cystatin C (CST3), a physiological inhibitor of cysteine protease, is negatively correlated with the severity of diabetic macular edema. However, its function in the retina has not been clearly elucidated. In this study, we found a significant decrease in the aqueous humor concentration of CST3 with DR progression. Furthermore, we found that CST3 was expressed in retinal endothelial cells and that its expression was significantly downregulated in high glucose-treated human retinal microvascular endothelial cells (HRMECs) and the retinal vessels of oxygen-induced retinopathy (OIR) mice. Silencing CST3 expression resulted in decreased HRMEC migration and tubule formation ability. Exogenous addition of the CST3 protein significantly improved HRMEC migration and tubular formation. In-vivo experiments demonstrated that CST3 silencing induced retinal vascular leakage in WT mice, while its intravitreal injection significantly reduced retinal leakage in OIR mice. Mechanistically, CST3 promoted the expression of the downstream adhesion molecules, claudin5, VE-cadherin, and ZO-1, in retinal vascular cells by regulating the Rap1 signaling pathway. Therefore, this study revealed a novel mechanism by which CST3 improves retinal vascular function and provided evidence that it is a potential therapeutic target for retinal vascular leakage., Competing Interests: Declaration of Competing interest The authors declare that no confict of interest exists., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Extensive bodies of research are dedicated to the study of seed aging with a particular focus on the roles of reactive oxygen species (ROS), and the ensuing oxidative damage during storage, as a primary cause of seed vigor decreasing. ROS diffuse to the nucleus and damage the telomeres, resulting in a loss of genetic integrity. Protection of telomeres 1 (POT1) is a telomeric protein that binds to the telomere region, and plays an essential role in maintaining genomic stability in plants. In this study, there were totally four MsPOT1 genes obtained from alfalfa genome. Expression analysis of four MsPOT1 genes in germinated seed presented the different expressions. Four MsPOT1 genes displayed high expression levels at the early stage of seed germination, Among the four POT1 genes, it was found that MS. gene040108 was significantly up-regulated in the early germination stage of CK seeds, but down-regulated in aged seeds. RT-qPCR assays and RNA-seq data revealed that MsPOT1-X gene was significantly induced by seed aging treatment. Transgenic seeds overexpressing MsPOT1-X gene in Arabidopsis thaliana and Medicago trunctula exhibited enhanced seed vigor, telomere length, telomerase activity associated with reduced H 2 O 2 content. These results would provide a new way to understand aging stress-responsive MsPOT1 genes for genetic improvement of seed vigor. Although a key gene regulating seed vigor was identified in this study, the specific mechanism of MsPOT1-X gene regulating seed vigor needs to be further explored., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To investigate the potential role of shelterin dysfunction in naïve CD4+ T cells in the pathogenesis of Behçet's disease (BD)., Methods: Naïve CD4+ T cells were isolated from 40 BD patients and 40 sex- and age-matched healthy controls (HC). Senescent profiles, shelterin subunits expression, telomere length, telomerase activity and critical DNA damage response (DDR) were evaluated. Telomere repeat factor-2 (TRF2) silencing was conducted for further validation., Results: Compared with HC, BD patients had significantly decreased naïve CD4+ T cells, increased cell apoptosis, senescence, and productions of TNF-α and IFN-γ upon activation. Notably, BD naïve CD4+ T cells had shortened telomere, impaired telomerase activity, and expressed lower levels of shelterin subunits TRF2, TRF1- and TRF2-Interacting Nuclear Protein 2 (TIN2) and Repressor/Activator Protein 1 (RAP1). Furthermore, BD naïve CD4+ T cells exhibited significantly increased DDR, evidenced by elevated phosphorylated ataxia telangiectasia (AT) mutated (pATM), phosphorylated p53 (pp53) and p21. Finally, TRF2 silencing markedly upregulated DDR, apoptosis and proinflammatory cytokines production in HC naïve CD4+ T cells., Conclusion: Our study demonstrated that TRF2 deficiency in BD naïve CD4+ T cells promoted cell apoptosis and senescence, leading to proinflammatory cytokines overproduction. Therefore, restoring TRF2 might be a promising therapeutic strategy for BD., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Telomeres, the ends of eukaryotic linear chromosomes, are composed of repeated DNA sequences and specialized proteins, with the conserved telomeric Cdc13/CTC1-Stn1-Ten1 (CST) complex providing chromosome stability via telomere end protection and the regulation of telomerase accessibility. In this study, SIZ1 , coding for a SUMO E3 ligase, and TOP2 (a SUMO target for Siz1 and Siz2) were isolated as extragenic suppressors of Saccharomyces cerevisiae CST temperature-sensitive mutants. ten1 - sz , stn1 - sz and cdc13 - sz mutants were isolated next due to being sensitive to intracellular Siz1 dosage. In parallel, strong negative genetic interactions between mutants of CST and septins were identified, with septins being noticeably sumoylated through the action of Siz1. The temperature-sensitive arrest in these new mutants of CST was dependent on the G2/M Mad2-mediated and Bub2-mediated spindle checkpoints as well as on the G2/M Mec1-mediated DNA damage checkpoint. Our data suggest the existence of yet unknown functions of the telomeric Cdc13-Stn1-Ten1 complex associated with mitotic spindle positioning and/or assembly that could be further elucidated by studying these new ten1 - sz , stn1 - sz and cdc13 - sz mutants.

Background: It has long been observed that there are families in which non-medullary thyroid cancer (NMTC) occurs, but few syndromes and genes have been described to date. Proteins in the shelterin complex have been implied in cancer. Here, we have studied shelterin genes in families affected by NMTC (FNMTC)., Methods: We performed whole-exome sequencing (WES) in 10 affected individuals from four families with at least three affected members. Polymerase chain reaction (PCR) and Sanger sequencing were performed to search for variants in the TINF2 gene in 40 FNMTC families. TINF2 transcripts and loss of heterozygosity (LOH) were studied in several affected patients of one family., Results: We found the c.507G>T variant in heterozygosis in the TINF2 gene in one family, co-segregating in all five affected members. This variant affects the normal splicing. LOH was not observed., Conclusions: Our results reinforce the TINF2 gene as a susceptibility cause of FNMTC suggesting the importance of location of frameshift variants in TINF2 . According to our data and previous literature, TINF2 pathogenic variants appear to be a significant risk factor for the development of NMTC and/or melanoma., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Telomeric repeat containing RNA (TERRA) is a noncoding RNA that is transcribed from telomeres. Previous study showed that TERRA trans anneals by invading into the telomeric duplex to form an R-loop in mammalian cells. Here, we elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins, RAD51 and RNase H using single molecule (sm) assays. We demonstrate that TERRA trans annealing into telomeric DNA exhibits dynamic movement that is stabilized by TRF2. TERRA annealing to the telomeric duplex results in the formation of a stable triplex structure which differs from a conventional R-loop. We identified that the presence of a sub-telomeric DNA and a telomeric overhang in the form of a G-quadruplex significantly enhances TERRA annealing to telomeric duplex. We also demonstrate that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Additionally, TRF2 increases TERRA affinity to telomeric duplex and protects it from RNase H digestion. In contrast, TRF1 represses TERRA annealing to telomeric duplex and fails to provide protection against RNase H digestion. Our findings provide an in-depth molecular mechanism underpinning TERRA recruitment and annealing to the telomere., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Premature telomere shortening or telomere instability is associated with a group of rare and heterogeneous diseases collectively known as telomere biology disorders (TBDs). Here we identified two unrelated individuals with clinical manifestations of TBDs and short telomeres associated with the identical monoallelic variant c.767A>G; Y256C in RPA2 Although the replication protein A2 (RPA2) mutant did not affect ssDNA binding and G-quadruplex-unfolding properties of RPA, the mutation reduced the affinity of RPA2 with the ubiquitin ligase RFWD3 and reduced RPA ubiquitination. Using engineered knock-in cell lines, we found an accumulation of RPA at telomeres that did not trigger ATR activation but caused short and dysfunctional telomeres. Finally, both patients acquired, in a subset of blood cells, somatic genetic rescue events in either POT1 genes or TERT promoters known to counteract the accelerated telomere shortening. Collectively, our study indicates that variants in RPA2 represent a novel genetic cause of TBDs. Our results further support the fundamental role of the RPA complex in regulating telomere length and stability in humans., (© 2024 Kochman et al.; Published by Cold Spring Harbor Laboratory Press.)

Single-particle imaging and tracking can be combined with colocalization analysis to study the dynamic interactions between macromolecules in living cells. Indeed, single-particle tracking has been extensively used to study protein-DNA interactions and dynamics. Still, unbiased identification and quantification of binding events at specific genomic loci remains challenging. Herein, we describe CoPixie, a new software that identifies colocalization events between a theoretically unlimited number of imaging channels, including single-particle movies. CoPixie is an object-based colocalization algorithm that relies on both pixel and trajectory overlap to determine colocalization between molecules. We employed CoPixie with live-cell single-molecule imaging of telomerase and telomeres, to test the model that cancer-associated POT1 mutations facilitate telomere accessibility. We show that POT1 mutants Y223C, D224N or K90E increase telomere accessibility for telomerase interaction. However, unlike the POT1-D224N mutant, the POT1-Y223C and POT1-K90E mutations also increase the duration of long-lasting telomerase interactions at telomeres. Our data reveal that telomere elongation in cells expressing cancer-associated POT1 mutants arises from the dual impact of these mutations on telomere accessibility and telomerase retention at telomeres. CoPixie can be used to explore a variety of questions involving macromolecular interactions in living cells, including between proteins and nucleic acids, from multicolor single-particle tracks., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Inappropriate homology-directed repair (HDR) of telomeres results in catastrophic telomere loss and aberrant chromosome fusions, leading to genome instability. We have previously shown that the TRF2-RAP1 heterodimer protects telomeres from engaging in aberrant telomere HDR. Cells lacking the basic domain of TRF2 and functional RAP1 display HDR-mediated telomere clustering, resulting in the formation of ultrabright telomeres (UTs) and massive chromosome fusions. Using purified proteins, we uncover three distinct molecular pathways that the TRF2-RAP1 heterodimer utilizes to protect telomeres from engaging in aberrant HDR. We show mechanistically that TRF2-RAP1 inhibits RAD51-initiated telomeric D-loop formation. Both the TRF2 basic domain and RAP1-binding to TRF2 are required to block RAD51-mediated homology search. TRF2 recruits the BLM helicase to telomeres through its TRFH domain to promote BLM-mediated unwinding of telomere D-loops. In addition, TRF2-RAP1 inhibits BLM-DNA2-mediated 5' telomere end resection, preventing the generation of 3' single-stranded telomere overhangs necessary for RAD51-dependent HDR. Importantly, cells expressing BLM mutants unable to interact with TRF2 accumulate telomere D-loops and UTs. Our findings uncover distinct molecular mechanisms coordinated by TRF2-RAP1 to protect telomeres from engaging in aberrant HDR., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

POT1 is the second most frequently reported gene (after CDKN2A ) in familial melanoma. Pathogenic variants are associated with earlier onset and/or multiple primary melanomas (MPMs). To date, POT1 phenotypical reports have been largely restricted to associated malignancies, and description of the dermatological landscape has been limited. We identified 10 variants in n=18 of 384 (4.7%) unrelated individuals (n=13 MPMs; n=5 single primary melanomas) of European ancestry. Five variants were rare (minor allele frequency <0.001) or novel (two loss-of-function (LOF), one splice acceptor and two missense) and were predicted to be functionally significant, in five unrelated probands with MPMs (≥3 melanomas). We performed three-dimensional total body photography on both individuals with confirmed pathogenic LOF variants to characterise the dermatological phenotype. Total body naevus counts (≥2 mm diameter) were significantly higher (p=7.72 ×10-12 ) in carriers compared with a control population. Majority of naevi were on the probands' back and lower limb regions, where only mild to moderate ultraviolet (UV) damage was observed. Conversely, the head/neck region, where both probands exhibited severe UV damage, had comparably fewer naevi. We hypothesise that carriage of functionally significant POT1 variants is associated with increased naevus counts generally, and naevi >5 mm in diameter specifically and the location of these are independent of UV damage., Competing Interests: Competing interests: PS is a shareholder of MoleMap Limited and e-derm consult and undertakes regular teledermatological reporting for both companies. PS is a medical consultant for Canfield Scientific and Blaze Bioscience MoleMap Australia Limited, and a medical advisor for First Derm. No other authors have conflicts to declare., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol β, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The Drosophila Id gene extramacrochaetae (emc) is required during Drosophila eye development for proper cell fate specification within the R7 equivalence group. Without emc, R7 cells develop like R1/6 cells, and there are delays and deficits in differentiation of non-neuronal cone cells. Although emc encodes an Inhibitor of DNA-binding (Id) protein that is known to antagonize proneural bHLH protein function, no proneural gene is known for R7 or cone cell fates. These fates are also independent of daughterless (da), which encodes the ubiquitous E protein heterodimer partner of proneural bHLH proteins. We report here that the effects of emc mutations disappear in the absence of da, and are partially mimicked by forced expression of Da dimers, indicating that emc normally restrains da from interfering with R7 and cone cell specification, as occurs in emc mutants. emc, and da, regulate three known contributors to R7 fate, which are Notch signaling, Rap1, and Sevenless. R7 specification is partially restored to emc mutant cells by mutation of RapGap1, confirming that Rap1 activity, in addition to Notch activity, is a critical target of emc. These findings exemplify how mutations of an Id protein gene can affect processes that do not require any bHLH protein, by restraining Da activity within physiological bounds., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Although previous studies have reported that pre-mRNA splicing factors (SFs) are involved in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR), their exact role in promoting HR remains poorly understood. Here, we showed that SART1, an SF upregulated in several types of cancer, promotes DSB end resection, an essential first step of HR. The resection-promoting function of SART1 requires phosphorylation at threonine 430 and 695 by ATM/ATR. SART1 is recruited to DSB sites in a manner dependent on transcription and its RS domain. SART1 is epistatic with BRCA1, a major HR factor, in the promotion of resection, especially transcription-associated resection in the G2 phase. SART1 and BRCA1 accumulate at DSB sites in an interdependent manner, and epistatically counteract the resection blockade posed by 53BP1 and RIF1. Furthermore, chromosome analysis demonstrated that SART1 and BRCA1 epistatically suppressed genomic alterations caused by DSB misrepair in the G2 phase. Collectively, these results indicate that SART1 and BRCA1 cooperatively facilitate resection of DSBs arising in transcriptionally active genomic regions in the G2 phase, thereby promoting faithful repair by HR, and suppressing genome instability., (© 2024. The Author(s).)

To assess the immediate responses of the yeast cells to telomere defects, we employed the auxin-inducible degron (AID) enabling rapid depletion of essential (Rap1, Tbf1, Cdc13, Stn1) and non-essential (Est1, Est2, Est3) telomeric proteins. Using two variants of AID systems, we show that most of the studied proteins are depleted within 10-30 min after the addition of auxin. As expected, depletion of essential proteins yields nondividing cells, provided that the strains are cultivated in an appropriate carbon source and at temperatures lower than 28°C. Cells with depleted Cdc13 and Stn1 exhibit extension of the single-stranded overhang as early as 3 h after addition of auxin. Notably, prolonged incubation of strains carrying AID-tagged essential proteins in the presence of auxin resulted in the appearance of auxin-resistant clones, caused at least in part by mutations within the OsTIR1 gene. Upon assessing the length of telomeres in strains carrying AID-tagged non-essential telomeric proteins, we found that the depletion of Est1 and Est3 leads to auxin-dependent telomere shortening. However, the EST3-AID strain had slightly shorter telomeres even in the absence of auxin. Furthermore, a strain with the AID-tagged version of Est2 (catalytic subunit of telomerase) not only had shorter telomeres in the absence of auxin but also did not exhibit auxin-dependent telomere shortening. Our results demonstrate that while AID can be useful in assessing immediate cellular responses to telomere deprotection, each strain must be carefully evaluated for the effect of AID-tag on the properties of the protein of interest., (© 2024 John Wiley & Sons Ltd.)

Protection of Telomeres Protein 1 (POT1) protein is an essential subunit of the shelterin telomere binding complex, regulating telomere length. Some POT1 gene pathogenic variants (PV) lead to telomere elongation, genomic instability and higher risk of cancer. POT1 tumour predisposition syndrome (POT1-TPD) has autosomal dominant inheritance and unknown penetrance. It is associated with increased risk of cutaneous melanoma, chronic lymphocytic leukaemia, angiosarcoma and gliomas. In this work, we aim to describe a broader cancer phenotype related to POT1-TPD, in three families (two with a four generation pedigree, one with a five generation pedigree). The three index cases were referred to our oncogenetic centre for genetic counselling due to their personal history of cancer. Two underwent clinical exome sequencing of 4,867 genes associated with Mendelian genetic diseases, and another underwent gene panel sequencing including POT1, which identified three different POT1 PV: NC&#95;000007.14(NM&#95;015450.2):c.349C>T; NC&#95;000007.14(NM&#95;015450.2):c.233T>C and NC&#95;000007.14(NM&#95;015450.2):c.818G>A; already described in the literature. Referenced relatives, did a target genetic test (according to the POT1 PV identified in the family). In total, 37 individuals were tested (51.4% females), median age of 46 (22-81) years, with POT1 PV detected in 22. POT1-TPD was observed, but also a higher incidence of other cancers (other sarcomas, papillary thyroid cancer, early onset prostate cancer and leukaemia). These findings contribute to an increase in our knowledge about POT1 PV, and it can play a role in the definition of future POT1 PV screening criteria, POT1 carrier surveillance protocols (possibly considering screening for all types of sarcomas) and in genetic counselling., (© 2024. The Author(s).)

The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.

Recently in Cell, Cai et al. 1 reported how phosphorylation of human shelterin protein POT1 allows it to recruit the telomeric C-rich strand replication machinery, providing mechanistic insights into an understudied area of telomere biology with implications for telomere biology disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Telomeres prevent damage to coding DNA as end-nucleotides are lost during mitosis. Mutations in telomere maintenance genes cause excessive telomere shortening, a condition known as short telomere syndrome (STS). One hepatic manifestation documented in STS is porto-sinusoidal vascular disorder (PSVD)., Methods: As the etiology of many cases of PSVD remains unknown, this study explored the extent to which short telomeres are present in patients with idiopathic PSVD., Results: This monocentric cross-sectional study included patients with histologically defined idiopathic PSVD. Telomere length in 6 peripheral blood leukocyte subpopulations was assessed using fluorescent in situ hybridization and flow cytometry. Variants of telomere-related genes were identified using high-throughput exome sequencing. In total, 22 patients were included, of whom 16 (73%) had short (9/22) or very short (7/22) telomeres according to age-adjusted reference ranges. Fourteen patients (64%) had clinically significant portal hypertension. Shorter telomeres were more frequent in males (p = 0.005) and patients with concomitant interstitial lung disease (p < 0.001), chronic kidney disease (p < 0.001), and erythrocyte macrocytosis (p = 0.007). Portal hypertension (p = 0.021), low serum albumin level (p < 0.001), low platelet count (p = 0.007), and hyperbilirubinemia (p = 0.053) were also associated with shorter telomeres. Variants in known STS-related genes were identified in 4 patients with VSTel and 1 with STel., Conclusions: Short and very short telomeres were highly prevalent in patients with idiopathic PSVD, with 31% presenting with variants in telomere-related genes. Telomere biology may play an important role in vascular liver disease development. Clinicians should consider measuring telomeres in any patient presenting with PSVD., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Telomeres protect chromosome ends from unscheduled DNA repair, including from the MRN (MRE11, RAD50, NBS1) complex, which processes double-stranded DNA breaks (DSBs) via activation of the ATM kinase, promotes DNA end-tethering aiding the non-homologous end-joining (NHEJ) pathway, and initiates DSB resection through the MRE11 nuclease. A protein motif (MIN, for MRN inhibitor) inhibits MRN at budding yeast telomeres by binding to RAD50 and evolved at least twice, in unrelated telomeric proteins Rif2 and Taz1. We identify the iDDR motif of human shelterin protein TRF2 as a third example of convergent evolution for this telomeric mechanism for binding MRN, despite the iDDR lacking sequence homology to the MIN motif. CtIP is required for activation of MRE11 nuclease action, and we provide evidence for binding of a short C-terminal region of CtIP to a RAD50 interface that partly overlaps with the iDDR binding site, indicating that the interaction is mutually exclusive. In addition, we show that the iDDR impairs the DNA binding activity of RAD50. These results highlight direct inhibition of MRN action as a crucial role of telomeric proteins across organisms and point to multiple mechanisms enforced by the iDDR to disable the many activities of the MRN complex., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Although DNA repair mechanisms function to maintain genomic integrity, in cancer cells these mechanisms may negatively affect treatment efficiency. The strategy of targeting cancer cells via inhibiting DNA damage repair has been successfully used in breast and ovarian cancer using PARP inhibitors. Unfortunately, such strategies have not yet yielded results in liver cancer. Hepatocellular carcinoma (HCC), the most common type of liver cancer, is a treatment-resistant malignancy. Despite the development of guided therapies, treatment regimens for advanced HCC patients still fall short of the current need and significant problems such as cancer relapse with resistance still exist. In this paper, we targeted telomeric replication protein CTC1, which is responsible for telomere maintenance., Methods: CTC expression was analyzed using tumor and matched-tissue RNA-sequencing data from TCGA and GTEx. In HCC cell lines, q-RT-PCR and Western blotting were used to detect CTC1 expression. The knock-down of CTC1 was achieved using lentiviral plasmids. The effects of CTC1 silencing on HCC cells were analyzed by flow cytometry, MTT, spheroid and colony formation assays., Results: CTC1 is significantly downregulated in HCC tumor samples. However, CTC1 protein levels were higher in sorafenib-resistant cell lines compared to the parental groups. CTC1 inhibition reduced cell proliferation in sorafenib-resistant HCC cell lines and diminished their spheroid and colony forming capacities. Moreover, these cells were more sensitive to single and combined drug treatment with G4 stabilizer RHPS4 and sorafenib., Conclusion: Our results suggest that targeting CTC1 might be a viable option for combinational therapies designed for sorafenib resistant HCC patients., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Plant-specific transcriptional regulators called TELOMERE REPEAT BINDING proteins (TRBs) combine two DNA-binding domains, the GH1 domain, which binds to linker DNA and is shared with H1 histones, and the Myb/SANT domain, which specifically recognizes the telobox DNA-binding site motif. TRB1, TRB2, and TRB3 proteins recruit Polycomb group complex 2 (PRC2) to deposit H3K27me3 and JMJ14 to remove H3K4me3 at gene promoters containing telobox motifs to repress transcription. Here, we demonstrate that TRB4 and TRB5, two related paralogs belonging to a separate TRB clade conserved in spermatophytes, regulate the transcription of several hundred genes involved in developmental responses to environmental cues. TRB4 binds to several thousand sites in the genome, mainly at transcription start sites and promoter regions of transcriptionally active and H3K4me3-marked genes, but, unlike TRB1, it is not enriched at H3K27me3-marked gene bodies. However, TRB4 can physically interact with the catalytic components of PRC2, SWINGER, and CURLY LEAF (CLF). Unexpectedly, we show that TRB4 and TRB5 are required for distinctive phenotypic traits observed in clf mutant plants and thus function as transcriptional activators of several hundred CLF-controlled genes, including key flowering genes. We further demonstrate that TRB4 shares multiple target genes with TRB1 and physically and genetically interacts with members of both TRB clades. Collectively, these results reveal that TRB proteins engage in both positive and negative interactions with other members of the family to regulate plant development through both PRC2-dependent and -independent mechanisms., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In fission yeast lacking the telomere binding protein, Taz1, replication forks stall at telomeres, triggering deleterious downstream events. Strand invasion from one taz1Δ telomeric stalled fork to another on a separate (nonsister) chromosome leads to telomere entanglements, which are resolved in mitosis at 32°C; however, entanglement resolution fails at ≤20°C, leading to cold-specific lethality. Previously, we found that loss of the mitotic function of Rif1, a conserved DNA replication and repair factor, suppresses cold sensitivity by promoting resolution of entanglements without affecting entanglement formation. To understand the underlying pathways of mitotic entanglement resolution, we performed a series of genome-wide synthetic genetic array screens to generate a comprehensive list of genetic interactors of taz1Δ and rif1Δ. We modified a previously described screening method to ensure that the queried cells were kept in log phase growth. In addition to recapitulating previously identified genetic interactions, we find that loss of genes encoding components of the nuclear pore complex (NPC) promotes telomere disentanglement and suppresses taz1Δ cold sensitivity. We attribute this to more rapid anaphase midregion nuclear envelope (NE) breakdown in the absence of these NPC components. Loss of genes involved in lipid metabolism reverses the ability of rif1+ deletion to suppress taz1Δ cold sensitivity, again pinpointing NE modulation. A rif1+ separation-of-function mutant that specifically loses Rif1's mitotic functions yields similar genetic interactions. Genes promoting membrane fluidity were enriched in a parallel taz1+ synthetic lethal screen at permissive temperature, cementing the idea that the cold specificity of taz1Δ lethality stems from altered NE homeostasis., Competing Interests: Conflicts of interest The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. Nevan Krogan has a financially compensated consulting agreement with Maze Therapeutics. Nevan is the President and is on the Board of Directors of Rezo Therapeutics, and he is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The function of TERRA in the regulation of telomerase in human cells is still debated. While TERRA interacts with telomerase, how it regulates telomerase function remains unknown. Here, we show that TERRA colocalizes with the telomerase RNA subunit hTR in the nucleoplasm and at telomeres during different phases of the cell cycle. We report that TERRA transcripts relocate away from chromosome ends during telomere lengthening, leading to a reduced number of telomeric TERRA-hTR molecules and consequent increase in "TERRA-free" telomerase molecules at telomeres. Using live-cell imaging and super-resolution microscopy, we show that upon transcription, TERRA relocates from its telomere of origin to long chromosome ends. Furthermore, TERRA depletion by antisense oligonucleotides promoted hTR localization to telomeres, leading to increased residence time and extended half-life of hTR molecules at telomeres. Overall, our findings indicate that telomeric TERRA transcripts inhibit telomere elongation by telomerase acting in trans, impairing telomerase access to telomeres that are different from their chromosome end of origin.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy. 10% to 15% of individuals show familial clustering with three or more affected members, but the factors underlying this risk are unknown. In a group of recently studied individuals with POT1 pathogenic variants and ultra-long telomere length, PTC was the second most common solid tumor. We tested whether variants in POT1 and four other telomere-maintenance genes associated with familial cancer underlie PTC susceptibility. Among 470 individuals, we identified pathogenic or likely pathogenic variants in three genes encoding telomere-binding proteins: POT1, TINF2, and ACD. They were found in 4.5% and 1.5% of familial and unselected cases, respectively. Individuals harboring these variants had ultra-long telomere length, and 15 of 18 (83%) developed other cancers, of which melanoma, lymphoma, and sarcoma were most common. Among individuals with PTC and melanoma, 22% carried a deleterious germline variant, suggesting that a long telomere syndrome might be clinically recognizable. Successive generations had longer telomere length than their parents and, at times, developed more cancers at younger ages. Tumor sequencing identified a single oncogenic driver, BRAF p.Val600Glu, in 10 of 10 tumors studied, but no telomere-maintenance mechanism, including at the TERT promoter. These data identify a syndromic subset of PTCs with locus heterogeneity and telomere lengthening as a convergent mechanism. They suggest these germline variants lower the threshold to cancer by obviating the need for an acquired telomere-maintenance mechanism in addition to sustaining the longevity of oncogenic mutations., Competing Interests: Declaration of interests M.A. has a pending provisional patent application on the use of telomere length to assess cancer risk., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork (RF) stalling and consequently, telomere entanglements that stretch between segregating mitotic chromosomes. At ≤20 °C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, hinders the resolution of telomere entanglements without affecting their formation. At mitosis, local nuclear envelope (NE) breakdown occurs in the cell's midregion. Here we demonstrate that entanglement resolution occurs in the cytoplasm following this NE breakdown. However, in response to taz1Δ telomeric entanglements, Rif1 delays midregion NE breakdown at ≤20 °C, in turn disfavoring entanglement resolution. Moreover, Rif1 overexpression in an otherwise wild-type setting causes cold-specific NE defects and lethality, which are rescued by membrane fluidization. Hence, NE properties confer the cold-specificity of taz1Δ lethality, which stems from postponement of NE breakdown. We propose that such postponement promotes clearance of simple stalled RFs, but resolution of complex entanglements (involving strand invasion between nonsister telomeres) requires rapid exposure to the cytoplasm., (© 2024. The Author(s).)

The telomere sequence, TTAGGG, is conserved across all vertebrates and plays an essential role in suppressing the DNA damage response by binding a set of proteins termed shelterin. Changes in the telomere sequence impair shelterin binding, initiate a DNA damage response, and are toxic to cells. Here we identify a family with a variant in the telomere template sequence of telomerase, the enzyme responsible for telomere elongation, that led to a non-canonical telomere sequence. The variant is inherited across at least one generation and one family member reports no significant medical concerns despite ~9% of their telomeres converting to the novel sequence. The variant template disrupts telomerase repeat addition processivity and decreased the binding of the telomere-binding protein POT1. Despite these disruptions, the sequence is readily incorporated into cellular chromosomes. Incorporation of a variant sequence prevents POT1-mediated inhibition of telomerase suggesting that incorporation of a variant sequence may influence telomere addition. These findings demonstrate that telomeres can tolerate substantial degeneracy while remaining functional and provide insights as to how incorporation of a non-canonical telomere sequence might alter telomere length dynamics., (© 2024. The Author(s).)

Introduction: Search for new clinical biomarkers targets in prostate cancer (PC) is urgent. Telomeres might be one of these targets. Telomeres are the extremities of linear chromosomes, essential for genome stability and control of cell divisions. Telomere homeostasis relies on the proper functioning of shelterin and CST complexes. Telomeric dysfunction and abnormal expression of its components are reported in most cancers and are associated with PC. Despite this, there are only a few studies about the expression of the main telomere complexes and their relationship with PC progression. We aimed to evaluate the role of shelterin (POT1, TRF2, TPP1, TIN2, and RAP1) and CST (CTC1, STN1, and TEN1) genes and telomere length in the progression of PC., Methods: We evaluated genetic alterations of shelterin and CST by bioinformatics in samples of localized (n = 499) and metastatic castration-resistant PC (n = 444). We also analyzed the expression of the genes using TCGA (localized PC n = 497 and control n = 152) and experimental approaches, with surgical specimens (localized PC n = 81 and BPH n = 10) and metastatic cell lines (LNCaP, DU145, PC3 and PNT2 as control) by real-time PCR. Real-time PCR also determined the telomere length in the same experimental samples. All acquired data were associated with clinical parameters., Results: Genetic alterations are uncommon in PC, but POT1, TIN2, and TEN1 showed significantly more amplifications in the metastatic cancer. Except for CTC1 and TEN1, which are differentially expressed in localized PC samples, we did not detect an expression pattern relative to control and cell lines. Nevertheless, except for TEN1, the upregulation of all genes is associated with a worse prognosis in localized PC. We also found that increased telomere length is associated with disease aggressiveness in localized PC., Conclusion: The upregulation of shelterin and CST genes creates an environment that favors telomere elongation, giving selective advantages for localized PC cells to progress to more aggressive stages of the disease., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Telomeres are inert DNA sequences (TTAGGG) at the end of chromosomes that protect genetic information and maintain DNA integrity. Emerging evidence has demonstrated that telomere alteration can be closely related to occupational exposure and the development of various disease conditions, including cancer. However, the functions and underlying molecular mechanisms of telomere alteration and shelterin dysregulation after welding fume exposures have not been broadly defined. In this study, we analyzed telomere length and shelterin complex proteins in peripheral blood mononuclear cells (PBMCs) and in lung tissue recovered from male Sprague-Dawley rats following exposure by intratracheal instillation (ITI) to 2 mg/rat of manual metal arc-stainless steel (MMA-SS) welding fume particulate or saline (vehicle control). PBMCs and lung tissue were harvested at 30 d after instillation. Our study identified telomere elongation and shelterin dysregulation in PBMCs and lung tissue after welding fume exposure. Mechanistically, telomere elongation was independent of telomerase reverse transcriptase (TERT) activation. Collectively, our findings demonstrated that welding fume-induced telomere elongation was (a) TERT-independent and (b) associated with shelterin complex dysregulation. It is possible that an alteration of telomere length and its regulatory proteins may be utilized as predictive biomarkers for various disease conditions after welding fume exposure. This needs further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Telomeres, potential biomarkers of aging, are known to shorten with continued cigarette smoke exposure. In order to further investigate this process and its impact on cellular stress and inflammation, we used an in vitro model with cigarette smoke extract (CSE) and observed the downregulation of telomere stabilizing TRF2 and POT1 genes after CSE treatment. hTERT is a subunit of telomerase and a well-known oncogenic marker, which is overexpressed in over 85% of cancers and may contribute to lung cancer development in smokers. We also observed an increase in hTERT and ISG15 expression levels after CSE treatment, as well as increased protein levels revealed by immunohistochemical staining in smokers' lung tissue samples compared to non-smokers. The effects of ISG15 overexpression were further studied by quantifying IFN-γ , an inflammatory protein induced by ISG15 , which showed greater upregulation in smokers compared to non-smokers. Similar changes in gene expression patterns for TRF2 , POT1 , hTERT , and ISG15 were observed in blood and buccal swab samples from smokers compared to non-smokers. The results from this study provide insight into the mechanisms behind smoking causing telomere shortening and how this may contribute to the induction of inflammation and/or tumorigenesis, which may lead to comorbidities in smokers.

Background: Gastric cancer (GC) is frequently diagnosed at advanced stages, when cancer cells have already metastasized. Therefore, patients with GC have a low survival rate and poor prognosis even after treatment., Methods: We downloaded GC-related RNA sequencing (RNA-Seq) data, copy number variation (CNV) data, and clinical data for bioinformatics analysis to screen prognostic genes of GC. Single-sample gene set enrichment analysis and survival analyses were performed on the RNA-Seq data, and differential and correlation analyses were conducted on the CNV data to obtain CNV-driven differentially expressed genes (DEGs). Prognostic genes were identified through univariate Cox analyses of the CNV-driven DEGs, combined with the clinical data. F2R like thrombin or trypsin receptor 3 ( F2RL3 ) was finally selected for verification after functional and survival analyses of the prognostic genes., Results: F2RL3 expression was lower in paracancer tissue than in GC tissue, and lower in GES-1 gastric epithelial cells than in GC cells. The cell culture supernatants from F2RL3 -knockdown GC cells were collected and used to culture human umbilical vein endothelial cells (HUVECs). It was observed that F2RL3 enhanced the activity, metastasis, invasion, and angiogenesis of GC cells; promoted the epithelial-mesenchymal transition (EMT) of GC cells; and impacted the Ras-associated protein 1 (Rap1)/mitogen-activated protein kinase (MAPK) pathway. To further explore the involvement of the Rap1/MAPK pathway in GC development, a pathway activator was added to GC cells with knockdown of F2RL3 expression. This pathway activator not only enhanced the activity, invasion, and migration of GC cells but also promoted the EMT and blood vessel formation., Conclusions: F2RL3 regulates the angiogenesis and EMT of GC cells through the Rap1/MAPK pathway, thus influencing the onset and progression of GC., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

It is unclear how telomere-binding protein TPP1 interacts with human telomerase reverse transcriptase (hTERT) and influences cervical cancer development and progression. This study included all eligible 156 cervical cancers diagnosed during 2003-2008 and followed up through 2014, 102 cervical intraepithelial neoplasia (CIN) patients, and 16 participants with normal cervix identified at the same period. Correlation of expression of TPP1 and hTERT in these lesions was assessed using Kappa statistics. TPP1 was knocked down by siRNA in three cervical cancer cell lines. We assessed mRNA expression using quantitative real-time polymerase chain reaction and protein expression using tissue microarray-based immunohistochemical staining. We further analyzed the impact of TPP1 expression on the overall survival of cervical cancer patients by calculating the hazard ratio (HR) with 95% confidence intervals (CIs) using the multivariable-adjusted Cox regression model. Compared to the normal cervix, high TPP1expression was significantly associated with CIN 3 and cervical cancers (P<0.001 for both). Expressions of TPP1 and hTERT were highly correlated in CIN 3 (Kappa statistics = 0.50, P = 0.005), squamous cell carcinoma (Kappa statistics = 0.22, P = 0.011), and adenocarcinoma/adenosquamous carcinoma (Kappa statistics = 0.77, P = 0.001). Mechanistically, knockdown of TPP1 inhibited the expression of hTERT in both mRNA and protein levels. High expression of TPP1 (HR = 2.61, 95% CI 1.23-5.51) and co-high expression of TPP1 and hTERT (HR = 2.38, 95% CI 1.28-4.43) were independently associated with worse survival in cervical cancer patients. TPP1 and hTERT expression was correlated and high expression of TPP1 was associated with high risk of CIN 3 and cervical cancer and could predict a worse survival in cervical cancer., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang 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.)

Background:  Neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive, inherited, lysosomal, and neurodegenerative diseases that causes progressive dementia, seizures, movement disorders, language delay/regression, progressive visual failure, and early death. Neuronal ceroid lipofuscinosis type 2 (CLN2), caused by biallelic pathogenic variants of the TPP1 gene, is the only NCL with an approved targeted therapy. The laboratory diagnosis of CLN2 is established through highly specific tests, leading to diagnostic delays and eventually hampering the provision of specific treatment for patients with CLN2. Epilepsy is a common and clinically-identifiable feature among NCLs, and seizure onset is the main driver for families to seek medical care., Objective:  To evaluate the results of the Latin America Epilepsy and Genetics Program, an epilepsy gene panel, as a comprehensive tool for the investigation of CLN2 among other genetic causes of epilepsy., Methods:  A total of 1,284 patients with epilepsy without a specific cause who had at least 1 symptom associated with CLN2 were screened for variants in 160 genes associated with epilepsy or metabolic disorders presenting with epilepsy through an epilepsy gene panel., Results:  Variants of the TPP1 gene were identified in 25 individuals (1.9%), 21 of them with 2 variants. The 2 most frequently reported variants were p.Arg208* and p.Asp276Val, and 2 novel variants were detected in the present study: p.Leu308Pro and c.89 + 3G > C Intron 2., Conclusion:  The results suggest that these genetic panels can be very useful tools to confirm or exclude CLN2 diagnosis and, if confirmed, provide disease-specific treatment for the patients., Competing Interests: EB and DRFV are BioMarin Brasil Farmacêutica LTDA employees and/or hold company stocks. FK is founder and shareholder at Mendelics Análise Genômica SA. AMP and AP reported speaker fees/payments from BioMarin Brasil Farmacêutica LTDA., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution 4.0 International License, permitting copying and reproduction so long as the original work is given appropriate credit (https://creativecommons.org/licenses/by/4.0/).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate and overcome replication stress, enabling them to continue replication. One effective strategy to overcome stalled replication involves skipping the DNA lesion using a specialized polymerase known as PrimPol, which reinitiates DNA synthesis downstream of the damage. However, the mechanism regulating PrimPol repriming is largely unclear. In this study, we observe that knockdown of STN1 or CTC1, components of the CTC1/STN1/TEN1 complex, leads to enhanced replication progression following UV exposure. We find that such increased replication is dependent on PrimPol, and PrimPol recruitment to stalled forks increases upon CST depletion. Moreover, we find that p21 is upregulated in STN1-depleted cells in a p53-independent manner, and p21 depletion restores normal replication rates caused by STN1 deficiency. We identify that p21 interacts with PrimPol, and STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. Our findings reveal a previously undescribed interplay between CST, PrimPol and p21 in promoting repriming in response to stalled replication, and shed light on the regulation of PrimPol repriming at stalled forks., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ovarian cancer, more precisely high-grade serous ovarian cancer, is one of the most lethal age-independent gynecologic malignancies in women worldwide, regardless of age. There is mounting evidence that there is a link between telomeres and the RIF1 protein and the proliferation of cancer cells. Telomeres are hexameric (TTAGGG) tandem repeats at the tip of chromosomes that shorten as somatic cells divide, limiting cell proliferation and serving as an important barrier in preventing cancer. RIF1 (Replication Time Regulation Factor 1) plays, among other factors, an important role in the regulation of telomere length. Interestingly, RIF1 appears to influence the DNA double-strand break (DSB) repair pathway. However, detailed knowledge regarding the interplay between RIF1 and telomeres and their degree of engagement in epithelial ovarian cancer (EOC) is still elusive, despite the fact that such knowledge could be of relevance in clinical practice to find novel biomarkers. In this review, we provide an update of recent literature to elucidate the relation between telomere biology and the RIF1 protein during the development of ovarian cancer in women.

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., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

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., (© 2024. The Author(s).)

Telomeres act as the protective caps of eukaryotic linear chromosomes; thus, proper telomere maintenance is crucial for genome stability. Successful telomere replication is a cornerstone of telomere length regulation, but this process can be fraught due to the many intrinsic challenges telomeres pose to the replication machinery. In addition to the famous "end replication" problem due to the discontinuous nature of lagging strand synthesis, telomeres require various telomere-specific steps for maintaining the proper 3' overhang length. Bulk telomere replication also encounters its own difficulties as telomeres are prone to various forms of replication roadblocks. These roadblocks can result in an increase in replication stress that can cause replication forks to slow, stall, or become reversed. Ultimately, this leads to excess single-stranded DNA (ssDNA) that needs to be managed and protected for replication to continue and to prevent DNA damage and genome instability. RPA and CST are single-stranded DNA-binding protein complexes that play key roles in performing this task and help stabilize stalled forks for continued replication. The interplay between RPA and CST, their functions at telomeres during replication, and their specialized features for helping overcome replication stress at telomeres are the focus of this review.

Mammalian telomeres have evolved safeguards to prevent their recognition as DNA double-stranded breaks by suppressing the activation of various DNA sensing and repair proteins. We have shown that the telomere-binding proteins TRF2 and RAP1 cooperate to prevent telomeres from undergoing aberrant homology-directed recombination by mediating t-loop protection. Our recent findings also suggest that mammalian telomere-binding proteins interact with the nuclear envelope to maintain chromosome stability. RAP1 interacts with nuclear lamins through KU70/KU80, and disruption of RAP1 and TRF2 function result in nuclear envelope rupture, promoting telomere-telomere recombination to form structures termed ultrabright telomeres. In this review, we discuss the importance of the interactions between shelterin components and the nuclear envelope to maintain telomere homeostasis and genome stability., (© 2023 Wiley Periodicals LLC.)

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.

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.

Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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 Mg 2+ , 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., (© 2023. The Author(s).)