Telomere Length Control Research Articles

Acclimation during Embryogenesis Remodulates Telomerase Activity and Gene Expression in Baikal Whitefish Larvae, Mitigating the Effects of Acute Temperature Stress.

Acclimation through the hormesis effect increases the plasticity of organisms, which has been shown for many ectothermic animals, including fish. We investigated the effect of temperature acclimation in Baikal whitefish Coregonus baicalensis (Dybowski, 1874). Telomere length, telomerase activity, and the expression of genes, whose products are involved in the regulation of telomere length and defense against reactive oxygen species, were selected to assess the state of the larvae. Acclimation and acute temperature stress (+12 °C) had no effect on telomere length, but altered telomerase activity (acclimation decreased it; stress increased it) and the levels of genes expression. Under stress, the expression of superoxide dismutase genes was increased in acclimated larvae and that of glutathione peroxidases in non-acclimated larvae, which may indicate lower reactive oxygen species formation and slower antioxidant responses in acclimated fish. The expression of some telomere-related genes was reduced under temperature stress, but the expression of the tzap and smg genes, whose products improve the control of telomere length by preventing them from lengthening or shortening, was increased in acclimated individuals. The data obtained indicate a positive effect of acclimation on the state of the Baikal whitefish larvae by remodulation of their telomerase activity and the transcriptional profile.

Profiling Tel1 Signaling Reveals a Non-Canonical Motif Targeting DNA Repair and Telomere Control Machineries.

The stability of the genome relies on Phosphatidyl Inositol 3-Kinase-related Kinases (PIKKs) that sense DNA damage and trigger elaborate downstream signaling responses. In S. cerevisiae, the Tel1 kinase (ortholog of human ATM) is activated at DNA double strand breaks (DSBs) and short telomeres. Despite the well-established roles of Tel1 in the control of telomere maintenance, suppression of chromosomal rearrangements, activation of cell cycle checkpoints, and repair of DSBs, the substrates through which Tel1 controls these processes remain incompletely understood. Here we performed an in depth phosphoproteomic screen for Tel1-dependent phosphorylation events. To achieve maximal coverage of the phosphoproteome, we developed a scaled-up approach that accommodates large amounts of protein extracts and chromatographic fractions. Compared to previous reports, we expanded the number of detected Tel1-dependent phosphorylation events by over 10-fold. Surprisingly, in addition to the identification of phosphorylation sites featuring the canonical motif for Tel1 phosphorylation (S/T-Q), the results revealed a novel motif (D/E-S/T) highly prevalent and enriched in the set of Tel1-dependent events. This motif is unique to Tel1 signaling and not shared with the Mec1 kinase, providing clues to how Tel1 plays specialized roles in DNA repair and telomere length control. Overall, these findings define a Tel1-signaling network targeting numerous proteins involved in DNA repair, chromatin regulation, and telomere maintenance that represents a framework for dissecting the molecular mechanisms of Tel1 action.

Arabidopsis AN3 and OLIGOCELLULA genes link telomere maintenance mechanisms with cell division and expansion control.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a -deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

Telomere maintenance and the DNA damage response: a paradoxical alliance.

Telomeres are the protective caps at the ends of linear chromosomes of eukaryotic organisms. Telomere binding proteins, including the six components of the complex known as shelterin, mediate the protective function of telomeres. They do this by suppressing many arms of the canonical DNA damage response, thereby preventing inappropriate fusion, resection and recombination of telomeres. One way this is achieved is by facilitation of DNA replication through telomeres, thus protecting against a "replication stress" response and activation of the master kinase ATR. On the other hand, DNA damage responses, including replication stress and ATR, serve a positive role at telomeres, acting as a trigger for recruitment of the telomere-elongating enzyme telomerase to counteract telomere loss. We postulate that repression of telomeric replication stress is a shared mechanism of control of telomerase recruitment and telomere length, common to several core telomere binding proteins including TRF1, POT1 and CTC1. The mechanisms by which replication stress and ATR cause recruitment of telomerase are not fully elucidated, but involve formation of nuclear actin filaments that serve as anchors for stressed telomeres. Perturbed control of telomeric replication stress by mutations in core telomere binding proteins can therefore cause the deregulation of telomere length control characteristic of diseases such as cancer and telomere biology disorders.

Control of telomere length in yeast by SUMOylated PCNA and the Elg1 PCNA unloader.

Telomeres cap and protect the linear eukaryotic chromosomes. Telomere length is determined by an equilibrium between positive and negative regulators of telomerase activity. A systematic screen for yeast mutants that affect telomere length maintenance in the yeast Saccharomyces cerevisiae revealed that mutations in any of ~500 genes affects telomere length. One of the genes that, when mutated, causes telomere elongation is ELG1, which encodes an unloader of PCNA, the processivity factor for replicative DNA polymerases. PCNA can undergo SUMOylation on two conserved residues, K164 and K127, or ubiquitination at lysine 164. These modifications have already been implicated in genome stability processes. We report that SUMOylated PCNA acts as a signal that positively regulates telomerase activity. We also uncovered physical interactions between Elg1 and the CST (Cdc13-Stn1-Ten) complex and addressed the mechanism by which Elg1 and Stn1 negatively regulates telomere elongation, coordinated by SUMO. We discuss these results with respect to how chromosomal replication and telomere elongation are coordinated.

Control of telomere length in yeast by SUMOylated PCNA and the Elg1 PCNA unloader

Telomeres cap and protect the linear eukaryotic chromosomes. Telomere length is determined by an equilibrium between positive and negative regulators of telomerase activity. A systematic screen for yeast mutants that affect telomere length maintenance in the yeast Saccharomyces cerevisiae revealed that mutations in any of ~500 genes affects telomere length. One of the genes that, when mutated, causes telomere elongation is ELG1, which encodes an unloader of PCNA, the processivity factor for replicative DNA polymerases. PCNA can undergo SUMOylation on two conserved residues, K164 and K127, or ubiquitination at lysine 164. These modifications have already been implicated in genome stability processes. We report that SUMOylated PCNA acts as a signal that positively regulates telomerase activity. We also uncovered physical interactions between Elg1 and the CST (Cdc13-Stn1-Ten) complex and addressed the mechanism by which Elg1 and Stn1 negatively regulates telomere elongation, coordinated by SUMO. We discuss these results with respect to how chromosomal replication and telomere elongation are coordinated.

CST-Polα/Primase: the second telomere maintenance machine.

It has been known for decades that telomerase extends the 3' end of linear eukaryotic chromosomes and dictates the telomeric repeat sequence based on the template in its RNA. However, telomerase does not mitigate sequence loss at the 5' ends of chromosomes, which results from lagging strand DNA synthesis and nucleolytic processing. Therefore, a second enzyme is needed to keep telomeres intact: DNA polymerase α/Primase bound to Ctc1-Stn1-Ten1 (CST). CST-Polα/Primase maintains telomeres through a fill-in reaction that replenishes the lost sequences at the 5' ends. CST not only serves to maintain telomeres but also determines their length by keeping telomerase from overelongating telomeres. Here we discuss recent data on the evolution, structure, function, and recruitment of mammalian CST-Polα/Primase, highlighting the role of this complex and telomere length control in human disease.

Compilation and functional classification of telomere length-associated genes in humans and other animal species.

Telomeres are the terminal regions of chromosomes that ensure their stability while cell division. Telomere shortening initiates cellular senescence, which can lead to degeneration and atrophy of tissues, so the process is associated with a reduction in life expectancy and predisposition to a number of diseases. An accelerated rate of telomere attrition can serve as a predictor of life expectancy and health status of an individual. Telomere length is a complex phenotypic trait that is determined by many factors, including the genetic ones. Numerous studies (including genome-wide association studies, GWAS) indicate the polygenic nature of telomere length control. The objective of the present study was to characterize the genetic basis of the telomere length regulation using the GWAS data obtained during the studies of various human and other animal populations. To do so, a compilation of the genes associated with telomere length in GWAS experiments was collected, which included information on 270 human genes, as well as 23, 22, and 9 genes identified in the cattle, sparrow, and nematode, respectively. Among them were two orthologous genes encoding a shelterin protein (POT1 in humans and pot-2 in C. elegans). Functional analysis has shown that telomere length can be influenced by genetic variants in the genes encoding: (1) structural components of telomerase; (2) the protein components of telomeric regions (shelterin and CST complexes); (3) the proteins involved in telomerase biogenesis and regulating its activity; (4) the proteins that regulate the functional activity of the shelterin components; (5) the proteins involved in telomere replication and/or capping; (6) the proteins involved in the alternative telomere lengthening; (7) the proteins that respond to DNA damage and are responsible for DNA repair; (8) RNA-exosome components. The human genes identified by several research groups in populations of different ethnic origins are the genes encoding telomerase components such as TERC and TERT as well as STN1 encoding the CST complex component. Apparently, the polymorphic loci affecting the functions of these genes may be the most reliable susceptibility markers for telomere-related diseases. The systematized data about the genes and their functions can serve as a basis for the development of prognostic criteria for telomere length-associated diseases in humans. Information about the genes and processes that control telomere length can be used for marker-assisted and genomic selection in the farm animals, aimed at increasing the duration of their productive lifetime.

Thymidine nucleotide metabolism controls human telomere length.

Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed genome-wide CRISPR-Cas9 functional telomere length screening and identified thymidine (dT) nucleotide metabolism as a limiting factor in human telomere maintenance. Targeted genetic disruption using CRISPR-Cas9 revealed multiple telomere length control points across the thymidine nucleotide metabolism pathway: decreasing dT nucleotide salvage via deletion of the gene encoding nuclear thymidine kinase (TK1) or de novo production by knockout of the thymidylate synthase gene (TYMS) decreased telomere length, whereas inactivation of the deoxynucleoside triphosphohydrolase-encoding gene SAMHD1 lengthened telomeres. Remarkably, supplementation with dT alone drove robust telomere elongation by telomerase in cells, and thymidine triphosphate stimulated telomerase activity in a substrate-independent manner in vitro. In induced pluripotent stem cells derived from patients with genetic telomere biology disorders, dT supplementation or inhibition of SAMHD1 promoted telomere restoration. Our results demonstrate a critical role of thymidine metabolism in controlling human telomerase and telomere length, which may be therapeutically actionable in patients with fatal degenerative diseases.

POT-3 preferentially binds the terminal DNA-repeat on the telomeric G-overhang.

Eukaryotic chromosomes typically end in 3' telomeric overhangs. The safeguarding of telomeric single-stranded DNA overhangs is carried out by factors related to the protection of telomeres 1 (POT1) protein in humans. Of the three POT1-like proteins in Caenorhabditis elegans, POT-3 was the only member thought to not play a role at telomeres. Here, we provide evidence that POT-3 is a bona fide telomere-binding protein. Using a new loss-of-function mutant, we show that the absence of POT-3 causes telomere lengthening and increased levels of telomeric C-circles. We find that POT-3 directly binds the telomeric G-strand in vitro and map its minimal DNA binding site to the six-nucleotide motif, GCTTAG. We further show that the closely related POT-2 protein binds the same motif, but that POT-3 shows higher sequence selectivity. Crucially, in contrast to POT-2, POT-3 prefers binding sites immediately adjacent to the 3' end of DNA. These differences are significant as genetic analyses reveal that pot-2 and pot-3 do not function redundantly with each other in vivo. Our work highlights the rapid evolution and specialisation of telomere binding proteins and places POT-3 in a unique position to influence activities that control telomere length.

Biological Effects of Melatonin on Telomere Length in Breast Cancer: A Review Article

There has been increasing interest in studying the effects of dietary factors on telomere length. The telomere is a noncoding DNA sequence including "TTAGGG" at the ends of chromosomes of vertebrates. The stability of telomere length is an important factor as a survival signal for cells and cancer prevention. Telomerase is a multi-subunit DNA polymerase that plays a crucial role in maintaining the telomere length, which is critical for the age-related pathogenesis of breast neoplasm. Some regulatory factors interfere with telomerase activity and therefore promote breast tumorigenesis. High telomerase activity and restoring telomere lengths are determined as key factors in progressing tumors to advanced stages of malignancies, which are highly estrogen-dependent in breast carcinogenesis. Melatonin is a hormone-like substance secreted by the pineal gland and has been reported to downregulate telomerase. It may therefore control telomere length in cancer cells. Certain malignancy-related biological pathways have recently been linked to telomere length, and this review provides new insights regarding the effects of melatonin on telomere length by reviewing the anticarcinogenic mechanisms underlying melatonin in relation to telomerase activity in breast carcinogenesis. Experimental insights presenting the effects of melatonin alone or in combination with drugs on enhancing therapeutic protocols were also reviewed, which could assist our understanding of this hormone-like substance and telomeres as prognostic and therapeutic biomarkers in breast cancer.

Manipulation of Thymidine Nucleotide Metabolism Controls Human Telomere Length and Promotes Telomere Elongation in Dyskeratosis Congenita Patient Derived Cells

Inherited mutations in telomere maintenance genes are associated with short telomeres and can cause severe diseases including the congenital bone marrow failure syndrome dyskeratosis congenita, liver cirrhosis, and pulmonary fibrosis, collectively referred to as telomere biology disorders (TBDs). Current approaches to treat TBDs rely on bone marrow and organ transplantation, and patients face poor prognoses. New systemic therapies to promote telomere elongation and treat TBDs throughout the body are needed. Here, we performed the first genome-wide CRISPR/Cas9 functional telomere length screen using flow cytometry-FISH (flow-FISH) in human cells to identify novel telomere maintenance pathways that might be manipulated for therapeutic benefit. Validating our approach, we found guide RNAs targeting positive (e.g. TERT) and negative (e.g. POT1 and TRF1) regulators of telomere length enriched in sorted cells with short and long telomeres, respectively. Unbiased pathway analysis of screening hits demonstrated nucleotide metabolism genes to be highly associated with telomere length, consistent with recent genome-wide association studies, and which in follow-up screening we narrowed down to multiple control points specifically along the thymidine (dT) nucleotide metabolism pathway. Targeted genetic disruption using CRISPR/Cas9 revealed that decreasing dT nucleotide salvage by thymidine kinase (TK1) or de novo production by thymidylate synthase (TYMS) reduced telomere length, whereas inactivation of the dNTPase-encoding gene SAMHD1 lengthened telomeres. We next asked whether nucleotide precursor manipulation could alter telomere length dynamics. Remarkably, we found supplementation with dT nucleoside alone drove rapid telomere elongation in human cells (Figure 1). Telomere elongation after dT treatment required conversion to thymidine monophosphate (dTMP) by TK1, was telomerase dependent, and could be achieved at doses that did not inhibit cell cycle progression. In cells with TYMS deletion or after treatment with the TYMS inhibitor 5-fluorouracil, dT supplementation was sufficient to bypass the de novo nucleotide synthesis defect and promoted robust telomere elongation. SAMHD1 overexpression blunted the degree of telomere elongation from dT treatment in a manner requiring its dNTPase activity, suggesting that telomere length control occurs at the level of dTTP in human cells. To assess whether nucleotide manipulation could overcome the genetic causes of telomere insufficiency which drive TBDs, we tested dT on induced pluripotent stem cell (iPSC) lines derived from patients harboring mutations in a spectrum of TBD-associated genes including DKC1, TERC, TERT, TINF2, RTEL1, and PARN. We found dT supplementation or inhibition of SAMHD1 drove robust telomere elongation in patient derived iPSCs spanning genotypes that cause telomere maintenance defects both by telomerase-dependent and telomerase-independent mechanisms. In preliminary experiments using primary human hematopoietic stem and progenitor cells, dT treatment resulted in telomere elongation in vitro, pointing to a potential treatment for TBD-associated bone marrow failure. Collectively, our results demonstrate a critical role of thymidine nucleotide metabolism in regulating human telomere length. Based on our findings, we propose there may be a therapeutic window to modulate telomere length by manipulating dT metabolism in patients with TBDs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Exercise regulates shelterin genes and microRNAs implicated in ageing in Thoroughbred horses

Ageing causes a gradual deterioration of bodily functions and telomere degradation. Excessive telomere shortening leads to cellular senescence and decreases tissue vitality. Six proteins, called shelterin, protect telomere integrity and control telomere length through telomerase-dependent mechanisms. Exercise training appears to maintain telomeres in certain somatic cells, although the underlying molecular mechanisms are incompletely understood. Here, we examined the influence of a single bout of vigorous exercise training on leukocyte telomerase reverse transcriptase (TERT) and shelterin gene expression, and the abundance of three microRNAs (miRNAs) implicated in biological ageing (miRNA-143, -223 and -486-5p) in an elite athlete and large animal model, Thoroughbred horses. Gene and miRNA expression were analysed using primer-based and TaqMan Assay qPCR. Leukocyte TRF1, TRF2 and POT1 expression were all significantly increased whilst miR-223 and miR-486-5p were decreased immediately after vigorous exercise (all p < 0.05), and tended to return to baseline levels 24 h after training. Relative to the young horses (~ 3.9 years old), middle-aged horses (~ 14.8 years old) exhibited reduced leukocyte TERT gene expression, and increased POT1 and miR-223 abundance (all p < 0.05). These data demonstrate that genes transcribing key components of the shelterin-telomere complex are influenced by ageing and dynamically regulated by a single bout of vigorous exercise in a large, athletic mammal — Thoroughbred horses. Our findings also implicate TERT and shelterin gene transcripts as potential targets of miR-223 and miR-486-5p, which are modulated by exercise and may have a role in the telomere maintenance and genomic stability associated with long-term aerobic training.

An epigenetic clock model for assessing the human biological age of healthy aging

Objective: To construct an epigenetic clock model for assessing and calibrating human biological age. Methods: Convenience sampling was used to select 186 subjects from the longevity cohort of Guangxi Zhuang Antonornous Region from July 1 to November 30, 2019, and 124 subjects from the physical examination population of the Seventh Medical Center of the PLA General Hospital from October 1 to December 31, 2020. Self-designed questionnaire was applied to collect demographic characteristics and family history of disease. Physical examination was applied to determine heart rate and blood pressure. Fasting peripheral venous blood was drawn for determination of fasting plasma glucose, plasma total cholesterol, triglyceride, plasma high-density lipoprotein cholesterol, plasma low-density lipoprotein cholesterol and telomere length. Methylation levels of EDARADD cg09809672, IPO8 cg19722847, NHLRC1 cg22736354, P2RX6 cg05442902 and SCGN cg06493994 were detected by targeted methylation site sequencing. A total of 54 subjects with unqualified quality control of DNA methylation and telomere length were excluded, and 256 subjects' data were finally analyzed. Trend test was used for the change of methylation level among different ages groups, multiple linear regression method was used to build prediction models of biological age. Kendal rank correlation analysis was used to evaluate the correlation of age gap (Gregorian calendar age minus biological age) with telomere length. Independent sample t-test was used to compare the health-related indicators between subjects with different age gap within different age groups. Results: The M(Q1, Q3)of age of subjects were 67 (51, 91) years old, including 166 females (64.84%). With increase of age, the methylation levels of gene loci were decreased (EDARADD cg09809672, IPO8 cg19722847 and P2RX6 cg05442902) and increased (NHLRC1 cg22736354 and SCGN cg06493994) (all P values<0.05). The established biological age prediction model was as follows: Y=-53.121×EDARADD cg09809672-137.564×IPO8 cg19722847+141.040×NHLRC1 cg22736354-67.893×P2RX6 cg05442902+149.547×SCGNcg06493994+4.592×sex+64.185 (R2=0.86, P<0.001), where Y was the biological age, and the items in the equation were methylation level, sex (male =1, female =2) and intercept in sequence. The Kendall rank correlation coefficient between age gap and telomere length was 0.731 (P<0.001). Compared with the subjects whose age gaP<0, the subjects with age gaP≥0 had higher systolic blood pressure in adolescence [(88.50±8.89) and (109.83±9.48) mmHg, respectively, 1 mmHg=0.133 kPa]; lower TC [(5.48±0.23) and (3.98±0.54) mmol/L, respectively, ] and TG [(3.51±0.32) and (3.41±0.20) mmol/L] in young adults; lower fasting blood glucose in middle age [(6.17±0.67) and (5.37±0.79) mmol/L, respectively, ] and higher diastolic blood pressure in nonagenarian age [(76.99±6.78) and (83.97±9.36) mmHg, respectively, ] (all P values<0.05). Conclusion: The constructed epigenetic clock model can be used to evaluate and calibrate human biological age.

Possible Involvement of Hsp90 in the Regulation of Telomere Length and Telomerase Activity During the Leishmania amazonensis Developmental Cycle and Population Proliferation.

The Leishmania developmental cycle comprises three main life forms in two hosts, indicating that the parasite is continually challenged due to drastic environmental changes. The disruption of this cycle is critical for discovering new therapies to eradicate leishmaniasis, a neglected disease that affects millions worldwide. Telomeres, the physical ends of chromosomes, maintain genome stability and cell proliferation and are potential antiparasitic drug targets. Therefore, understanding how telomere length is regulated during parasite development is vital. Here, we show that telomeres form clusters spread in the nucleoplasm of the three parasite life forms. We also observed that amastigotes telomeres are shorter than metacyclic and procyclic promastigotes and that in parasites with continuous in vitro passages, telomere length increases over time. These observed differences in telomere length among parasite’s life stages were not due to lack/inhibition of telomerase since enzyme activity was detected in all parasite life stages, although the catalysis was temperature-dependent. These data led us to test if, similar to other eukaryotes, parasite telomere length maintenance could be regulated by Hsp83, the ortholog of Hsp90 in trypanosomatids, and Leishmania (LHsp90). Parasites were then treated with the Hsp90 inhibitor 17AAG. The results showed that 17AAG disturbed parasite growth, induced accumulation into G2/M phases, and telomere shortening in a time-dependent manner. It has also inhibited procyclic promastigote’s telomerase activity. Besides, LHsp90 interacts with the telomerase TERT component as shown by immunoprecipitation, strongly suggesting a new role for LHsp90 as a parasite telomerase component involved in controlling telomere length maintenance and parasite life span.

Lrrc34 Interacts with Oct4 and Has an Impact on Telomere Length in Mouse Embryonic Stem Cells.

Telomere length maintenance in pluripotent stem cells (PSCs) is a main characteristic and a major premise for their undifferentiated long-term survival. However, little is known about the factors that control telomere length and elongation in these cells. Here, I describe Lrrc34 (leucine-rich repeat 34) as a novel telomere length regulating gene in murine embryonic stem cells. Downregulation of Lrrc34 results in significant reduction of telomerase activity and telomere length over time while also influencing the expression of known telomere length-associated genes. Generating induced PSCs (iPSCs) with Lrrc34 as a fifth factor in classical Yamanaka reprogramming increases the efficiency but did not have an impact on telomere length in the resulting iPSCs. Moreover, Lrrc34 was found to interact with Oct4, connecting the pluripotency network to telomere length regulation.

Metal co-exposures and telomere length in Bangladeshi children

BACKGROUND AND AIM: Telomeres are non-coding DNA sequences that protect the ends of chromosomes and preserve genetic material during cell division. Telomere length is associated with the occurrence and progression of common chronic diseases. In younger populations, telomere length may represent a biomarker of disease susceptibility. Growing evidence suggests that environmental factors, such as metals, can substantially impact the mechanisms that control telomere length. METHODS: We investigated the association between a panel of metals measured in toenail samples by inductively-coupled plasma mass spectrometry and leukocyte relative telomere length (RTL) measured by Luminex assay, among 472 five- to seven-year-old children enrolled in the Bangladesh Environmental Research in Children's Health (BiRCH) cohort. RESULTS:Of the 18 metals tested, statistically significant correlations with RTL were observed with toenail arsenic (tAs) and mercury (tHg). In linear regression models adjusted for child age, sex, and father’s age at child’s birth, a doubling of log2-transformed tAs was associated with a -0.022 (95% CI: -0.033, -0.011; p=0.0001) decrease in RTL. Similarly, a doubling of log2-transformed tHg was associated with a -0.020 (95% CI: -0.032, -0.007; p=0.002) decrease in RTL. Similar associations were observed using co-exposure regression models including both tAs and tHg, adjusted for covariates. In this model, we observed independent, negative associations of both log2-transformed tAs and tHg with RTL, such that a doubling of tAs was associated with a -0.019 (95% CI: -0.031, -0.007; p=0.003) decrease in RTL and a doubling of tHg was associated with a -0.015 (95% CI: -0.028, -0.002; p=0.02) decrease in RTL. CONCLUSIONS:Our study suggests that both As and Hg may independently influence RTL, with detectable associations in early- to mid-childhood. Further studies are needed to confirm our findings and investigate the potential long-term impacts of As- and Hg- exposure associated telomere shortening in childhood on health outcomes in adult life. KEYWORDS: metals, telomeres, toenails, Bangladesh, children

The cooperative assembly of shelterin bridge provides a kinetic gateway that controls telomere length homeostasis.

Shelterin is a six-protein complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1 and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.

RBL2 bi-allelic truncating variants cause severe motor and cognitive impairment without evidence for abnormalities in DNA methylation or telomeric function.

RBL2/p130, a member of the retinoblastoma family of proteins, is a key regulator of cell division and propagates irreversible senescence. RBL2/p130 is also involved in neuronal differentiation and survival, and eliminating Rbl2 in certain mouse strains leads to embryonic lethality accompanied by an abnormal central nervous system (CNS) phenotype. Conflicting reports exist regarding a role of RBL2/p130 in transcriptional regulation of DNA methyltransferases (DNMTs), as well as the control of telomere length. Here we describe the phenotype of three patients carrying bi-allelic RBL2-truncating variants. All presented with infantile hypotonia, severe developmental delay and microcephaly. Malignancies were not reported in carriers or patients. Previous studies carried out on mice and human cultured cells, associated RBL2 loss to DNA methylation and telomere length dysregulation. Here, we investigated whether patient cells lacking RBL2 display related abnormalities. The study of primary patient fibroblasts did not detect abnormalities in expression of DNMTs. Furthermore, methylation levels of whole genome DNA, and specifically of pericentromeric repeats and subtelomeric regions, were unperturbed. RBL2-null fibroblasts show no evidence for abnormal elongation by telomeric recombination. Finally, gradual telomere shortening, and normal onset of senescence were observed following continuous culturing of RBL2-mutated fibroblasts. Thus, this study resolves uncertainties regarding a potential non-redundant role for RBL2 in DNA methylation and telomere length regulation, and indicates that loss of function variants in RBL2 cause a severe autosomal recessive neurodevelopmental disorder in humans.

Alternative splicing is a developmental switch for hTERT expression

Telomere length control is critical for cellular lifespan and tumor suppression. Telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere reserves. Its silencing upon differentiation leads to gradual telomere shortening in somatic cells. Here, we report that transcriptional regulation through cis-regulatory elements only partially accounts for telomerase activation in pluripotent cells. Instead, developmental control of telomerase is primarily driven by an alternative splicing event, centered around hTERT exon 2. Skipping of exon 2 triggers hTERT mRNA decay in differentiated cells, and conversely, its retention promotes telomerase accumulation in pluripotent cells. We identify SON as a regulator of exon 2 alternative splicing and report a patient carrying a SON mutation and suffering from insufficient telomerase and short telomeres. In summary, our study highlights a critical role for hTERT alternative splicing in the developmental regulation of telomerase and implicates defective splicing in telomere biology disorders.