The DDR at telomeres lacking intact shelterin does not require substantial chromatin decompaction.

Telomeres are protected by shelterin, a six-subunit protein complex that represses the DNA damage response (DDR) at chromosome ends. Extensive data suggest that TRF2 in shelterin remodels telomeres into the t-loop structure, thereby hiding telomere ends from double-stranded break repair and ATM signaling, whereas POT1 represses ATR signaling by excluding RPA. An alternative protection mechanism was suggested recently by which shelterin subunits TRF1, TRF2, and TIN2 mediate telomeric chromatin compaction, which was proposed to minimize access of DDR factors. We performed superresolution imaging of telomeres in mouse cells after conditional deletion of TRF1, TRF2, or both, the latter of which results in the complete loss of shelterin. Upon removal of TRF1 or TRF2, we observed only minor changes in the telomere volume in most of our experiments. Upon codeletion of TRF1 and TRF2, the telomere volume increased by varying amounts, but even those samples exhibiting small changes in telomere volume showed DDR at nearly all telomeres. Upon shelterin removal, telomeres underwent 53BP1-dependent clustering, potentially explaining at least in part the apparent increase in telomere volume. Furthermore, chromatin accessibility, as determined by ATAC-seq (assay for transposase-accessible chromatin [ATAC] with high-throughput sequencing), was not substantially altered by shelterin removal. These results suggest that the DDR induced by shelterin removal does not require substantial telomere decompaction.

Different requirements of functional telomeres in neural stem cells and terminally differentiated neurons.

Telomeres have been studied extensively in peripheral tissues, but their relevance in the nervous system remains poorly understood. Here, we examine the roles of telomeres at distinct stages of murine brain development by using lineage-specific genetic ablation of TRF2, an essential component of the shelterin complex that protects chromosome ends from the DNA damage response machinery. We found that functional telomeres are required for embryonic and adult neurogenesis, but their uncapping has surprisingly no detectable consequences on terminally differentiated neurons. Conditional knockout of TRF2 in post-mitotic immature neurons had virtually no detectable effect on circuit assembly, neuronal gene expression, and the behavior of adult animals despite triggering massive end-to-end chromosome fusions across the brain. These results suggest that telomeres are dispensable in terminally differentiated neurons and provide mechanistic insight into cognitive abnormalities associated with aberrant telomere length in humans.

The telomeric DNA damage response occurs in the absence of chromatin decompaction.

Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.

Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells with strong immunosuppressive activity that promote tumor growth. In this study, we describe a mechanism by which cancer cells control MDSCs in human cancers by upregulating TRF2, a protein required for telomere stability. Specifically, we showed that the TRF2 upregulation in cancer cells has extratelomeric roles in activating the expression of a network of genes involved in the biosynthesis of heparan sulfate proteoglycan, leading to profound changes in glycocalyx length and stiffness, as revealed by atomic force microscopy. This TRF2-dependent regulation facilitated the recruitment of MDSCs, their activation via the TLR2/MyD88/IL-6/STAT3 pathway leading to the inhibition of natural killer recruitment and cytotoxicity, and ultimately tumor progression and metastasis. The clinical relevance of these findings is supported by our analysis of cancer cohorts, which showed a correlation between high TRF2 expression and MDSC infiltration, which was inversely correlated with overall patient survival.

TRF2 recruits RTEL1 to telomeres in S phase to promote t-loop unwinding.

The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1(R1264H). Conversely, we define a TRF2(I124D) substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1(R1264H) mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase.

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.

Transcription factor Sp1 prevents TRF2(ΔBΔM)-induced premature senescence in human diploid fibroblasts.

Telomere uncapping is thought to be the fundamental cause of replicative cellular senescence, but the cellular machineries mediating this process have not been fully understood. In the present study, we present the role of Sp1 transcription factor in the state of telomere uncapping using the TRF2(ΔBΔM)-induced senescence model in human diploid fibroblasts. We observed that the expression of Sp1 is down-regulated in the TRF2(ΔBΔM)-induced senescence, which was mediated by ATM and p38 MAPK. In addition, overexpression of Sp1 prevented the TRF2(ΔBΔM)-induced senescence. Among transcriptional targets of Sp1, expression levels of nuclear transport genes such as karyopherin α, Nup107, and Nup50 were down-regulated in the TRF2(ΔBΔM)-induced senescence, which was prevented by Sp1 overexpression. Moreover, inhibition of the nuclear transport by wheat germ agglutinin (an import inhibitor) and leptomycin B (an export inhibitor) induced premature senescence. These results suggest that Sp1 is an anti-senescence transcription factor in the telomere uncapping-induced senescence and that down-regulation of Sp1 leads to the senescence via down-regulation of the nuclear transport.

TRF1 and TRF2 differentially modulate Rad51-mediated telomeric and nontelomeric displacement loop formation in vitro.

A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and nontelomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90-nucleotide telomeric and nontelomeric substrates into telomeric and nontelomeric plasmid templates. We report that preincubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation upon preincubation with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated nontelomeric D-loop formation, possibly explaining how TRF2 promotes nontelomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication.

[Telomerase and telomere: their structure and dynamics in health and disease]. / Telomerasa y telómero: su estructura y dinámica en salud y enfermedad.

Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.

Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination.

Ku70-Ku80 heterodimers promote the non-homologous end-joining (NHEJ) of DNA breaks and, as shown here, the fusion of dysfunctional telomeres. Paradoxically, this heterodimer is also located at functional mammalian telomeres and interacts with components of shelterin, the protein complex that protects telomeres. To determine whether Ku contributes to telomere protection, we analysed Ku70(-/-) mouse cells. Telomeres of Ku70(-/-) cells had a normal DNA structure and did not activate a DNA damage signal. However, Ku70 repressed exchanges between sister telomeres - a form of homologous recombination implicated in the alternative lengthening of telomeres (ALT) pathway. Sister telomere exchanges occurred at approximately 15% of the chromosome ends when Ku70 and the telomeric protein TRF2 were absent. Combined deficiency of TRF2 and another NHEJ factor, DNA ligase IV, did not elicit this phenotype. Sister telomere exchanges were not elevated at telomeres with functional TRF2, indicating that TRF2 and Ku70 act in parallel to repress recombination. We conclude that mammalian chromosome ends are highly susceptible to homologous recombination, which can endanger cell viability if an unequal exchange generates a critically shortened telomere. Therefore, Ku- and TRF2-mediated repression of homologous recombination is an important aspect of telomere protection.

Quantitative theory of telomere length regulation and cellular senescence.

In normal somatic cells, telomere length shortens with each cell replication. This progressive shortening is associated with cellular senescence and apoptosis. Germ cells, stem cells, and the majority of cancer cells express telomerase, an enzyme that extends telomere length and, when expressed at sufficient levels, can immortalize or extend the life span of a cell line. It is believed that telomeres switch between two states: capped and uncapped. The telomere state determines its accessibility to telomerase and also the onset of senescence. One hypothesis is that the t loop, a large lariat-like structure, represents the capped state. In this paper we model a telomere state on the basis of the biophysics of t-loop formation, allowing us to develop a single mathematical model that accounts for two processes: telomere length regulation for telomerase positive cells and cellular senescence in somatic cells. The model predicts the steady-state length distribution for telomerase positive cells, describes the time evolution of telomere length, and computes the life span of a cell line on the basis of the levels of TRF2 and telomerase expression. The model reproduces a wide range of experimental behavior and fits data from immortal cell lines (HeLa S3 and 293T) and somatic cells (human diploid fibroblasts) well. We conclude that the t loop as the capped state is a quantitatively reasonable model of telomere length regulation and cellular senescence.

Overexpression of ethylene response factor TERF2 confers cold tolerance in rice seedlings.

Rice (Oryza sativa L.) is a warm-season plant exposed to various stresses. Low temperature is an important factor limiting extension of rice cultivation areas and productivity. Previously, we have demonstrated that tomato ERF protein TERF2 enhances freezing tolerance of transgenic tobacco and tomato plants. Herein, we report that overexpression of TERF2 enhances transgenic rice tolerance to cold without affecting growth or agronomic traits. Physiological assays revealed that TERF2 could not only increase accumulation of osmotic substances and chlorophyll, but also reduce reactive oxygen species (ROS) and malondialdehyde (MDA) content and decrease electrolyte leakage in rice under cold stress. Further analysis of gene expression showed that TERF2 could activate expression of cold-related genes, including OsMyb, OsICE1, OsCDPK7, OsSODB, OsFer1, OsTrx23, and OsLti6, in transgenic rice plants under natural condition or cold stress. Thus, our findings demonstrated that TERF2 modulated expression of stress-related genes and a series of physiological adjustments under cold stress, indicating that TERF2 might have important regulatory roles in response to abiotic stress in rice and possess potential utility in improving crop cold tolerance.

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle.

Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV-304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially positioned at the interface of chromatin domains. TRF1 and TRF2 are abundantly present in these territories but not firmly bound. At the onset of mitosis, the bulk of TRF protein dissociates from telomere regions, territories disintegrate and individual telomeres become faintly visible. The combination of stable cell lines, CLEM and cytometry proved essential in providing novel insights in compartment-based nuclear organization and may serve as a model approach for investigating telomere-driven genome-instability and studying long-term nuclear dynamics.

Mouse models in modeling aging and cancer.

Mouse models have been widely used in the research of human diseases. Aging, just as cancer, is influenced by the interaction of various genetic and environmental factors. Currently, aging could be induced by many mechanism, including telomere dysfunction, oxidase stress, DNA damage and epigenetic changes. Many of these genetic pathways are also shared by aging and cancer. The mouse models generated to study these pathways might manifest either aging or cancer phenotypes, sometimes both, which in deed has worked as a good model system in understanding the correlation between aging and cancer. Here, we reviewed these mouse models that were generated to model aging or cancer. These mouse models might help us put those related pathways in context and discover essential interactions in cancer and aging regulation.

Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference.

Telomerase-negative cancer cells can maintain their telomeres by a recombination-mediated alternative lengthening of telomeres (ALT) process. We reported previously that sequestration of MRE11/RAD50/NBS1 complexes represses ALT-mediated telomere length maintenance, and suppresses formation of ALT-associated promyelocytic leukemia (PML) bodies (APBs). APBs are PML bodies containing telomeric DNA and telomere-binding proteins, and are observed only in a small fraction of cells within asynchronously dividing ALT-positive cell populations. Here, we report that methionine restriction caused a reversible arrest in G0/G1 phase of the cell cycle and reversible induction of APB formation in most cells within an ALT-positive population. We combined methionine restriction with RNA interference to test whether the following proteins are required for APB formation: PML body-associated proteins, PML and Sp100; telomere-associated proteins, TRF1, TRF2, TIN2 and RAP1; and DNA repair proteins, MRE11, RAD50, NBS1 and 53BP1. APB formation was not decreased by depletion of Sp100 (as reported previously) or of 53BP1, although 53BP1 partially colocalizes with APBs. Depletion of the other proteins suppressed APB formation. Because of the close linkage between ALT-mediated telomere maintenance and ability to form APBs, the eight proteins identified by this screen as being required for APB formation are also likely to be required for the ALT mechanism.

[Functions of transcription factor TRF2 in Drosophila melanogaster].

A study was made of the function of the Drosophila melanogaster TRF2 protein. Expression analysis of the trf2(P1) mutation implicated TRF2 in the D. melanogaster embryo development. High-level expression of the trf2 gene was observed in female germline cells. A high level of TRF2 was detected in primary spermatocytes and trophocytes, characterized by intense transcription. In the female gonads, TRF2 was detected in both nurse cells with intense transcription and transcriptionally inactive oocyte nuclei. In addition, TRF2 proved to be necessary for premeiotic chromatin condensation and further differentiation of germline cells.

The role of telomere binding molecules for normal and abnormal hematopoiesis.

In order to maintain the homeostasis of the hematopoietic system, hematopoietic stem cells (HSCs) need to be maintained while slowly dividing over their lifetime. However, repeated cell divisions lead to the gradual accumulation of DNA damage and ultimately impair HSC function. Since telomeres are particularly fragile when subjected to replication stress, cells have several defense machinery to protect telomeres. Moreover, HSCs must protect their genome against possible DNA damage, while maintaining telomere length. A group of proteins called the shelterin complex are deeply involved in this two-way role, and it is highly resistant to the replication stress to which HSCs are subjected. Most shelterin-deficient experimental models suffer acute cytotoxicity and severe phenotypes, as each shelterin component is essential for telomere protection. The Tin2 point mutant mice show a dyskeratosis congenita (DC) like phenotype, and the Tpp1 deletion impairs the hematopoietic system. POT1/Pot1a is highly expressed in HSCs and contributes to the maintenance of the HSC pool during in vitro culture. Here, we discuss the role of shelterin molecules in HSC regulation and review current understanding of how these are regulated in the maintenance of the HSC pool and the development of hematological disorders.

The core-promoter factor TRF2 mediates a Fruitless action to masculinize neurobehavioral traits in Drosophila.

In fruit flies, the male-specific fruitless (fru) gene product FruBM plays a central role in establishing the neural circuitry for male courtship behavior by orchestrating the transcription of genes required for the male-type specification of individual neurons. We herein identify the core promoter recognition factor gene Trf2 as a dominant modifier of fru actions. Trf2 knockdown in the sexually dimorphic mAL neurons leads to the loss of a male-specific neurite and a reduction in male courtship vigor. TRF2 forms a repressor complex with FruBM, strongly enhancing the repressor activity of FruBM at the promoter region of the robo1 gene, whose function is required for inhibiting the male-specific neurite formation. In females that lack FruBM, TRF2 stimulates robo1 transcription. Our results suggest that TRF2 switches its own role from an activator to a repressor of transcription upon binding to FruBM, thereby enabling the ipsilateral neurite formation only in males.

TRF2 promotes multidrug resistance in gastric cancer cells.

The role of telomere in drug resistance has not been clearly understood. Recent studies have been focused on telomerase activity and telomere length, but the findings are still controversial. It's been found that DNA double-strand breaks induced by anticancer drugs or irradiation increase TRF2 expression as an early response to DNA damage, which inhibits activation of ATM-dependent DNA damage response network, indicating TRF2 might probably be a general DNA-repair factor rather than merely a telomere-binding factor. In this study, the possible involvement of telomerase, telomere and TRF2 in DNA damage response and drug resistance was investigated. Telomere length was found elongated in multidrug-resistant variants of gastric cancer cell line SGC7901 treated with adriamycin or etoposide, however, drug-treatment per se had no effect on telomere length. Telomerase activity and TRF2 expression were upregulated after treatment, but not TRF1. TRF2 upregulation was more dramatic in drug-resistant cells and occurred before the expression of ATM, gammaH2AX and p53. Moreover, TRF2 inhibited the expression of ATM-dependent DSB responsive genes. Inhibition of TRF2 expression by RNA interference in drug-resistant cells partially reversed its resistance phenotype and overexpression of TRF2 in SGC7901 promoted its resistance phenotype. Taken together, current results indicate that TRF2 plays an important role in DNA damage response, and is involved in drug resistance of gastric cancer. Further study of the biological functions of TRF2 might be helpful to dissect the molecular mechanism of multiple drug-resistance and generate novel target to overcome it.