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Reactive oxygen species (ROS) exposure triggers granulosa cells' (GCs) senescence, which is an important causal factor for premature ovarian failure (POF). However, underlying mechanism in this process remains unknown. In our study, we observed increased ROS levels in POF ovarian tissues, POF patient follicular GCs and cyclophosphamide (CTX) pretreated GCs. Correspondingly, increased SIAH1, reduced TRF2 and GC senescence were also found in these cases. Silencing of SIAH1 rescued ROS-induced TRF2 reduction and cell senescence in GCs. Moreover, SIAH1 co-localized with TRF2 in the cytoplasm, facilitating its ubiquitination degradation, further leading to telomere abnormalities in GCs. In conclusion, our findings indicate that ROS induces telomere abnormalities by augmenting SIAH1-mediated TRF2 degradation, leading to cell senescence in GCs in POF processing.

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2–TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein–DNA structures, and monitoring of DNA–DNA and DNA–RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA–dsDNA, dsDNA–ssDNA and dsDNA–ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

The mammalian telomeric shelterin complex—comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1—blocks the DNA damage response at chromosome ends and interacts with telomerase and the CST complex to regulate telomere length. The evolutionary origins of shelterin are unclear, partly because unicellular organisms have distinct telomeric proteins. Here, we describe the evolution of metazoan shelterin, showing that TRF1 emerged in vertebrates upon duplication of a TRF2-like ancestor. TRF1 and TRF2 diverged rapidly during vertebrate evolution through the acquisition of new domains and interacting factors. Vertebrate shelterin is also distinguished by the presence of an HJRL domain in the split C-terminal OB fold of POT1, whereas invertebrate POT1s carry inserts of variable nature. Importantly, the data reveal that, apart from the primate and rodent POT1 orthologs, all metazoan POT1s are predicted to have a fourth OB fold at their N termini. Therefore, we propose that POT1 arose from a four-OB-fold ancestor, most likely an RPA70-like protein. This analysis provides insights into the biology of shelterin and its evolution from ancestral telomeric DNA-binding proteins.

Telomeres protect chromosome ends from degradation. Telomere length (TL) can be altered by aging and environmental stress. Shortened TL has been observed in peripheral blood leukocytes of alcohol dependent subjects and ethanol-exposed somatic cells. To understand the impact of ethanol on telomeres in pluripotent stem cells, we investigated the influence of ethanol on TL and the expression of six Shelterin complex subunit or telomere-regulating genes (POT1, RAP1, TIN2, TPP1, TRF1, and TRF2) in human embryonic stem cells (hESCs), which were exposed to 0, 25, 50, or 100 mM of ethanol for 3, 7, or 14 days. Ethanol-induced TL and Shelterin complex subunit gene expression changes were examined by quantitative polymerase chain reactions. Two-way ANOVA tests indicated that TL variation and expression changes of four associated Shelterin complex subunit genes (POT1, TPP1, TIN2, and TRF2) were mainly dependent on the length of ethanol exposure, while TRF1 and RAP1expression was influenced by ethanol concentration, exposure time, and the interaction of ethanol concentration and exposure time. Tukey's multiple comparison tests showed that TL and the expression of POT1, RAP1, TIN2, TPP1, and TRF1 were decreased after a 7-day (versus a 3-day) ethanol exposure. However, the decreased expression of all six Shelterin complex subunit genes was recovered and TL was not further shortened after a 14-day (versus a 7-day) ethanol exposure, likely due to the adaptation of hESCs to ethanol-induced stress. Our study provided further evidence that TL is regulated and maintained by telomere-regulating genes in stem cells under ethanol stress.

Telomere maintenance is essential to preserve genomic stability and involves telomere-specific proteins, DNA replication and repair proteins. Lamins are key components of the nuclear envelope and play numerous roles, including maintenance of the nuclear integrity, regulation of transcription, and DNA replication. Elevated levels of lamin B1, one of the major lamins, have been observed in some human pathologies and several cancers. Yet, the effect of lamin B1 dysregulation on telomere maintenance remains unknown. Here, we unveil that lamin B1 overexpression drives telomere instability through the disruption of the shelterin complex. Indeed, lamin B1 dysregulation leads to an increase in telomere dysfunction-induced foci, telomeric fusions and telomere losses in human cells. Telomere aberrations were preceded by mislocalizations of TRF2 and its binding partner RAP1. Interestingly, we identified new interactions between lamin B1 and these shelterin proteins, which are strongly enhanced at the nuclear periphery upon lamin B1 overexpression. Importantly, chromosomal fusions induced by lamin B1 in excess were rescued by TRF2 overexpression. These data indicated that lamin B1 overexpression triggers telomere instability through a mislocalization of TRF2. Altogether our results point to lamin B1 as a new interacting partner of TRF2, that is involved in telomere stability.

Background and aims Hepatitis C virus (HCV) leads to a high rate of chronic infection and T cell dysfunction. Although it is well known that chronic antigenic stimulation is a driving force for impaired T cell functions, the precise mechanisms underlying immune activation-induced T cell dysfunctions during HCV infection remain elusive. Approach and results Here, we demonstrated that circulating CD4+ T cells from patients who are chronically HCV-infected exhibit an immune activation status, as evidenced by the overexpression of cell activation markers human leukocyte antigen-antigen D-related, glucose transporter 1, granzyme B, and the short-lived effector marker CD127- killer cell lectin-like receptor G1+ . In contrast, the expression of stem cell-like transcription factor T cell factor 1 and telomeric repeat-binding factor 2 (TRF2) are significantly reduced in CD4+ T cells from patients who are chronically HCV-infected compared with healthy participants (HP). Mechanistic studies revealed that CD4+ T cells from participants with HCV exhibit phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling hyperactivation on T cell receptor stimulation, promoting proinflammatory effector cell differentiation, telomeric DNA damage, and cellular apoptosis. Inhibition of Akt signaling during T cell activation preserved the precursor memory cell population and prevented inflammatory effector cell expansion, DNA damage, and apoptotic death. Moreover, knockdown of TRF2 reduced HP T cell stemness and triggered telomeric DNA damage and cellular apoptosis, whereas overexpression of TRF2 in CD4 T cells prevented telomeric DNA damage. Conclusions These results suggest that modulation of immune activation through inhibiting Akt signaling and protecting telomeres through enhancing TRF2 expression may open therapeutic strategies to fine tune the adaptive immune responses in the setting of persistent immune activation and inflammation during chronic HCV infection.

We investigated the role of telomerase and telomere repeat-binding factor 2 (TRF2 or TERF2) in T-cell dysfunction in chronic viral infection. We found that the expression and activity of telomerase in CD4+ T (CD4T) cells from patients with hepatitis C virus (HCV) infections or people living with HIV (PLWH) were intact, but TRF2 expression was significantly inhibited at the post-transcriptional level, suggesting that TRF2 inhibition is responsible for the CD4T cell dysfunction observed during chronic viral infection. Silencing TRF2 expression in CD4T cells derived from healthy subjects induced telomeric DNA damage and CD4T cell dysfunction without affecting telomerase activity or translocation – similar to what we observed in CD4T cells from HCV patients and PLWH. These findings indicate that premature T-cell aging and dysfunction during chronic HCV or HIV infection are primarily caused by chronic immune stimulation and T-cell overactivation and/or proliferation that induce telomeric DNA damage due to TRF2 inhibition, rather than telomerase disruption. This study suggests that restoring TRF2 presents a novel approach to prevent telomeric DNA damage and premature T-cell aging, thus rejuvenating T-cell functions during chronic viral infection.

Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.

Telomeres are transcribed into telomeric RNA termed as TERRA. However, the transcription itself and excessive TERRA may interfere with telomere replication during S phase. The mechanism that coordinates telomere transcription and replication is unknown. Here, we report that TCOF1 leaves the nucleolus and is recruited to telomeres specifically during S phase by interacting with TRF2. Therein, TCOF1 acts to suppress telomere transcription by binding and inhibiting Pol II. Thus, TERRA is limited to low levels in S phase. Depletion of TCOF1 leads to abnormally elevated TERRA and formation of DNA/RNA hybrids (R-loops) at telomeres, which induces replication fork stalling and fragile telomeres. Importantly, telomere replication defect induced by TCOF1 deficiency can be rescued by either masking TERRA or expressing an R-loop eraser RNase H1, demonstrating a critical role of TCOF1 in coordinating telomere transcription and replication. These findings link nucleolus to telomeres and uncover a novel function of TCOF1 on ensuring telomere integrity.

The shelterin protein complex is required for telomere protection in various eukaryotic organisms. In mammals, the shelterin subunit TRF2 is specialized in preventing ATM activation at telomeres and chromosome end fusion in somatic cells. Here, we demonstrate that the zebrafish ortholog of TRF2 (encoded by the terfa gene) is protecting against unwanted ATM activation genome-wide. The terfa-compromised fish develop a prominent and specific embryonic neurodevelopmental failure. The heterozygous fish survive to adulthood but exhibit a premature aging phenotype. The recovery from embryonic neurodevelopmental failure requires both ATM inhibition and transcriptional complementation of neural genes. Furthermore, restoring the expression of TRF2 in glial cells rescues the embryonic neurodevelopment phenotype. These results indicate that the shelterin subunit TRF2 evolved in zebrafish as a general factor of genome maintenance and transcriptional regulation that is required for proper neurodevelopment and normal aging. These findings uncover how TRF2 links development to aging by separate functions in gene expression regulation and genome stability control.

Telomeres, the ends of linear chromosomes, are composed of repetitive DNA sequences, histones and a protein complex called shelterin. How DNA is packaged at telomeres is an outstanding question in the field with significant implications for human health and disease. Here, we studied the architecture of telomeres and their spatial association with other chromatin domains in different cell types using correlative light and electron microscopy. To this end, the shelterin protein TRF1 or TRF2 was fused in tandem to eGFP and the peroxidase APEX2, which provided a selective and electron-dense label to interrogate telomere organization by transmission electron microscopy, electron tomography and scanning electron microscopy. Together, our work reveals, for the first time, ultrastructural insight into telomere architecture. We show that telomeres are composed of a dense and highly compacted mesh of chromatin fibres. In addition, we identify marked differences in telomere size, shape and chromatin compaction between cancer and non-cancer cells and show that telomeres are in direct contact with other heterochromatin regions. Our work resolves the internal architecture of telomeres with unprecedented resolution and advances our understanding of how telomeres are organized in situ. Ministry of Education (MOE) Published version Ministry of Education, Singapore [MOE 2012-T3-1-001 to S.S.; MOE 2017-T1-002-067 to S.S. and A.L.]; Vetenskapsradet, Sweden [National Microscopy Infrastructure ˚ (NMI) VR nr. 2019-00217; Molecular Infection Medicine in Sweden (MIMS) VR nr. 2016-06598]. Funding for open access charge: Ministry of Education, Singapore [MOE 2017- T1-002-067].

Type II alveolar epithelial cells (AEC2s) play an essential role in the function and maintenance of the pulmonary epithelium. Several transgenic mice have been developed to study the function of these cells in vivo by using the human SFTPC promoter to drive expression of Cre recombinase. The precise activity of each of these transgenic alleles has not been studied, and previous reports suggest that their activity can depend on breeding strategies. We bred mice with a conditional allele of the essential telomere capping protein TRF2 with two different SFTPC-Cre–transgenic strains and observed opposite phenotypes (100% lethality vs. 100% viability). We characterized the Cre recombinase activity in these two transgenic lines and found that the contrasting phenotypes were driven by difference in embryonic expression of the two transgenes, likely due to position effects or differences in the transgenic constructs. We also tested if SFTPC-Cre activity was dependent on maternal or paternal inheritance. When paternally inherited, both SFTPC-Cre alleles produced offspring with constitutive reporter activity independent of the inheritance of the Cre allele, suggesting that Cre recombinase was expressed in the male germline before meiosis. Immunohistochemical analysis of the testis showed reporter activity during spermatogenesis. Analysis of single-cell RNA sequencing data from murine and human testis demonstrated SFTPC expression uniquely during human spermatogenesis, suggesting that use of the human promoter in these constructs is responsible for male germline activity. Our data highlight the importance of careful analysis of transgenic allele activity and identify an SFTPC-Cre allele that is useful for panepithelial targeting in the mouse.

Reactive oxygen species (ROS) inflict multiple types of lesions in DNA, threatening genomic integrity. How cells respond to ROS-induced DNA damage at telomeres is still largely unknown. Here, we show that ROS-induced DNA damage at telomeres triggers R-loop accumulation in a TERRA- and TRF2-dependent manner. Both ROS-induced single- and double-strand DNA breaks (SSBs and DSBs) contribute to R-loop induction, promoting the localization of CSB and RAD52 to damaged telomeres. RAD52 is recruited to telomeric R-loops through its interactions with both CSB and DNA:RNA hybrids. Both CSB and RAD52 are required for the efficient repair of ROS-induced telomeric DSBs. The function of RAD52 in telomere repair is dependent on its ability to bind and recruit POLD3, a protein critical for break-induced DNA replication (BIR). Thus, ROS-induced telomeric R-loops promote repair of telomeric DSBs through CSB–RAD52–POLD3-mediated BIR, a previously unknown pathway protecting telomeres from ROS. ROS-induced telomeric SSBs may not only give rise to DSBs indirectly, but also promote DSB repair by inducing R-loops, revealing an unexpected interplay between distinct ROS-induced DNA lesions.

Human telomeres are protected by shelterin proteins, but how telomeres maintain a dynamic structure remains elusive. Here, we report an unexpected activity of POT1 in imparting conformational dynamics of the telomere overhang, even at a monomer level. Strikingly, such POT1-induced overhang dynamics is greatly enhanced when TRF2 engages with the telomere duplex. Interestingly, TRF2, but not TRF2ΔB, recruits POT1-bound overhangs to the telomere ds/ss junction and induces a discrete stepwise movement up and down the axis of telomere duplex. The same steps are observed regardless of the length of the POT1-bound overhang, suggesting a tightly regulated conformational dynamic coordinated by TRF2 and POT1. TPP1 and TIN2 which physically connect POT1 and TRF2 act to generate a smooth movement along the axis of the telomere duplex. Our results suggest a plausible mechanism wherein telomeres maintain a dynamic structure orchestrated by shelterin.

The incidence of depression among humans is growing worldwide, and so is the use of antidepressants. However, our fundamental understanding regarding the mechanisms by which these drugs function and their off-target effects against human sexuality remains poorly defined. The present study aimed to determine their differential toxicity on mouse spermatogenic cells and provide mechanistic data of cell-specific response to antidepressant and neuroleptic drug treatment. To directly test reprotoxicity, the spermatogenic cells (GC-1 spg and GC-2 spd cells) were incubated for 48 and 96 h with amitriptyline (hydrochloride) (AMI), escitalopram (ESC), fluoxetine (hydrochloride) (FLU), imipramine (hydrochloride) (IMI), mirtazapine (MIR), olanzapine (OLZ), reboxetine (mesylate) (REB), and venlafaxine (hydrochloride) (VEN), and several cellular and biochemical features were assessed. Obtained results reveal that all investigated substances showed considerable reprotoxic potency leading to micronuclei formation, which, in turn, resulted in upregulation of telomeric binding factor (TRF1/TRF2) protein expression. The TRF-based response was strictly dependent on p53/p21 signaling and was followed by irreversible G2/M cell cycle arrest and finally initiation of apoptotic cell death. In conclusion, our findings suggest that antidepressants promote a telomere-focused DNA damage response in germ cell lines, which broadens the established view of antidepressants’ and neuroleptic drugs’ toxicity and points to the need for further research in this topic with the use of in vivo models and human samples.

Telomere repeat binding factor 2 (TRF2) has a well-known function at the telomeres, which acts to protect the telomere end from being recognized as a DNA break or from unwanted recombination. This protection mechanism prevents DNA instability from mutation and subsequent severe diseases caused by the changes in DNA, such as cancer. Since TRF2 actively inhibits the DNA damage response factors from recognizing the telomere end as a DNA break, many more studies have also shown its interactions outside of the telomeres. However, very little has been discovered on the mechanisms involved in these interactions. This review aims to discuss the known function of TRF2 and its interaction with the DNA damage response (DDR) factors at both telomeric and non-telomeric regions. In this review, we will summarize recent progress and findings on the interactions between TRF2 and DDR factors at telomeres and outside of telomeres.

In addition to mediating telomerase recruitment, shelterin protein TPP1 also stimulates telomerase processivity. Assessing the in vivo significance of the latter role of TPP1 has been difficult, as TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. We sought to separate the two activities of TPP1 in regulating telomerase by considering a structure-guided mutagenesis study on the S. cerevisiae telomerase-associated Est3 protein, which revealed a TELR surface region on Est3 that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase (1). Here, we show that mutations within the structurally conserved TELR region on TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1, in a process separable from its role in recruiting telomerase to telomeres, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term cell viability.SignificanceTelomerase directs the synthesis of new telomeric repeats at chromosome ends, enabling cells to overcome the end replication problem and continue to divide. The shelterin protein TPP1 interacts with telomerase, promoting both telomerase recruitment and processivity (the addition of multiple telomeric repeats after a single substrate binding event). Here we show the identification of separation-of-function mutants of TPP1 that eliminate telomerase processivity but leave the telomerase recruitment function intact. When introduced into human cells in a homozygous manner, these mutations can induce critical telomere shortening and cellular senescence. Our observations therefore provide the first demonstration that telomerase processivity, in addition to telomerase recruitment, is a key regulatory step in vivo for continued human cell proliferation.

Senescent vascular cells are detected in atherosclerotic lesion, and its involvement in the development of atherosclerosis has been revealed; however, whether and the mechanism by which endothelial cell (EC) senescence is causally implicated in atherosclerosis remains unclear. We here investigate a role of EC senescence in atherosclerosis by utilizing EC-specific progeroid mice that overexpress the dominant negative form of telomeric repeat-binding factor 2 under the control of the Tie2 or vascular endothelial cadherin promoter. EC-specific progeria accelerated atherosclerosis in mice with target deletion of ApoE. Mechanistically, senescent ECs were markedly sensitive for inflammation-mediated VCAM-1 induction, leading to enhanced monocyte adhesion. Inhibition of NF-κB signaling abolished the enhanced inflammatory responses in senescent ECs, while NF-κB nuclear translocation in response to TNF-α were similar between young and senescent ECs. We found a higher association of VCAM-1 gene with active histone H3 trimethylated on lysine 4, leading to increased NF-κB accessibility in senescent ECs. Our data revealed that EC cellular senescence causes endothelial hyper-inflammability through epigenetic alteration, which consequently accelerates atherosclerosis. Therefore, EC senescence is a promising therapeutic target for the prevention and/or treatment of atherosclerotic disease in elderly population.

Recent studies have found that somatic gene mutations and environmental tumor‑promoting factors are both indispensable for tumor formation. Telomeric repeat‑binding factor (TRF)2 is the core component of the telomere shelterin complex, which plays an important role in chromosome stability and the maintenance of normal cell physiological states. In recent years, TRF2 and its role in tumor formation have gradually become a research hot topic, which has promoted in‑depth discussions into tumorigenesis and treatment strategies, and has achieved promising results. Some cells bypass elimination, due to either aging, apoptosis via mutations or abnormal prolongation of the mitotic cycle, and enter the telomere crisis period, where large‑scale DNA reorganization occurs repeatedly, which manifests as the precancerous cell cycle. Finally, at the end of the crisis cycle, the mutation activates either the expression level of telomerase or activates the alternative lengthening of telomere mechanism to extend the local telomeres. Under the protection of TRF2, chromosomes are gradually stabilized, immortal cells are formed and the stagewise mutation‑driven transformation of normal cells to cancer cells is completed. In addition, TRF2 also shares the characteristics of environmental tumor‑promoting factors. It acts on multiple signal transduction pathway‑related proteins associated with cell proliferation, and affects peripheral angiogenesis, inhibits the immune recognition and killing ability of the microenvironment, and maintains the stemness characteristics of tumor cells. TRF2 levels are abnormally elevated by a variety of tumor control proteins, which are more conducive to the protection of telomeres and the survival of tumor cells. In brief, the various regulatory mechanisms which tumor cells rely on to survive are organically integrated around TRF2, forming a regulatory network, which is conducive to the optimization of the survival direction of heterogeneous tumor cells, and promotes their survival and adaptability. In terms of clinical application, TRF2 is expected to become a new type of cancer prognostic marker and a new tumor treatment target. Inhibition of TRF2 overexpression could effectively cut off the core network regulating tumor cell survival, reduce drug resistance, or bypass the mutation under the pressure of tumor treatment selection, which may represent a promising therapeutic strategy for the complete eradication of tumors in the clinical setting. Based on recent research, the aim of the present review was to systematically elaborate on the basic structure and functional characteristics of TRF2 and its role in tumor formation, and to analyze the findings indicating that TRF2 deficiency or overexpression could cause severe damage to telomere function and telomere shortening, and induce DNA damage response and chromosomal instability.

RNA-protein interactions drive key cellular pathways such as protein translation, nuclear organization and genome stability maintenance. The human telomeric protein TRF2 binds to the long noncoding RNA TERRA through independent domains, including its N-terminal B domain. We previously demonstrated that TRF2 B domain binding to TERRA supports invasion of TERRA into telomeric double stranded DNA, leading to the formation of telomeric RNA:DNA hybrids. The other telomeric protein TRF1, which also binds to TERRA, suppresses this TRF2-associated activity by preventing TERRA-B domain interactions. Herein, we show that the binding of both TRF1 and TRF2 to TERRA depends on the ability of the latter to form G-quadruplex structures. Moreover, a cluster of arginines within the B domain is largely responsible for its binding to TERRA. On the other side, a patch of glutamates within the N-terminal A domain of TRF1 mainly accounts for the inhibition of TERRA-B domain complex formation. Finally, mouse TRF2 B domain binds to TERRA, similarly to its human counterpart, while mouse TRF1 A domain lacks the inhibitory activity. Our data shed further light on the complex crosstalk between telomeric proteins and RNAs and suggest a lack of functional conservation in mouse.

Telomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, upon changes in their protein composition, telomeres can cluster to form telomere-induced-foci (TIFs) or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.

Uterine leiomyoma is a benign smooth muscle tumor of the uterus that can exhibit histopathological traits that mimic malignancy. Telomere shortening is an early event in tumorigenesis and telomerase activation facilitates tumor progression later in the course of carcinogenesis. Telomeric repeat‑binding factor (TRF)1 and TRF2 protect telomeres, and their gene expression levels are dysregulated in various cancer types. However, the roles of telomeres and telomere protection proteins in uterine leiomyoma remain largely unknown. In this study, telomere length and the mRNA levels of various telomere‑related genes in normal tissues and leiomyoma were determined, and their relationships were evaluated. Uterine leiomyoma and normal myometrium were surgically obtained from 18 and 13 patients, respectively. Telomere length and gene expression were determined by Southern blot analysis and reverse transcription‑quantitative PCR, respectively. In matched samples, telomeres were consistently shorter in leiomyoma tissue than in adjacent normal tissue. TRF1, TRF2, PIN2‑interacting telomerase inhibitor 1 (PINX1), and telomerase RNA component were expressed at comparable levels in both leiomyoma and normal tissues. None of these genes were associated with telomere length in leiomyoma. All tested tissues were negative for telomerase reverse transcriptase, which encodes the catalytic component of telomerase, indicating that cells in uterine leiomyoma were not immortalized. In summary, telomere erosion, which reflects active proliferation during tumor evolution, was evident in uterine leiomyoma. Steady‑state expression of TRF1, TRF2 and PINX1 may be important for maintenance of telomere integrity in leiomyoma, where telomere length is shortened.

Cellular senescence triggers various types of heterochromatin remodeling that contribute to aging. However, the age-related mechanisms that lead to these epigenetic alterations remain elusive. Here, we asked how two key aging hallmarks, telomere shortening and constitutive heterochromatin loss, are mechanistically connected during senescence. We show that, at the onset of senescence, pericentromeric heterochromatin is specifically dismantled consisting of chromatin decondensation, accumulation of DNA breakages, illegitimate recombination and loss of DNA. This process is caused by telomere shortening or genotoxic stress by a sequence of events starting from TP53-dependent downregulation of the telomere protective protein TRF2. The resulting loss of TRF2 at pericentromeres triggers DNA breaks activating ATM, which in turn leads to heterochromatin decondensation by releasing KAP1 and Lamin B1, recombination and satellite DNA excision found in the cytosol associated with cGAS. This TP53–TRF2 axis activates the interferon response and the formation of chromosome rearrangements when the cells escape the senescent growth arrest. Overall, these results reveal the role of TP53 as pericentromeric disassembler and define the basic principles of how a TP53-dependent senescence inducer hierarchically leads to selective pericentromeric dismantling through the downregulation of TRF2.

Pt-ttpy (tolyl terpyridin-Pt complex) covalently binds to G-quadruplex (G4) structures in vitro and to telomeres in cellulo via its Pt moiety. Here, we identified its targets in the human genome, in comparison to Pt-tpy, its derivative without G4 affinity, and cisplatin. Pt-ttpy, but not Pt-tpy, induces the release of the shelterin protein TRF2 from telomeres concomitantly to the formation of DNA damage foci at telomeres but also at other chromosomal locations. γ-H2AX chromatin immunoprecipitation (ChIP-seq) after treatment with Pt-ttpy or cisplatin revealed accumulation in G- and A-rich tandemly repeated sequences, but not particularly in potential G4 forming sequences. Collectively, Pt-ttpy presents dual targeting efficiency on DNA, by inducing telomere dysfunction and genomic DNA damage at specific loci.

Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of multiple vascular pathologies, including following neointimal formation after injury and atherosclerosis. However, human VSMCs in advanced atherosclerotic lesions show reduced cell proliferation, extensive and persistent DNA damage, and features of premature cell senescence. Here, we report that stress-induced premature senescence (SIPS) and stable expression of a telomeric repeat-binding factor 2 protein mutant (TRF2T188A) induce senescence of human VSMCs, associated with persistent telomeric DNA damage. VSMC senescence is associated with formation of micronuclei, activation of cGAS-STING cytoplasmic sensing, and induction of multiple pro-inflammatory cytokines. VSMC-specific TRF2T188A expression in a multicolor clonal VSMC-tracking mouse model shows no change in VSMC clonal patches after injury, but an increase in neointima formation, outward remodeling, senescence and immune/inflammatory cell infiltration or retention. We suggest that persistent telomere damage in VSMCs inducing cell senescence has a major role in driving persistent inflammation in vascular disease., Anna Uryga and Mandy Grootaert et al. combine cell culture and animal models to examine how senescence of human vascular smooth muscle cells (VSMCs) and persistent telomere damage drive inflammation. Their results suggest that telomere injury can be the primary cause of premature senescence in VSMCs, and that DNA damage can be a major cause of persistent inflammation in vascular disease.

Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end joining. TPP1 facilitates end protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1’s functions. Our results extend current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with their selectively impacting end replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes.