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Telomere-binding proteins constituting the shelterin complex have been studied primarily for telomeric functions. However, mounting evidence shows non-telomeric binding and gene regulation by shelterin factors. This raises a key question—do telomeres impact binding of shelterin proteins at distal non-telomeric sites? Here we show that binding of the telomere-repeat-binding-factor-2 (TRF2) at promoters ~60 Mb from telomeres depends on telomere length in human cells. Promoter TRF2 occupancy was depleted in cells with elongated telomeres resulting in altered TRF2-mediated transcription of distal genes. In addition, histone modifications—activation (H3K4me1 and H3K4me3) as well as silencing marks (H3K27me3)—at distal promoters were telomere length-dependent. These demonstrate that transcription, and the epigenetic state, of telomere-distal promoters can be influenced by telomere length. Molecular links between telomeres and the extra-telomeric genome, emerging from findings here, might have important implications in telomere-related physiology, particularly ageing and cancer., Author summary Telomeres (special DNA-protein assemblies that protect chromosome ends) affect ageing and diseases such as cancer. Although this has been recognized for many years, biological processes that connect telomeres to ageing, cancer and other cellular functions remain to be fully understood. Certain proteins, believed to be only telomere-associated, engage DNA outside telomeres. This raises an interesting question. Does telomere length influence how telomere-binding proteins associate with DNA at regions distal from telomeres. If so, how does this impact function? Motivated by these questions, in the present studies we tested if extra-telomeric binding of the well-known telomere-repeat-binding-actor-2 (TRF2) depends on telomere length. Our results show that the level of DNA-bound TRF2 at telomere-distal sites changes as telomeres shorten or elongate. Consequently, TRF2-mediated gene regulation affects many genes. Notably, histone modifications that dictate chromatin compaction and access to regulatory factors, at sites distant from telomere ends also depended on telomere length. Together, this links the state of telomeres to gene regulation and epigenetics directly in ways not previously appreciated that might impact a more complete understanding of molecular processes underlying ageing and cancer.

TRF2 and TRF1 are a key component in shelterin complex that associates with telomeric DNA and protects chromosome ends. BRM is a core ATPase subunit of SWI/SNF chromatin remodeling complex. Whether and how BRM-SWI/SNF complex is engaged in chromatin end protection by telomeres is unknown. Here, we report that depletion of BRM does not affect heterochromatin state of telomeres, but results in telomere dysfunctional phenomena including telomere uncapping and replication defect. Mechanistically, expression of TRF2 and TRF1 is jointly regulated by BRM-SWI/SNF complex, which is localized to promoter region of both genes and facilitates their transcription. BRM-deficient cells bear increased TRF2-free or TRF1-free telomeres due to insufficient expression. Importantly, BRM depletion-induced telomere uncapping or replication defect can be rescued by compensatory expression of exogenous TRF2 or TRF1, respectively. Together, these results identify a new function of BRM-SWI/SNF complex in enabling functional telomeres for maintaining genome stability., Author summary Human telomeres consist of repetitive “TTAGGG” DNA sequences and associated shelterin complex, which maintain genomic stability by preventing linear chromosome ends from being recognized as broken DNA. TRF1 and TRF2, as key components of shelterin complex, directly associate with double strand telomeric DNA. In this study, we discovered that both TRF1 and TRF2 are jointly regulated by BRM-SWI/SNF complex. Depletion of BRM led to insufficient amount of TRF1 and TRF2, which is associated with telomere replication defect and telomere uncapping. More importantly, these phenomena can be rescued by ectopically expressed TRF1 and TRF2. Our work demonstrates a specific role of BRM-SWI/SNF complex on safeguarding genome stability by enabling functional telomeres.

Telomeric repeat-containing RNAs (TERRAs) are long noncoding RNAs transcribed from subtelomeres toward telomeric repeat tracts, which have been implicated in telomere protection and heterochromatin formation. Genotoxic stress leads to upregulation of TERRAs. However, the mechanism of DNA damage-mediated TERRA induction remains elusive. Here, we treated HeLa cells with etoposide, a DNA double-strand break-generating agent, for various times and monitored the levels of TERRAs. Etoposide treatment led to a gradual time-dependent increase in TERRAs. Etoposide-mediated induction was evident in many TERRAs arising from various chromosome loci, including 20q and XpYp. Chromatin immunoprecipitation assays revealed no significant changes in the occupancy of RNA polymerase II at telomeres upon etoposide treatment. Interestingly, TERRAs arising from 20q, XpYp, 10q, and 13q degraded at slower rates in cells treated with etoposide, while degradation rates of TERRAs from many loci tested were nearly identical in both etoposide- and mock-treated cells. Telomere damage occurred from early time points of etoposide treatment, but telomere lengths and abundance of telomeric repeat-binding factor 2 (TRF2) at telomeres remained unchanged. In summary, etoposide treatment led to telomere damage and TERRA accumulation, but telomere lengths and TRF2-mediated telomere integrity were maintained. Etoposide-mediated TERRA accumulation could be attributed partly to RNA stabilization. These findings may provide insight into the post-transcriptional regulation of TERRAs in response to DNA damage.

The general transcription factor TBP (TATA-box binding protein) and its associated factors (TAFs) together form the TFIID complex, which directs transcription initiation. Through RNAi and mutant analysis, we identified a specific TBP family protein, TRF2, and a set of TAFs that regulate lipid droplet (LD) size in the Drosophila larval fat body. Among the three Drosophila TBP genes, trf2, tbp and trf1, only loss of function of trf2 results in increased LD size. Moreover, TRF2 and TAF9 regulate fatty acid composition of several classes of phospholipids. Through RNA profiling, we found that TRF2 and TAF9 affects the transcription of a common set of genes, including peroxisomal fatty acid β-oxidation-related genes that affect phospholipid fatty acid composition. We also found that knockdown of several TRF2 and TAF9 target genes results in large LDs, a phenotype which is similar to that of trf2 mutants. Together, these findings provide new insights into the specific role of the general transcription machinery in lipid homeostasis., Author summary Lipid droplets (LD) are main lipid storage structures in most cells. The size of LDs varies greatly in different cell types or different metabolic states to accommodate cellular functions and metabolism demands. How cells regulate the lipid storage and LD dynamics is not fully understood. Here, we identified that general transcription factors, including a specific TBP (TATA-box binding protein) family protein TRF2 (TBP-related factor 2) and several TAFs (TBP-associated factors), regulate LD size in the fruitfly larval fat body. Moreover, quantitated lipid analysis reveals that TRF2 and TAF9 affect the fatty acid composition of several classes of phospholipids. We showed that TRF2 and TAF9 regulate transcription of several target genes, including peroxisomal fatty acid β-oxidation-related genes which likely mediate the effect of TRF2 and TAF9 on phospholipid fatty acid composition. We also found that overexpression of some target genes restores the LD phenotype in trf2 mutants. Our findings therefore reveal specific roles of general transcription factors in lipid homeostasis.

Telomeres are capped at the ends of eukaryotic chromosomes and are composed of TTAGGG repeats bound to the shelterin complex. Here we report that a replication-dependent histone H2A isotype, H2ac, was associated with telomeres in human cells and co-immunoprecipitates with telomere repeat factor 2 (TRF2) and protection of telomeres protein 1 (POT1), whereas other histone H2A isotypes and mutations of H2ac did not bind to telomeres or these two proteins. The amino terminal basic domain of TRF2 was necessary for the association with H2ac and for the recruitment of H2ac to telomeres. Depletion of H2ac led to loss of telomeric repeat sequences, the appearance of dysfunctional telomeres, and chromosomal instability, including chromosomal breaks and anaphase bridges, as well as accumulation of telomere-associated DNA damage factors in H2ac depleted cells. Additionally, knockdown of H2ac elicits an ATM-dependent DNA damage response at telomeres and depletion of XPF protects telomeres against H2ac-deficiency-induced G-strand overhangs loss and DNA damage response, and prevents chromosomal instability. These findings suggest that the H2A isotype, H2ac, plays an essential role in maintaining telomere functional integrity.

Trypanosoma brucei causes debilitating human African trypanosomiasis and evades the host’s immune response by regularly switching its major surface antigen, VSG, which is expressed exclusively from subtelomeric loci. We previously showed that two interacting telomere proteins, TbTRF and TbTIF2, are essential for cell proliferation and suppress VSG switching by inhibiting DNA recombination events involving the whole active VSG expression site. We now find that TbTIF2 stabilizes TbTRF protein levels by inhibiting their degradation by the 26S proteasome, indicating that decreased TbTRF protein levels in TbTIF2-depleted cells contribute to more frequent VSG switching and eventual cell growth arrest. Surprisingly, although TbTIF2 depletion leads to more subtelomeric DNA double strand breaks (DSBs) that are both potent VSG switching inducers and detrimental to cell viability, TbTRF depletion does not increase the amount of DSBs inside subtelomeric VSG expression sites. Furthermore, expressing an ectopic allele of F2H-TbTRF in TbTIF2 RNAi cells allowed cells to maintain normal TbTRF protein levels for a longer frame of time. This resulted in a mildly better cell growth and partially suppressed the phenotype of increased VSG switching frequency but did not suppress the phenotype of more subtelomeric DSBs in TbTIF2-depleted cells. Therefore, TbTIF2 depletion has two parallel effects: decreased TbTRF protein levels and increased subtelomeric DSBs, both resulting in an acute increased VSG switching frequency and eventual cell growth arrest.

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication., Author Summary Chromosome ends are capped by specialized structures called telomeres, which protect chromosomes from deterioration, incomplete replication and end-to-end fusion. Defects in telomere structure and/or function may have a strong impact on human health, leading to premature aging and a variety of diseases including cancer. One of the most important tasks to understand and possibly prevent the consequences of telomere dysfunction is the identification and characterization of telomere-associated proteins. Here we show for the first time that human telomeric proteins can be identified on the basis of their homology with those that protect the telomeres of the fruit fly Drosophila melanogaster. Although flies and humans elongate their telomeres through different mechanisms, our studies suggested that a subset of Drosophila telomere-associated proteins have conserved human counterparts. Based on this hypothesis we identified and characterized a novel human telomeric protein called AKTIP. We show that AKTIP binds the components of the shelterin multiprotein complex, which caps and protects the human telomeres. AKTIP-depleted chromosomes exhibit an accumulation of DNA repair factors at their ends (telomere dysfunction foci), which are diagnostic of telomere damage. Loss of AKTIP results in a general impairment of DNA synthesis and in defective telomere replication. Collectively, our results indicate that AKTIP cooperates with the shelterin component TRF1 to ensure proper telomere replication.

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ., Author Summary The ends of chromosomes, called telomeres, prevent chromosome ends from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion by forming a specialized nucleo-protein complex. The critical function of telomeres in end protection has a downside, in that it interferes with the repair of DSBs that occur near telomeres. DSBs are critical DNA lesions, because if they are not repaired correctly they can result in gross chromosome rearrangements (GCRs). As a result, the deficiency in DSB repair near telomeres has now been implicated in ageing, by promoting cell senescence, and cancer, by promoting telomere dysfunction due to oncogene-induced replication stress. The studies presented here demonstrate that DSBs near telomeres commonly result in GCRs in a human tumor cell line. Moreover, our results demonstrate that the mechanism of repair of telomeric DSBs is very different from the mechanism of repair of DSBs at other locations, supporting our hypothesis that the deficiency in repair of DSBs near telomeres is a result of the abnormal processing of DSBs due to the presence of telomeric proteins. Understanding the mechanism responsible for the deficiency in DSB repair near telomeres will provide important insights into critical human disease pathways.

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidence towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.

Telomere binding factors viz. TRF1 and TRF2 are a part of sheltrin complex that are present exclusively at the ends of chromosomes. These factors play an important role in maintaining chromosomal integrity at the ends. However, their status and role are not clear in renal cell carcinoma (RCC). Therefore, the present study was conducted to evaluate TRF1 and TRF2 expressions in RCC tissues. Further, the role of these factors involved in tumorigenesis was elucidated by gene silencing using siRNA in RCC cell line (A498). The present study documented a significant over-expression of TRF1 (P = 0.005) and TRF2 (P = 0.0048) mRNAs by real time PCR in RCC tissues as compared with adjacent normal kidney tissues. Immunohistochemistry studies also revealed higher expression of TRF1 and TRF2 proteins in RCC. Moreover, TRF1 or TRF2 gene silencing using siRNA showed marked reduction in proliferation of RCC cells (P = 0.000). Further, significantly induced cell cycle arrest (P = 0.000) and apoptosis of RCC cells (P = 0.000) was documented upon TRF1 or TRF2 gene silencing. Henceforth, the results deduce that TRF1 or TRF2 inhibitions play an important role in the induction of apoptosis in A498 cells, which may serve as a potential therapeutic target in RCC.

Background Telomeres alteration during carcinogenesis and tumor progression has been described in several cancer types. Telomeres length is stabilized by telomerase (h-TERT) and controlled by several proteins that protect telomere integrity, such as the Telomere Repeat-binding Factor (TRF) 1 and 2 and the tankyrase-poli-ADP-ribose polymerase (TANKs-PARP) complex. Objective To investigate telomere dysfunction in astroglial brain tumors we analyzed telomeres length, telomerase activity and the expression of a panel of genes controlling the length and structure of telomeres in tissue samples obtained in vivo from astroglial brain tumors with different grade of malignancy. Materials and Methods Eight Low Grade Astrocytomas (LGA), 11 Anaplastic Astrocytomas (AA) and 11 Glioblastoma Multiforme (GBM) samples were analyzed. Three samples of normal brain tissue (NBT) were used as controls. Telomeres length was assessed through Southern Blotting. Telomerase activity was evaluated by a telomere repeat amplification protocol (TRAP) assay. The expression levels of TRF1, TRF2, h-TERT and TANKs-PARP complex were determined through Immunoblotting and RT-PCR. Results LGA were featured by an up-regulation of TRF1 and 2 and by shorter telomeres. Conversely, AA and GBM were featured by a down-regulation of TRF1 and 2 and an up-regulation of both telomerase and TANKs-PARP complex. Conclusions In human astroglial brain tumours, up-regulation of TRF1 and TRF2 occurs in the early stages of carcinogenesis determining telomeres shortening and genomic instability. In a later stage, up-regulation of PARP-TANKs and telomerase activation may occur together with an ADP-ribosylation of TRF1, causing a reduced ability to bind telomeric DNA, telomeres elongation and tumor malignant progression.

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms., Author Summary By performing genetic mutant screens using senescence-associated biomarkers, we show that the zebrafish is a tractable model system for the study of aging. In vertebrate organisms, it has not previously been possible to carry out systematic screens for genes that are important for stress responses and aging in an unbiased way. However, such vertebrate models are of considerable importance, given the provocative evidence of common biochemical and functional pathways modulating stress responses and lifespan as well as aging in a wide range of organisms. Our present study has successfully employed a colorimetric high-throughput method using a senescence-associated β-galactosidase-based assay to screen for mutations that alter the stress responses in zebrafish embryos, in the hope that these might represent potential aging mutants. Subsequently, the mutations identified by embryonic senescence have indeed displayed adult aging-related phenotypes in zebrafish. Hence, our method for the identification of mutant zebrafish has the immediate potential to accelerate the discovery of novel genes and new functions relevant for our understanding of aging processes in vertebrates. Such knowledge will be essential for the ultimate development of pharmacological, nutritional, and behavioral interventions for the amelioration of oxidative stress- and age-associated diseases and disabilities in humans.