Telomere-binding Protein Research Articles

Subtelomere-binding protein Tbf1 and telomere-binding protein Rap1 collaborate to inhibit localization of the Mre11 complex to DNA ends in budding yeast

Chromosome ends, known as telomeres, have to be distinguished from DNA double-strand breaks that activate DNA damage checkpoints. In budding yeast, the Mre11-Rad50-Xrs2 (MRX) complex associates with DNA ends and promotes checkpoint activation. Rap1 binds to double-stranded telomeric regions and recruits Rif1 and Rif2 to telomeres. Rap1 collaborates with Rif1 and Rif2 and inhibits MRX localization to DNA ends. This Rap1-Rif1-Rif2 function becomes attenuated at shortened telomeres. Here we show that Rap1 acts together with the subtelomere-binding protein Tbf1 and inhibits MRX localization to DNA ends. The placement of a subtelomeric sequence or TTAGGG repeats together with a short telomeric TG repeat sequence inhibits MRX accumulation at nearby DNA ends in a Tbf1-dependent manner. Moreover, tethering of both Tbf1 and Rap1 proteins decreases MRX and Tel1 accumulation at nearby DNA ends. This Tbf1- and Rap1-dependent pathway operates independently of Rif1 or Rif2 function. Depletion of Tbf1 protein stimulates checkpoint activation in cells containing short telomeres but not in cells containing normal-length telomeres. These data support a model in which Tbf1 and Rap1 collaborate to maintain genomic stability of short telomeres.

The change of telomere protein in BEAS-2B malignant transformation cell induced by coal tar pitch smoke extracts

By testing the changes of telomere binding protein in malignant transformation BEAS-2B cells induced by coal tar pitch smoke extracts, to study the role of protection of telomeres 1 (POT1), telomeric repeat binding factor 1 (TRF1) and TRF2 in tumorgenesis that contact with coal tar pitch. The BEAS-2B cells were induced by coal tar pitch smoke extracts to form malignant transformation cell model in vitro. The gene expression levels of mRNA were assessed by real-time quantitative RT-PCR, the protein expression variations were determined by cell culture overslip of immunohistochemical methods. In malignant transformation cells, the mRNA expression level (POT1: 0.63 ± 0.04, TRF1: 0.36 ± 0.01) and the protein expression level (POT1: 0.36 ± 0.05, TRF1: 0.09 ± 0.03) of POT1 and TRF1 was statistically significant decreased compared to that of BEAS-2B group (mRNA: POT1: 1.00 ± 0.04, TRF1: 1.01 ± 0.16; protein: POT1: 0.55 ± 0.07, TRF1: 0.27 ± 0.07) and DMSO group (mRNA: POT1: 0.89 ± 0.12, TRF1: 0.90 ± 0.08; protein: POT1: 0.55 ± 0.10, TRF1: 0.26 ± 0.04) (P < 0.05); mRNA expression level (1.45 ± 0.07) and the protein expression level (0.88 ± 0.06) of TRF2 was increased compared to that of BEAS-2B group (mRNA: 1.00 ± 0.07, protein: 0.48 ± 0.06) and DMSO group (mRNA: 1.00 ± 0.06, protein: 0.50 ± 0.06) (P < 0.05). The change of gene and protein expression level in POT1, TRF1, and TRF2 involved in the process that evolved into malignant transformation in bronchial epithelial cells BEAS-2B induced by coal tar pitch smoke extracts.

Palmitoylation controls the dynamics of budding-yeast heterochromatin via the telomere-binding protein Rif1

The posttranslational addition of palmitate to cysteines occurs ubiquitously in eukaryotic cells, where it functions in anchoring target proteins to membranes and in vesicular trafficking. Here we show that the Saccharomyces cerevisiae palmitoyltransferase Pfa4 enhanced heterochromatin formation at the cryptic mating-type loci HMR and HML via Rif1, a telomere regulatory protein. Acylated Rif1 was detected in extracts from wild-type but not pfa4Δ mutant cells. In a pfa4Δ mutant, Rif1-GFP dispersed away from foci positioned at the nuclear periphery into the nucleoplasm. Sir3-GFP distribution was also perturbed, indicating a change in the nuclear dynamics of heterochromatin proteins. Genetic analyses indicated that PFA4 functioned upstream of RIF1. Surprisingly, the pfa4Δ mutation had only mild effects on telomeric regulation, suggesting Rif1's roles at HM loci and telomeres were more complexly related than previously thought. These data supported a model in which Pfa4-dependent palmitoylation of Rif1 anchored it to the inner nuclear membrane, influencing its role in heterochromatin dynamics.

Silencing of the hPOT1 gene by RNA inference promotes apoptosis and inhibits proliferation and aggressive phenotype of gastric cancer cells, likely through up-regulating PinX1 expression

BackgroundThe human protection of telomeres 1 (hPOT1) protein, a single-strand telomeric DNA binding protein, plays an important role in telomere protection and telomere length regulation. However, its effect on invasion...

Expressional Profiling of Telomerase and Telomere-Associated Molecules in the Rat Testis and Seminal Vesicle during Postnatal Developmental Period

Maintenance of adequate telomere length in developing cells is the most important concern to preserve the integrity of the genome. The length of telomere is strictly regulated by numerous telomere-binding proteins and/or interacting factors. Even though the expression of telomerase in the male reproductive tract has been characterized, developmental expressional profiling of telomerase and other telomere-associated proteins has not been determined in detail. The present study was attempted to examine expression patterns of catalytic subunit (Tert) and RNA component (Terc) of telomerase and two telomerase associated factors, telomerase associated protein 1 (Tep1) and TERF1 (TRF1) interacting nuclear factor 2 (Tinf2) in the testis and seminal vesicle of male rat during postnatal development. The real-time PCR analysis was utilized to quantify mRNA expression of molecules. The abundance of Tep1 mRNA in the testis and seminal vesicle was the highest at 5 months of age. Expressional fluctuation of Tinf2 during postnatal development was found in the testis, while expression of Tinf2 in the seminal vesicle was gradually increased until 5 months of age and then significantly decreased later. mRNA level of Tert gene in the testis was significantly increased at the adult and the elder, while the highest expression of Tert gene in the seminal vesicle was found at 5 months of age. Expression of Terc transcript in the testis and seminal vesicle was the highest at 5 months of age, followed by significant reduction at 1 and 2 years of ages. Such differential gene expression of telomere-associated factors and telomerase components in different male reproductive tissues during postnatal development indicates that maintenance of telomere length would be regulated in tissue- and/or age-specific manners. (Key words : Telomerase, Testis, Seminal vesicle, Development, Telomerase-associated molecule)

Cyclin B-dependent kinase 1 regulates human TRF1 to modulate the resolution of sister telomeres

Cyclin B-Cdk1 is a key mediator of mitotic entry, however little is known about its role in the separation of sister chromatids. Here we report that upon mitotic entry, Cdk1 specifically phosphorylates threonine 371 of TRF1, a telomere binding protein implicated in the regulation of sister telomere cohesion. Such phosphorylation is removed in late mitosis when Cdk1 activity is inhibited, indicative of a tight regulation of T371 phosphorylation. We show that T371 phosphorylation by Cdk1 keeps TRF1 free of chromatin and this phosphorylation is associated with loss of telomere-bound TRF1 and TIN2, and a reduction in telomere heterochromatin. We find that a phosphomimic mutation at T371 of TRF1 induces telomere deprotection, resulting in telomere loss and the formation of telomere fusions whereas a non-phosphorylatable substitution of T371 blocks sister telomere resolution, promotes micronuclei formation and impairs cell proliferation. Our work suggests that Cdk1 controls TRF1 association with telomeres to facilitate temporal telomere de-protection essential for sister telomere resolution.

Transcriptional Activation of TINF2, a Gene Encoding the Telomere-Associated Protein TIN2, by Sp1 and NF-κB Factors

The expression of the telomere-associated protein TIN2 has been shown to be essential for early embryonic development in mice and for development of a variety of human malignancies. Recently, germ-line mutations in TINF2, which encodes for the TIN2 protein, have been identified in a number of patients with bone-marrow failure syndromes. Yet, the molecular mechanisms that regulate TINF2 expression are largely unknown. To elucidate the mechanisms involved in human TINF2 regulation, we cloned a 2.7 kb genomic DNA fragment containing the putative promoter region and, through deletion analysis, identified a 406 bp region that functions as a minimal promoter. This promoter proximal region is predicted to contain several putative Sp1 and NF-κB binding sites based on bioinformatic analysis. Direct binding of the Sp1 and NF-κB transcription factors to the TIN2 promoter sequence was demonstrated by electrophoretic mobility shift assay (EMSA) and/or chromatin immunoprecipitation (ChIP) assays. Transfection of a plasmid carrying the Sp1 transcription factor into Sp-deficient SL2 cells strongly activated TIN2 promoter-driven luciferase reporter expression. Similarly, the NF-κB molecules p50 and p65 were found to strongly activate luciferase expression in NF-κB knockout MEFs. Mutating the predicted transcription factor binding sites effectively reduced TIN2 promoter activity. Various known chemical inhibitors of Sp1 and NF-κB could also strongly inhibit TIN2 transcriptional activity. Collectively, our results demonstrate the important roles that Sp1 and NF-κB play in regulating the expression of the human telomere-binding protein TIN2, which can shed important light on its possible role in causing various forms of human diseases and cancers.

Suppression of telomere-binding protein gene expression represses seed and fruit development in tomato

Tomato ( Solanum lycopersicum L.) plants were transformed with an antisense construct of a cDNA encoding tomato telomere-binding protein (LeTBP1) to describe the role of a telomere-binding protein at the whole plant level. Fruit size decreased corresponding to the degree of suppression of LeTBP1 expression. This inhibition of fruit development was likely due to a decrease in the number of seeds in the LeTBP1 antisense plants. Pollen fertility and pollen germination rate decreased in accordance with the degree of suppression of LeTBP1 expression. Ovule viability was also reduced in the LeTBP1 antisense plants. Although plant height was somewhat reduced in the antisense plants compared to the control plants, the number and weight of leaves were unaffected by LeTBP1 suppression. The number and morphology of flowers were also normal in the antisense plants. These indicate that reduced fertility in the antisense plants is not an indirect effect of altered vegetative growth. LeTBP1 expression was sensitive to temperature stress in wild-type plants. We conclude that LeTBP1 plays a critical role in seed and fruit development rather than vegetative growth and flower formation.

Subtelomeric repetitive elements determine TERRA regulation by Rap1/Rif and Rap1/Sir complexes in yeast

Telomeric repeat-containing RNA (TERRA) has been implicated in the control of heterochromatin and telomerase. We demonstrate that yeast TERRA is regulated by telomere-binding proteins in a chromosome-end-specific manner that is dependent on subtelomeric repetitive DNA elements. At telomeres that contain only X-elements, the Rap1 carboxy-terminal domain recruits the Sir2/3/4 and Rif1/2 complexes to repress transcription in addition to promoting Rat1-nuclease-dependent TERRA degradation. At telomeres that contain Y' elements, however, Rap1 represses TERRA through recruitment of Rif1 and Rif2. Our work emphasizes the importance of subtelomeric DNA in the control of telomeric protein composition and telomere transcription.

Ku circles the telomere?

Ku circles the telomere?

The Telomere Binding Protein TRF2 Induces Chromatin Compaction

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.

Abstract 3886: The G-quadruplex induced DNA damage at telomeres is regulated by ATM

Abstract Quadruplex-forming DNA sequences are present throughout the genome, including in the telomeres. Our group has shown that quadruplex-forming oligonucleotides selectively inhibit proliferation of transformed cells. One of these oligonucleotides, AS1411, is currently in a Phase IIb clinical trial. We have recently shown that the c-myc promoter quadruplex-forming sequence, Pu-27, also inhibits growth of malignant cells. We have hypothesized that one potential mechanism of this effect is competition for telomere-binding proteins. If this is correct, we would expect this effect to be mediated by the DNA repair response. Telomere integrity is maintained by the shelterin protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks. Like many other DNA damage response proteins, ATM has been shown to play a role in the maintenance of telomere integrity. We have shown that ATM deficient cells are more sensitive to growth inhibition by Pu-27 whereas ATM proficient cells were relatively resistant to Pu-27. Co-immunostaining with γ-H2AX and TRF1 revealed that the sensitivity to Pu-27 in ATM deficient cells was associated with phosphorylation of H2AX, an indicator of DNA double strand break and induction of TIFs (Telomere-dysfunction Induced Foci). There was induction of γ-H2AX and downregulation of shelterin proteins TRF2 and TRF1 in Pu-27 treated ATM deficient cells (as determined by western blotting). U937 leukemia cells, which were sensitive to Pu-27, became relatively resistant when they were stably transfected with a vector expressing a dominant negative mutant of the shelterin protein TRF2 (dn-TRF2). This suggests that Pu-27 may act by altering the telomere-shelterin complex. This idea was further substantiated by the fact that ALT (Alternative Lengthening of Telomere) cells were also relatively resistant to Pu27. A549 cells, which were sensitive to Pu-27, also showed abundant presence of TIF compared to untreated cells. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, this data suggests that ATM plays a pivotal role in Pu-27 induced DNA damage at telomere ends, which occurs in response to a Pu-27 induced alteration in telomere-shelterin homeostasis. It is likely that this response is induced in a p53 independent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2011-3886

Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair

We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.

Roles of NtGTBP1 in telomere stability

Telomeres are protective nucleoprotein structures at the ends of linear eukaryotic chromosomes. In contrast to double-stranded-specific telomere-binding proteins, the cellular roles of single-stranded-specific telomeric proteins are not well understood in higher plants. Three highly conserved tobacco G-strand-specific telomere-binding protein paralogs (NtGTBP1, NtGTBP2, and NtGTBP3) were identified and characterized. All three NtGTBPs were able to bind specifically to the plant single-stranded telomeric repeat elements in vitro with similar affinities. Suppression of NtGTBP1 by means of the RNAi-mediated gene knock-down method resulted in developmental defects in transgenic tobacco plants accompanied by lengthened telomeres, extra-chromosomal telomeric circles, and abnormal anaphase bridges. These results suggest that the down-regulation of NtGTBP1 results in genome instability. NtGTBP1 prevented in vitro strand invasion, a prerequisite process for inter-chromosomal telomeric recombination. Therefore, tobacco NtGTBP1 is one of the essential factors for telomere stability. Because abnormal telomeric elongation and recombination due to the suppression of NtGTBP1 are reminiscent of the recombinational telomere lengthening mechanism that purportedly operates in telomerase negative cancer cells, it is of interest to investigate whether telomeric recombination is associated with cell death in animal systems.

Exercise modifies age‐related telomere dynamics in multiple tissues of CAST/Ei mice

Telomere length has recently been positively associated with physical activity and exercise training in white blood cells, but data in other tissues are limited. We sought to determine the effects of age and exercise on telomere dynamics in heart, liver and skeletal muscle in CAST/EiJ mice. Twenty‐seven CAST/EiJ mice were separated into three groups: baseline (BL; n = 6; 8 wk old), exercise (EX, n = 10), and sedentary (SED, n = 11). EX animals had prolonged access to a running wheel from 8 wks of age until 52 wks of age (6.2±2.3km/d). SED animals were housed individually without a running wheel until 52 wk old. Telomere length, telomerase enzyme activity, mRNA and protein levels of telomere‐associated genes were determined. In heart and liver, telomere length decreased with age (SED vs BL) while exercise attenuated the loss of telomere length (P≤0.01). In skeletal muscle, age had no effect on telomere length vs BL but EX significantly reduced telomere length. Telomerase enzyme activity was not different in SED or EX vs BL in heart or liver, but was elevated vs SED in EX skeletal muscle. In general mRNA levels of telomere binding proteins (Trf1/2, Pot1a/b) decreased in heart and skeletal muscle with age, and EX was able to significantly attenuate this reduction, particularly in the heart (P≤0.05); no changes were observed in the liver. We conclude that telomere dynamics in vivo respond to age and exercise in a tissue specific manner.Support provided by NIH/NIA AG‐00268 and the Dept. of Kinesiology Graduate Student Research Initiative Fund.

MAPK signaling is associated with acute exercise‐induced changes in mRNA levels of telomere‐related genes

We examined the effects of an acute exercise bout on mRNA levels of telomere related genes in cardiac muscle. A secondary aim was to investigate stress response pathways (p38 and Erk1/2 MAPKs) previously associated with shelterin proteins. Twenty female C57/BL6J mice were assigned to 3 groups: baseline animals (BL; N=4) not exposed to exercise; and two groups of animals exposed to 30 min of treadmill running (66% peak speed; 23±4m/min; 7° incline) and sacrificed immediately (EXIMD; N=8) and 1h following (EX1h; N=8) the running bout. Protein content and mRNA levels for various targets were analyzed via immunoblotting and semiquantitative RT-PCR. Compared to BL, EXIMD animals showed increases in mRNA levels of telomere-repeat binding factor 1 (Trf1; +61%; p = .01) and 2 (Trf2; +47%; p = .04), DNA damage response gene Ku80 (+76%; p = .01) and cell cycle gene Chk2 (+69%; p = .03). In EX1h animals, up-regulation of mRNA levels of protection of telomeres 1a (Pot1a; +35%; p = .04) and Ku80 (+69%; p = .02) were observed compared to BL. Trf1 mRNA levels differed from EXIMD (+61% vs +11%; p = .01). Preliminary experiments indicated increases in phosphorylated p38-MAPK and decreases in phosphorylated Erk1/2 in EXIMD animals compared to BL. These findings indicate a novel regulatory pathway for cardiac telomere binding proteins in response to acute exercise. Supported by UMD Graduate Research Initiative Fund and NIH training grant AG000268

Using the Telobox to Search for Plant Telomere Binding Proteins

Using the Telobox to Search for Plant Telomere Binding Proteins

Role of HMGB Proteins in Chromatin Dynamics and Telomere Maintenance in Arabidopsis thaliana

Chromosome stability is conditioned by functional chromatin structure of chromosome ends - telomeres. Organisation and regulation of telomere maintenance represent a complex process whose details still remain enigmatic, especially in plants. Several telomere-binding or telomere-associated proteins and distinct epigenetic marks have been shown to influence telomere length and telomerase activity. HMGB proteins play important role in dynamic changes of chromatin structure and are involved in regulation of cellular processes of key importance, such as replication, transcription, recombination and DNA-repair. HMGB proteins in plants are more diversified than in other eukaryotes. Here, we summarise the roles of plant HMGB proteins in regulation of chromatin structure and dynamics and report on the newly identified role of AtHMGB1 protein in the regulation of plant telomere length. Astonishingly, contrary to mice mHMGB1 homologue, AtHMGB1 does not affect telomerase activity and AtHMGB1 loss or overexpression does not cause any obvious changes in chromatin architecture.

Using the Telobox to Search for Plant Telomere Binding Proteins

Telobox is a Myb-related DNA-binding domain which is present in a number of yeast, plant and animal proteins. Its capacity to bind preferentially double-stranded telomeric DNA has been used in numerous studies to search for candidate telomeric proteins in various organisms, including plants. Here we provide an overview of these studies with a special emphasis on plants, where a specific subfamily of the proteins possessing the N-terminally positioned telobox is present in addition to more common C-terminal telobox proteins. We further demonstrate the presence of a telobox protein (CpTBP1) in Cestrum parqui, a plant lacking typical telomeres and telomerase. The protein shows nuclear localisation and association with chromatin. The role of this protein in ancestral and current telomere structure is discussed in the evolutionary context. Altogether, the present overview shows the importance of the telobox domain in a search for candidate telomere proteins but at the same time warns against oversimplified identification of any telobox protein with telomere structure without appropriate evidence of its telomeric localisation and function.

Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins

Mammalian telomeres are formed by tandem repeats of the TTAGGG sequence, which are progressively lost with each round of cell division. Telomere protection requires a minimal length of TTAGGG repeats to allow the binding of shelterin, which prevents the activation of a DNA damage response (DDR) at chromosome ends. Telomere elongation is carried out by telomerase. Telomerase can also act as a transcriptional modulator of the Wnt-β-catenin signalling pathway and has RNA-dependent RNA polymerase activity. Dysfunctional telomeres can lead to either cancer or ageing pathologies depending on the integrity of the DDR. This Review discusses the role of telomeric proteins in cancer and ageing through modulating telomere length and protection, as well as regulating gene expression by binding to non-telomeric sites.