Telomere Binding Research Articles

TRF2 recruits ORC through TRFH domain dimerization

Telomeres are specialized chromatin structures that prevent the degradation and instability of the ends of linear chromosomes. While telomerase maintains long stretches of the telomeric repeat, the majority of telomeric DNA is duplicated by conventional DNA replication. A fundamental step in eukaryotic DNA replication involves chromatin binding of the origin recognition complex (ORC). In human cells, telomeric repeat binding factor 2 (TRF2) is thought to play a role in the recruitment of ORC onto telomeres. To better understand the mechanism of TRF2-mediated ORC recruitment, we utilized a lacO-LacI protein tethering system in U2OS cells and found that ectopically targeted TRF2, but not TRF1, can recruit ORC onto the lacO array. We further found that the TRF homology (TRFH) dimerization domain of TRF2, but not its mutant defective in dimerization, is sufficient for ORC and minichromosome maintenance (MCM) recruitment. Mutations impairing the dimerization also compromised ORC recruitment by full-length TRF2. Similar results were obtained using immunoprecipitation and GST pull-down assays. Together, these results suggest that dimerized TRF2 recruits ORC and stimulates pre-replication complex (pre-RC) formation at telomeres through the TRFH domain.

Evolution of Arabidopsis protection of telomeres 1 alters nucleic acid recognition and telomerase regulation.

Protection of telomeres (POT1) binds chromosome ends, recognizing single-strand telomeric DNA via two oligonucleotide/oligosaccharide binding folds (OB-folds). The Arabidopsis thaliana POT1a and POT1b paralogs are atypical: they do not exhibit telomeric DNA binding, and they have opposing roles in regulating telomerase activity. AtPOT1a stimulates repeat addition processivity of the canonical telomerase enzyme, while AtPOT1b interacts with a regulatory lncRNA that represses telomerase activity. Here, we show that OB1 of POT1a, but not POT1b, has an intrinsic affinity for telomeric DNA. DNA binding was dependent upon a highly conserved Phe residue (F65) that in human POT1 directly contacts telomeric DNA. F65A mutation of POT1aOB1 abolished DNA binding and diminished telomerase repeat addition processivity. Conversely, AtPOT1b and other POT1b homologs from Brassicaceae and its sister family, Cleomaceae, naturally bear a non-aromatic amino acid at this position. By swapping Val (V63) with Phe, AtPOT1bOB1 gained the capacity to bind telomeric DNA and to stimulate telomerase repeat addition processivity. We conclude that, in the context of DNA binding, variation at a single amino acid position promotes divergence of the AtPOT1b paralog from the ancestral POT1 protein.

Expression of Telomere Binding Proteins (RAP1 and POT1) in Renal Cell Carcinoma and Their Correlation with Clinicopathological Parameters.

Telomere stability is indispensable for continuous proliferation of cells. Telomere structure is maintained by group of six proteins termed as shelterin. RAP1 and POT1 proteins are significant members of shelterin complex. Expression of RAP1 and POT1 are crucial for telomere maintenance and hence uncontrolled division of cells. Notably, expression of RAP1 and POT1 is unknown in renal cell carcinoma (RCC). In view of these facts, the present study was initiated to investigate the expression of RAP1 and POT1 in RCC and their relationships with clinicopathological features. In total 65 histopathologically confirmed RCC cases and their adjacent normal renal parenchyma were analyzed for gene expression. The mRNA expression of telomere binding proteins RAP1 and POT1 were measured using RT-PCR. Expression of RAP1 was observed to be significantly increased in tumour tissues as compared to corresponding normal renal tissues (P=0.004). The gene expression of RAP1 was documented to be related to grades of RCC (P=0.002) and subtypes of RCC (P=0.01). Although, POT1 expression was up-regulated in RCC tissue, however it was not statistically significant. Also, POT1 expression was not related to grades, stages and subtypes of RCC. This is the first study which shows correlation RAP1 with grades and subtypes of RCC.

Advance in research on the function of telomeric shelterin component TPP1 and its relationship with characteristics of tumors

As an important telomere binding protein, TPP1 protects the ends of telomeres and maintains the stability and integrity of its structure and function by interacting with other five essential core proteins (POT1, TRF1, TRF2, TIN2, and RAP1) to form a complex called Shelterin. Recently, researchers have discovered that TPP1 participates in protection of telomeres and regulation of telomerase activity. The relationship between TPP1 and tumorigenesis, tumor progression and treatment has also been investigated. This paper reviews the latest findings of TPP1 regarding to its structure, function and interaction with other proteins involved in tumorigenesis.

Effect of concurrent training on telomere length in patients with myocardial infarction: Randomised clinical trial of cardiac rehabilitation

Telomeres are structures at the ends of DNA and protect chromosome ends. The length of DNA telomeres is an important parameter of the proliferative potential of tissues. Shorter telomeres have been seen in myocardial infarction patients. TRF1 (Telomeric repeat binding factor 1) and TRF2 (Telomeric repeat binding factor 2) are the most important Shelterin complex proteins. The purpose of this study was to investigate the effect of concurrent training on telomere length in Patients with myocardial infarction. In this Quasi-experimental pre-post intervention study, twenty male patients from Taleghani Hospital selected and randomly assigned to the training and control group. Training protocol was eight weeks of concurrent training, 3 times per week. Blood samples were taken a half hour before the first training session and 24h after last training session. The findings of the present study revealed that concurrent training prevents telomere shortening (p=0.001) and increases telomerase activity (p=0.002). In addition, there was a significant increase in TFR1 and TFR2 levels after training in patients with myocardial infarction (p=0.05 and p=0.001, respectively). Therefore, it seems that Eight weeks of concurrent training favorably affected telomere biology in Patients with myocardial infarction. Hence, concurrent training exercise is suggested during rehabilitation for myocardial infracted patients.

ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2.

Telomere stability is a hallmark of immortalized cells, including cancer cells. While the telomere length is maintained in most cases by the telomerase, the activity of a protein complex called Shelterin is required to protect telomeres against unsuitable activation of the DNA damage response pathway. Within this complex, telomeric repeat binding factor 2 (TRF2) plays an essential role by blocking the ataxia telangiectasia-mutated protein (ATM) signaling pathway at telomeres and preventing chromosome end fusion. We showed that TRF2 was phosphorylated in vitro and in vivo on serine 323 by extracellular signal-regulated kinase (ERK1/2) in both normal and cancer cells. Moreover, TRF2 and activated ERK1/2 unexpectedly interacted in the cytoplasm of tumor cells and human tumor tissues. The expression of non-phosphorylatable forms of TRF2 in melanoma cells induced the DNA damage response, leading to growth arrest and tumor reversion. These findings revealed that the telomere stability is under direct control of one of the major pro-oncogenic signaling pathways (RAS/RAF/MEK/ERK) via TRF2 phosphorylation.

Telomere- and Telomerase-Associated Proteins and Their Functions in the Plant Cell.

Telomeres, as physical ends of linear chromosomes, are targets of a number of specific proteins, including primarily telomerase reverse transcriptase. Access of proteins to the telomere may be affected by a number of diverse factors, e.g., protein interaction partners, local DNA or chromatin structures, subcellular localization/trafficking, or simply protein modification. Knowledge of composition of the functional nucleoprotein complex of plant telomeres is only fragmentary. Moreover, the plant telomeric repeat binding proteins that were characterized recently appear to also be involved in non-telomeric processes, e.g., ribosome biogenesis. This interesting finding was not totally unexpected since non-telomeric functions of yeast or animal telomeric proteins, as well as of telomerase subunits, have been reported for almost a decade. Here we summarize known facts about the architecture of plant telomeres and compare them with the well-described composition of telomeres in other organisms.

Telomerase RNA is more than a DNA template

ABSTRACTThe addition of telomeric DNA to chromosome ends is an essential cellular activity that compensates for the loss of genomic DNA that is due to the inability of the conventional DNA replication apparatus to duplicate the entire chromosome. The telomerase reverse transcriptase and its associated RNA bind to the very end of the telomere via a sequence in the RNA and specific protein-protein interactions. Telomerase RNA also provides the template for addition of new telomeric repeats by the reverse-transcriptase protein subunit. In addition to the template, there are 3 other conserved regions in telomerase RNA that are essential for normal telomerase activity. Here we briefly review the conserved core regions of telomerase RNA and then focus on a recent study in fission yeast that determined the function of another conserved region in telomerase RNA called the Stem Terminus Element (STE).1 The STE is distant from the templating core of telomerase in both the linear and RNA secondary structure, but, nonetheless, affects the fidelity of telomere sequence addition and, in turn, the ability of telomere binding proteins to bind and protect chromosome ends. We will discuss possible mechanisms of STE action and the suitability of the STE as an anti-cancer target.

Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA–DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA–DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.

Application of Created Restriction Site PCR-RFLP to Identify POT1 Gene Polymorphism.

Protection of telomeres protein 1 (POT1) plays pivotal roles in protection of chromosome ends and regulation of telomere length with other telomere binding proteins; its genetic polymorphisms are associated with many diseases. In this study, we explored a novel PCR-RFLP method for typing the single nucleotide polymorphism (SNP) rs1034794 of the human POT1 gene. A new restriction enzyme site was introduced into a POT1 gene amplification product by created restriction site PCR (CRS-PCR). One primer was designed based on changed sequence; after PCR amplification, a new restriction enzyme site for AluI was introduced into the PCR products. One hundred and seventy eight samples from Han Chinese individuals were tested to evaluate this new method. The 3'-end of the forward primer was next to the polymorphic site, and the third base from the 3'-end was the mismatched base A. The final PCR product contained the AGCT sequence (AluI recognition site) when the ancestral POT1 alleles were amplified. The data obtained with the new method perfectly matched those obtained with the sequencing method. Thus, CRS-PCR is a new low-cost and high-efficiency alternative for rs1034794 typing.

Detection of the rs10250202 polymorphism in protection of telomeres 1 gene through introducing a new restriction enzyme site for PCR-RFLP assay.

Human protection of telomeres 1 (POT1) gene is a single stranded telomere binding proteins with a critical role in ensuring chromosome stability. There have been variants of POT1 gene, and the polymorphisms of POT1 gene were associated with some diseases. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) is a traditional method to detect the single nucleotide polymorphism (SNP), and it can be used to detect the polymorphism of rs10250202. But the restriction enzymes required for the detection of the polymorphism of rs10250202 are expensive. So we designed a novel PCR–RFLP assay for genotyping the POT1 rs10250202 SNP. In the study, a new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and the restriction enzyme BclI for CRS-PCR was cheaper than other enzymes. After detecting Han Chinese workers, Allele frequencies were found to be 51.54 % for allele A and 48.46 % for allele C respectively. The PCR results were confirmed by DNA sequencing. CRS-PCR provides a simple, low-cost, practical, and reproducible method.

Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins.

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.

Editorial: The Evolving Telomeres.

Editorial: The Evolving Telomeres.

Hypothesis: Paralog Formation from Progenitor Proteins and Paralog Mutagenesis Spur the Rapid Evolution of Telomere Binding Proteins.

Through elegant studies in fungal cells and complex organisms, we propose a unifying paradigm for the rapid evolution of telomere binding proteins (TBPs) that associate with either (or both) telomeric DNA and telomeric proteins. TBPs protect and regulate telomere structure and function. Four critical factors are involved. First, TBPs that commonly bind to telomeric DNA include the c-Myb binding proteins, OB-fold single-stranded binding proteins, and G-G base paired Hoogsteen structure (G4) binding proteins. Each contributes independently or, in some cases, cooperatively, to provide a minimum level of telomere function. As a result of these minimal requirements and the great abundance of homologs of these motifs in the proteome, DNA telomere-binding activity may be generated more easily than expected. Second, telomere dysfunction gives rise to genome instability, through the elevation of recombination rates, genome ploidy, and the frequency of gene mutations. The formation of paralogs that diverge from their progenitor proteins ultimately can form a high frequency of altered TBPs with altered functions. Third, TBPs that assemble into complexes (e.g., mammalian shelterin) derive benefits from the novel emergent functions. Fourth, a limiting factor in the evolution of TBP complexes is the formation of mutually compatible interaction surfaces amongst the TBPs. These factors may have different degrees of importance in the evolution of different phyla, illustrated by the apparently simpler telomeres in complex plants. Selective pressures that can utilize the mechanisms of paralog formation and mutagenesis to drive TBP evolution along routes dependent on the requisite physiologic changes.

Telomere-binding protein regulates the chromosome ends through the interaction with histone deacetylases in Arabidopsis thaliana.

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes. Many telomere-binding proteins bind to telomeric repeat sequences and further generate T-loops in animals. However, it is not clear if they regulate telomere organization using epigenetic mechanisms and how the epigenetic molecules are involved in regulating the telomeres. Here, we show direct interactions between the telomere-binding protein, AtTRB2 and histone deacetylases, HDT4 and HDA6, in vitro and in vivo. AtTRB2 mediates the associations of HDT4 and HDA6 with telomeric repeats. Telomere elongation is found in AtTRB2, HDT4 and HDA6 mutants over generations, but also in met1 and cmt3 DNA methyltransferases mutants. We also characterized HDT4 as an Arabidopsis H3K27 histone deacetylase. HDT4 binds to acetylated peptides at residue K27 of histone H3 in vitro, and deacetylates this residue in vivo. Our results suggest that AtTRB2 also has a role in the regulation of telomeric chromatin as a possible scaffold protein for recruiting the epigenetic regulators in Arabidopsis, in addition to its telomere binding and length regulation activity. Our data provide evidences that epigenetic molecules associate with telomeres by direct physical interaction with telomere-binding proteins and further regulate homeostasis of telomeres in Arabidopsis thaliana.

PfGBP: una proteína de unión al telómero de Plasmodium falciparum

Los telómeros son estructuras complejas de ADN y proteína localizadas en el extremo de los cromosomas eucariotes. Su principal función es proteger el extremo cromosomal de ser reconocido y procesado como ADNs fracturado, evitando así eventos de recombinación y fusión que conducen a inestabilidad cromosomal. El ADN telomérico consta de secuencias cortas, repetidas una tras otra, ricas en guanina; la cadena rica en guanina se extiende formando una región de cadena sencilla denominada extremo 3´ protuberante. Las proteínas por su parte, se pueden clasificar en: dsBPs, o proteínas de unión a la cadena doble, GBPs aquellas que reconocen específicamente el extremo protuberante y, proteínas que las interconectan mediante interacciones proteína-proteína. El gen PF3D7_1006800 de <em>Plasmodium falciparum</em> codifica para una proteína putativa similar a una GBP de <em>Criptosporidium parvum</em>, con el fin de establecer si esta proteína de <em>P. falciparum</em> presenta la capacidad de unión al ADN telomérico del parásito, se produjo una proteína recombinante a partir de la región codificante del gen, se purificó y se utilizó en ensayos de unión a ADN, y en la generación de anticuerpos policlonales específicos contra PfGBP. Nuestros resultados indican que la proteína de <em>P. falciparum</em> es una proteína nuclear con capacidad de unión al ADN telomérico <em>in vitro, </em>por lo<em> </em>que podría ser<em> </em>parte del complejo proteico encargado de proteger y/o mantener el telómero <em>in vivo</em>.

Testosterone Antagonizes Doxorubicin-Induced Senescence of Cardiomyocytes.

BackgroundChronic cardiotoxicity is less common in male than in female patients receiving doxorubicin and other anthracyclines at puberty and adolescence. We hypothesized that this sex difference might be secondary to distinct activities of sex hormones on cardiomyocyte senescence, which is thought to be central to the development of long‐term anthracycline cardiomyopathy.Methods and ResultsH9c2 cells and neonatal mouse cardiomyocytes were exposed to doxorubicin with or without prior incubation with testosterone or 17β‐estradiol, the main androgen and estrogen, respectively. Testosterone, but not 17β‐estradiol, counteracted doxorubicin‐elicited senescence. Downregulation of telomere binding factor 2, which has been pinpointed previously as being pivotal to doxorubicin‐induced senescence, was also prevented by testosterone, as were p53 phosphorylation and accumulation. Pretreatment with the androgen receptor antagonist flutamide, the phosphatidylinositol 3 kinase inhibitor LY294002, and the nitric oxide synthase inhibitor L‐NG‐nitroarginine methyl ester abrogated the reduction in senescence and the normalization of telomere binding factor 2 levels attained by testosterone. Consistently, testosterone enhanced the phosphorylation of AKT and nitric oxide synthase 3. In H9c2 cells, doxorubicin‐stimulated senescence was still observed up to 21 days after treatment and increased further when cells were rechallenged with doxorubicin 14 days after the first exposure to mimic the schedule of anthracycline‐containing chemotherapy. Remarkably, these effects were also inhibited by testosterone.ConclusionsTestosterone protects cardiomyocytes against senescence caused by doxorubicin at least in part by modulating telomere binding factor 2 via a pathway involving the androgen receptor, phosphatidylinositol 3 kinase, AKT, and nitric oxide synthase 3. This is a potential mechanism by which pubescent and adolescent boys are less prone to chronic anthracycline cardiotoxicity than girls.

Using Centromere Mediated Genome Elimination to Elucidate the Functional Redundancy of Candidate Telomere Binding Proteins in Arabidopsis thaliana.

Proteins that bind to telomeric DNA form the key structural and functional constituents of telomeres. While telomere binding proteins have been described in the majority of organisms, their identity in plants remains unknown. Several protein families containing a telomere binding motif known as the telobox have been previously described in Arabidopsis thaliana. Nonetheless, functional evidence for their involvement at telomeres has not been obtained, likely due to functional redundancy. Here we performed genetic analysis on the TRF-like family consisting of six proteins (TRB1, TRP1, TRFL1, TRFL2, TRFL4, and TRF9) which have previously shown to bind telomeric DNA in vitro. We used haploid genetics to create multiple knock-out plants deficient for all six proteins of this gene family. These plants did not exhibit changes in telomere length, or phenotypes associated with telomere dysfunction. This data demonstrates that this telobox protein family is not involved in telomere maintenance in Arabidopsis. Phylogenetic analysis in major plant lineages revealed early diversification of telobox proteins families indicating that telomere function may be associated with other telobox proteins.

The Insertion in Fingers Domain in Human Telomerase Can Mediate Enzyme Processivity and Telomerase Recruitment to Telomeres in a TPP1-Dependent Manner.

In most human cancer cells, cellular immortalization relies on the activation and recruitment of telomerase to telomeres. The telomere-binding protein TPP1 and the TEN domain of the telomerase catalytic subunit TERT regulate telomerase recruitment. TERT contains a unique domain, called the insertion in fingers domain (IFD), located within the conserved reverse transcriptase domain. We report the role of specific hTERT IFD residues in the regulation of telomerase activity and processivity, recruitment to telomeres, and cell survival. One hTERT IFD variant, hTERT-L805A, with reduced activity and processivity showed impaired telomere association, which could be partially rescued by overexpression of TPP1-POT1. Another previously reported hTERT IFD mutant enzyme with similarly low levels of activity and processivity, hTERT-V791Y, displayed defects in telomere binding and was insensitive to TPP1-POT1 overexpression. Our results provide the first evidence that the IFD can mediate enzyme processivity and telomerase recruitment to telomeres in a TPP1-dependent manner. Moreover, unlike hTERT-V791Y, hTERT-V763S, a variant with reduced activity but increased processivity, and hTERT-L805A, could both immortalize limited-life-span cells, but cells expressing these two mutant enzymes displayed growth defects, increased apoptosis, DNA damage at telomeres, and short telomeres. Our results highlight the importance of the IFD in maintaining short telomeres and in cell survival.

Cdk-dependent phosphorylation regulates TRF1 recruitment to PML bodies and promotes C-circle production in ALT cells.

TRF1, a duplex telomeric DNA binding protein, is implicated in homologous-recombination-based alternative lengthening of telomeres, known as ALT. However, how TRF1 promotes ALT activity has yet to be fully characterized. Here we report that Cdk-dependent TRF1 phosphorylation on T371 acts as a switch to create a pool of TRF1, referred to as (pT371)TRF1, which is recruited to ALT-associated PML bodies (APBs) in S and G2 phases independently of its binding to telomeric DNA. We find that phosphorylation of T371 is essential for APB formation and C-circle production, both of which are hallmarks of ALT. We show that the interaction of (pT371)TRF1 with APBs is dependent upon ATM and homologous-recombination-promoting factors Mre11 and BRCA1. In addition, (pT371)TRF1 interaction with APBs is sensitive to transcription inhibition, which also reduces DNA damage at telomeres. Furthermore, overexpression of RNaseH1 impairs (pT371)TRF1 recruitment to APBs in the presence of campothecin, an inhibitor that prevents topoisomerase I from resolving RNA-DNA hybrids. These results suggest that transcription-associated DNA damage, perhaps arising from processing RNA-DNA hybrids at telomeres, triggers (pT371)TRF1 recruitment to APBs to facilitate ALT activity.