EPAS1 prevents telomeric damage-induced senescence by enhancing transcription of TRF1,TRF2,and RAD50

Telomeres are nucleoprotein structures located at the end of each chromosome,which function in terminal protection and genomic stability.Telomeric damage is closely related to replicative senescence in vitro and physical aging in vivo.As relatively long-lived mammals based on body size,bats display unique telomeric patterns,including the upregulation of genes involved in alternative lengthening of telomeres(ALT),DNA repair,and DNA replication.At present,however,the relevant molecular mechanisms remain unclear.In this study,we performed cross-species comparison and identified EPAS1,a well-defined oxygen response gene,as a key telomeric protector in bat fibroblasts.Bat fibroblasts showed high expression of EPAS1,which enhanced the transcription of shelterin components TRF1 and TRF2,as well as DNA repair factor RAD50,conferring bat fibroblasts with resistance to senescence during long-term consecutive expansion.Based on a human single-cell transcriptome atlas,we found that EPAS1 was predominantly expressed in the human pulmonary endothelial cell subpopulation.Using in vitro-cultured human pulmonary endothelial cells,we confirmed the functional and mechanistic conservation of EPAS1 in telomeric protection between bats and humans.In addition,the EPAS1 agonist M1001 was shown to be a protective compound against bleomycin-induced pulmonary telomeric damage and senescence.In conclusion,we identified a potential mechanism for regulating telomere stability in human pulmonary diseases associated with aging,drawing insights from the longevity of bats.

Telomeric associations of chromosomes in patients with esophageal squamous cell carcinomas

AIM To investigate the role of telomeric association in the development of esophageal cancer. METHODS Using chromosome R banding technique, telomeric association of chromosome in peripheral blood lymphocytes from 16 untreated patients with esophageal squamous cell carcinoma were observed and 16 healthy adults served as controls. RESULTS The telomeric association frequencies of cell and chromosomes were significantly higher than those of controls (x 2=9.56,P<0.01), but its distribution on the chromosome showed no significant difference (x 2=1.01, P>0.05) between the two groups. CONCLUSION Chromosomal instability can be initiated by telomeric associations, and sequential chromosome analysis can aid the understanding of the tumor occurrence and progression.

Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut

Interspecific hybridization is an important approach to improve cultivated peanut varieties. Cytological markers such as tandem repeats will facilitate alien gene introgression in peanut. Telomeric repeats have also been frequently used in chromosome research. Most plant telomeric repeats are（TTTAGGG）n that are mainly distributed at the chromosome ends, although interstitial telomeric repeats（ITRs） are also commonly identified. In this study, the telomeric repeat was chromosomally localized in 10 Arachis species through sequential GISH（genomic in situ hybridization） and FISH（fluorescence in situ hybridization） combined with 4＇,6-diamidino-2-phenylindole（DAPI） staining. Six ITRs were identified such as in the centromeric region of chromosome Bi5 in Arachis ipa？nsis, pericentromeric regions of chromosomes As5 in A. stenosperma, Bho7 in A. hoehnei and Av5 in A. villosa, nucleolar organizer regions of chromosomes As3 in A. stenosperma and Adi3 in A. diogoi, subtelomeric regions of chromosomes Bho9 in A. hoehnei and Adu7 in A. duranensis, and telomeric region of chromosome Es7 in A. stenophylla. The distributions of the telomeric repeat, 5S r DNA, 45 S r DNA and DAPI staining pattern provided not only ways of distinguishing different chromosomes, but also karyotypes with a higher resolution that could be used in evolutionary genome research. The distribution of telomeric repeats, 5S r DNA and 45 S r DNA sites in this study, along with inversions detected on the long arms of chromosomes Kb10 and Bho10, indicated frequent chromosomal rearrangements during evolution of Arachis species.

Study on the expression and mutation of human telomeric repeat binding factor (hTRF1) in 10 malignant hematopoietic cell lines

Objective： Detecting the expression and mutation of human telomeric repeat binding factor （hTRF1） in 10 malignant hematopoietic cell line cells on the base of determining its genomic structure and its four pseudogenes to clarify ifhTRF1 mutation is one of the factors of the activation of telomerase. Methods： hTRFlcDNA sequences were obtained from GenBank, its genome structure and pseudogenes were forecasted by BLAST and other biology information programs and then testified by sequencing. Real-time RT-PCR was used to detect the expression of h TRFlmRNA in 10 cell line cells, including myelogenous leukemia cell lines K562, HL-60, U-937, NB4, THP-I, HEL and Dami; lymphoblastic leukemia cell lines 6T-CEM, Jurkat and Raji. Telomerase activities of cells were detected by using telomeric repeat amplification （TRAP）-ELISA protocol. PCR and sequencing were used to detect mutation of each exon ofhTRF1 in 10 cell line cells. Results： hTRF1 gene, mapped to 8q13, was divided into 10 exons and spans 38.6 kb. Four processed pseudogenes ofhTRF1 located on chromosome 13, 18, 21 and X respectively, was named as ψhTRFI-13, ψhTRFI-18, ψhTRF1-21 and ψhTRFI-X respectively. All cell line cells showed positive telomerase activity. The expression of hTRF1 was significantly lower in malignant hematopoietic cell lines cells （0.0338, 0.0108-0.0749） than in normal mononuclear cells （0.0493, 0.0369-0.128） （P=0.004）. But no significant mutation was found in all exons of hTRF1 in 10 cell line cells. Four variants were found in part ofintron 1, 2 and 8 ofhTRF1. Their infection on gene function is unknown and needs further studies. Conclusion： hTRF1 mutation is probably not one of the main factors for telomerase activation in malignant hematopoietic disease.

Ten1p promotes the telomeric DNA-binding activity of Cdc13p： implication for its function in telomere length regulation

In Saccharomyces cerevisiae, the essential gene CDC13 encodes a telomeric single-stranded DNA-binding protein that interacts with Stnlp and Tenlp genetically and physically, and is required for telomere end protection and telomere length control. The molecular mechanism by which Tenl participates in telomere length regulation and chromosome end protection remains elusive. In this work, we observed a weak interaction of Cdc13p and Tenlp in a gelfiltration analysis using purified recombinant Cdc13p and Tenlp. Tenlp itself exhibits a weak DNA-binding activity, but enhances the telomeric TG1-3 DNA-binding ability of Cdc13p. Cdc13p is communoprecipitated with Tenlp. In the mutant ten1-55 or ten1-66 cells, the impaired interaction between Tenlp and Cdc13p results in much longer telomeres, as well as a decreased association of Cdc13p with telomeric DNA. Consistently, the Ten1-55 and Ten1-66 mutant proteins fail to stimulate the telomeric DNA-binding activity of Cdc13p in vitro. These results suggest that Tenlp enhances the telomeric DNA-binding activity of Cdc13p to negatively regulate telomere length.

ILF3 safeguards telomeres from aberrant homologous recombination as a telomeric R-loop reader

Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability.The telomeric repeat-containing RNA(TERRA)that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop.In tumor cells,R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres(ALT)pathway.Dysregulated R-loops can cause stalled replication forks and telomere instability.However,how R-loops are recognized and regulated,particularly at telomeres,is not well understood.We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination.Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses.In addition,ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway.Using the proximity-dependent biotin identification(BioID)technology,we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases,including DHX9.Importantly,ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9.Our findings suggest that ILF3 may function as a reader of telomeric R-loops,helping to prevent abnormal homologous recombination and maintain telomere homeostasis.

The restriction endonuclease Tru9I is a useful tool to analyze telomere sequences separately from interstitial telomeric sequences in <i>Arabidopsis thaliana</i>

We have previously described a simple method to analyze the chromatin structure of Arabidopsis telomeres independently of that of Interstitial Telomeric Sequences (ITSs). By using this method, we found that, whereas ITSs are heterochromatic, Arabidopsis telomeres exhibit euchromatic features [1]. Some concerns have been recently raised about the accuracy of this procedure [2]. Here, we summarize these concerns and justify our experimental approaches and interpretation of results.

Cancer Prohibitor-Telomeric DNA Repair Pathways

Telomeres are DNA-protein structures that form protective caps at the end of eukaryotic chromosomes,safeguarding the chromosomes from degradation and maintaining the genomic integrity.When DNA damage occurs,the cell will activate its repair system to fix the errors to prevent cancer.There are three major molecular mechanisms of DNA repair:base excision repair(BER),nucleotide excision repair(NER),and mismatch repair(MMR).In this review article,we discuss the three canonical repair pathways at the telomeres and their functions in cancer prevention and therapy.

Detection of telomerase activity in human cells with telomeric repeat amplification protocol by silver staining

Telomerase is a ribonucleoprotein enzyme, which synthesizes telomeric repeats (TTAGGG) n.While germline cells and most malignant tumor cells express telomerase activity, normal somatic cells aregenerally deficient in telomerase activity. Our objective was to detect telomerase activity of human cells bysilver staining with telorneric repeat amplification protocol (TRAP) which is easy and quick. Comparing withradioisotopic TRAP, we examined the telomerase activity in telomerase-positive 293-cell and RNase-pretreated and heat-pretreated negative controls by silver staining TRAP. We detected telomerase activity in 2 strainsof human liver tumor cells (QGY7701 and SMMC7721 ). The 293 cells (only 10 cells) and the 2 strains ofhuman liver tumor cells were all positive. while telomerase activity was not detected in the negative controls.These data suggest that non-radioisotopic silver staining TRAP is a specific, sensitive and fast assay fortelomerase activity. It was verified that the 2 strains of human liver tumor cells express telomerase activity.

Telomeric DNA breaks in human induced pluripotent stem cells trigger ATR-mediated arrest and telomerase-independent telomere damage repair

Although mechanisms of telomere protection are well-defined in differentiated cells,how stem cells sense and respond to telomere dysfunction,in particular telomeric double-strand breaks(DSBs),is poorly characterized.Here,we report the DNA damage signaling,cell cycle,and transcriptome changes in human induced pluripotent stem cells(iPSCs)in response to telomere-internal DSBs.We engineer human iPSCs with an inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres.Using this model,we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DNA damage response,which leads to p53-independent cell cycle arrest in G2.Using CRISPR–Cas9 to cripple the catalytic domain of telomerase reverse transcriptase,we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres,which instead are effectively repaired by robust homologous recombination(HR).In contrast to HR-based telomere maintenance in mouse embryonic stem cells,where HR causes ZSCAN4-dependent extension of telomeres beyond their initial lengths,HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length,which is compatible with sustained survival of the cells over several days of TRF1-FokI induction.Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric DNA damage.

Conservation and characterization of unique porcine interstitial telomeric sequences

Telomeres are composed of TTAGGG repeats and located at the ends of chromosomes. Telomeres protect chromosomes from instability in mammals, including mice and humans. Repetitive TTAGGG sequences are also found at intrachromosomal sites, where they are named as interstitial telomeric sequences （ITSs）. Aberrant ITSs are implicated in chromosomal instability and found in cancer cells. Interestingly, in pigs, vertebrate telomere sequences TTAGGG （vlTSs） are also localized at the centro- meric region of chromosome 6, in addition to the end of all chromosomes. Surprisingly, we found that botanic telomere se- quences, TTTAGGG （bITSs）, also localize with vITSs at the centromeric regions of pig chromosome 6 using telomere fluo- rescence in situ hybridization （FISH） and by comparisons between several species. Furthermore, the average lengths of vITSs are highly correlated with those of the terminal telomeres （TTS）. Also, pig ITSs show a high incidence of telomere doublets, suggesting that pig ITSs might be unstable and dynamic. Together, our results show that pig cells maintain the conserved te- lomere sequences that are found at the ITSs from of plants and other vertebrates. Further understanding of the function and regulation of pig ITSs may provide new clues for evolution and chromosomal instability.

A Study of Radiation-Induced Telomere Instability Using Multiplex Ligation-Dependent Probe Amplification (MLPA)

The integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiation was analyzed by Multiplex Ligation-Dependent Probe Amplification (MLPA). The TK6 cell line has the native p53 tumor-suppressor gene, whereas WTK1 cells contain a p53 mutation. Each cell line was isolated pre- and post-irradiation (2 and 3 Gy) and analyzed by MLPA. The impact of irradiation on these two cell lines was investigated using probes that target specific regions on chromosomes associated with subtelomeric regions. Results indicate that WTK1 and TK6 are impacted differently after irradiation, and that each cell line presents its own unique MLPA profile. The most notable differences are the appearance of a number of probes in the post-irradiated MLPA profile that are not present in the controls, and two unique probe signals only seen in WTK1 cells. These results build on our previous studies that indicate how different human cell lines can be affected by radiation in significantly different ways depending on the presence or absence of wild type p53.

Direct Correlation among Telomere Length, Cellular Aging, and Rejuvenation Effects of Honey Child Powder

Purpose: Telomere length (TL) is an indicator of age;however, hormonal influences complicate individual aging. It remains unclear whether TL shortening is a direct factor in both individual and cellular aging. Therefore, we examined the direct relationship between TL and cellular senescence at the cellular level. Methods: Telomerase activity, TL, and gene expression were measured in cultured human lung-, fetal-, and skin-derived fibroblasts, human skin keratinocytes, and telomerase reverse transcriptase (TERT) gene-immortalized cells using detection kits, Cawthon’s method, and reverse transcription-quantitative polymerase chain reaction, respectively. Novel substances that elongate telomeres were screened to confirm cell rejuvenation effects. Results: Long-term cell culture of TIG-1-20 normal human fibroblasts resulted in TL shortening, decreased division rate, and senescence progression, whereas in OUMS-36T-2 cells, TL elongation via TERT gene transfer increased the division rate, reduced endoplasmic reticulum stress, and upregulated genes associated with young individuals, indicating that cellular rejuvenation occurs via TL elongation. In addition, a honey child powder (HCP) extract was found through screening, and the HCP extract strongly suppressed the menin gene, resulting in increased telomerase activity and extended cell lifespan. Upon addition of the HCP extract to skin fibroblasts, gene expression of moisturizing components, including collagen, hyaluronic acid, and elastin, increased, and exhibited a rejuvenating effect with an increase in elastin amount. Conclusions: TL elongation or shortening is involved in cell proliferation rate and cellular aging, and TL elongation rejuvenates cells. In addition, HCP extract has a rejuvenating effect on cells and is expected to be a rejuvenating compound.

Analysis and location of a rice BAC clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG) n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescence in situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.

NDI-induced Topological Conversion of Human Telomeric G-Quadruplexes from Hybrid-2 to Parallel Form

G-Quadruplexes(GQs),which are formed by G-rich DNA sequences in human telomeres,have become an attractive target for cancer treatment.The ligands to stabilize the conformation of human telomeric GQs in vivo are particularly important for structure-based ligand design and drug development targeting the noncanonical DNA structure.Here we report the conformational conversion of Tel26 induced by a naphthalene diimide(NDI)ligand in K^(+)buffer,even at cellular physiological temperature(37℃)and under mimetic cellular crowding conditions created by Ficoll 70.We provide an insight into the dynamic conversion from initial hybrid-2 GQ topology to final parallel GQ topology.These results are helpful for the design of ligands with GQ conformation regulation.

Circular dichroism spectroscopic studies on structures formed by telomeric DNA sequences in vitro

Telomere plays an important role in cellular processes, such as cell aging, death and carcinogenisis. Having special sequences, it can form quadrupiex structure in vitro. Circular dichroism (CD) spectroscopic studies show that TTAGGG, (TTAGGG)2 and (TTAGGG)4 can all form quadrupiex in vitro and exist mainly as parallel quadrupiex without metal ions. Both K+ and Na+ can stabilize the tetrameric structure and facilitate the forming of anti-parallel conformation. Furthermore, the conformations of quadrupiex can also be affected by sequence length, the nature and concentration of metal ions.

Identification of the New Type of G-Quadruplex with Multiple Vacant Sites in Human Telomeric DNA

To date,few studies have reported on the folding mechanism of tandem G-quadruplexes in human telomeric DNA.Hence,the control of the biofunctions of G-quadruplex,which requires a thorough understanding of its dynamic behavior,is limited.Here,we investigated the folding/unfolding behavior of human telomeric sequences with lengths over 10 kilonucleotide(knt)by circular dichroism(CD)spectroscopy,UV melting assay,and atomic force microscopy(AFM)-based single-molecule force spectroscopy.

Single-molecule investigation of human telomeric G-quadruplex interactions with Thioflavin T

G-quadruplex ligands have been accepted as potential therapeutic agents for anticancer treatment. Thioflavin T （ThT）, a highly selective G-quadruplex ligand, can bind G-quadruplex with a fluorescent light-up signal change and high specificity against DNA duplex. However, there are still different opinions that ThT induces/stabilizes G-quadruplex foldings/topologies in human telomere sequence. Here, a sensitive single-molecule nanopore technology was utilized to analyze the interactions between human telomeric DNA （Tel DNA） and ThT. Both translocation time and current blockade were measured to investigate the translocation behaviors. Furthermore, the effects of metal ion （K~ and Na~） and pH on the translocation behaviors were studied. Based on the single-molecule level analysis, there are some conclusions： （1） In the electrolyte solution containing 50 mmol/L I（Cl and 450 mmol/L NaCl, ThT can bind strongly with Tel DNA but nearly does not change the G-quadruplex form; （2） in the presence of high concentration K~, the ThT binding induces the structural change of hybrid G-quadruplex into antiparallel topology with an enhanced structural stability; （3） In either alkaline or acidic buffer, G-quadruplex form will change in certain degree. All above conclusions are helpful to deeply understand the interaction behaviors between Tel DNA and ThT. This nanopore platform, investigating G-quadruplex ligands at the single-molecule level, has great potential for the design of new drugs and sensors.

How will telomeric complex be further contributed to our longevity?—The potential novel biomarkers of telomere complex counteracting both aging and cancer

With the smooth move towards the coming expected clinical reports of anticancer pharmaceutical molecules targeting telomeres and telomerase,and also with the exciting success in the extension of lifespan by regulating telomerase activity without increased onset of oncogenesis in laboratory mouse models(Garcia-Cao et al.,2006;Jaskelioff et al.,2011),we are convinced that targeting telomeres based on telomerase will be a potential approach to conquer both aging and cancer and the idea of longevity seems to be no more mysterious.More interestingly,emerging evidences from clinical research reveal that other telomeric factors,like specifi c telomeric binding proteins and nonspecific telomere associated proteins also show crucial importance in aging and oncogenesis.This stems from their roles in the stability of telomere structure and in the inhibition of DNA damage response at telomeres.Uncapping these proteins from chromosome ends leads to dramatic telomere loss and telomere dysfunction which is more abrupt than those induced by telomerase inactivation.Abnormal expression of these factors results in developmental failure,aging and even oncogenesis evidenced by several experimental models and clinical cases,indicating telomere specifi c proteins and its associated proteins have complimentary roles to telomerase in telomere protection and controlling cellular fate.Thus,these telomeric factors might be potential clinical biomarkers for early detection or even therapeutic targets of aging and cancer.Future studies to elucidate how these proteins function in telomere protection might benefit patients suffering aging or cancer who are not sensitive to telomerase mediation.

Spectroscopic investigation on the telomeric DNA base sequence repeat

Telomeres are protein-DNA complexes at the terminals of linear chromosomes, which protect chromoso-mal integrity and maintain cellular replicative capacity. From single-cell organisms to advanced animals and plants, structures and functions of telomeres are both very conser-vative. In cells of human and vertebral animals, telomeric DNA base sequences all are (TTAGGG)n. In the present work, we have obtained absorption and fluorescence spectra measured from seven synthesized oligonucleotides to simu-late the telomeric DNA system and calculated their relative fluorescence quantum yields on which not only telomeric DNA characteristics are predicted but also possibly the shortened telomeric sequences during cell division are im-plied. Oligonucleotide 5’- TTAGGGTTAGGG holds a low relative fluorescence quantum yield and remarkable excitation energy innerconversion, which tallies with the telomeric sequence of (TTAGGG)n. This result shows that telomeric DNA has a strong non-radiative or innerconvertible capabil-