Telomerase Components Research Articles

Clear view of telomerase at last

The enzyme telomerase, which restores chromosome ends shortened by cell division, was discovered over three decades ago, but no view of the human enzyme at atomic resolution, approximately 3 Å, has been captured. The problem is that telomerase is present in cells in tiny quantities and is not easily purified. Now, using refined purification methods, Kathleen Collins, Eva Nogales, Kelly Nguyen, and coworkers at the University of California, Berkeley, have taken a giant leap toward the atomic-resolution goal (Nature 2018, DOI: 10.1038/s41586-018-0062-x). With cryo-electron microscopy, they analyzed the enzyme at 7 to 8 Å. A previous 30 Å electron microscopy structure was so fuzzy that it left the components of telomerase’s two lobes uncertain. The new structure reveals that one lobe, the catalytic core, contains telomerase reverse transcriptase, which catalyzes DNA addition to chromosome end-caps, and telomerase RNA, which templates the addition and links the two lobes. The second lobe,

Comprehensive analysis of telomerase inhibition by gallotannin

Gallotannin (or tannic acid) is a naturally occurring compound that inhibits cell growth and activity of different DNA-polymerases, including telomerase. The purpose of the present study was to gain insight into the mechanism of telomerase inhibition by gallotannin. We determined that gallotannin inhibits telomerase in vitro with an half maximal inhibitory concentrations value of 130 nM, but it does not affect telomerase complex assembly and component levels in vivo. The inhibitory activity of gallotannin against telomerase provides an additional explanation for the anti-cancer activities of this compound.

The age of heterozygous telomerase mutant parents influences the adult phenotype of their offspring irrespective of genotype in zebrafish.

Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested. Here we use a zebrafish telomerase mutant which harbours a nonsense mutation in tert to investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths. We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate that tert homozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents. These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation.

Abstract P2-10-08: A predictive test for neoadjuvant chemotherapy in breast cancer identifies a subset of triple negative patients with resistant disease and the poorest prognosis

Abstract Prediction of pathological complete response (pCR) for neoadjuvant treatment is an area of unmet clinical need, especially for triple negative breast cancer (TNBC) as pCR is correlated with better outcomes. Predicting which patients will have residual disease (RD) provides an opportunity to improve treatment planning. We developed a test to predict which patients are likely to achieve pCR or RD to the standard of care (taxane-based) neoadjuvant chemotherapy using gene expression profiling of 325 previously identified novel biomarkers. Three microarray datasets were used (GSE22226, GSE25055, and GSE25065) including a total of 594 stage II-III breast cancer patients of which 125 (21%) achieved pCR, and 469 (79%) RD. ER+ tumors were present in 57% of the patients and 52% were PGR+. Almost 90% of the patients were Her2-. Of 231 TNBC, 78 (33.8%) achieved pCR, while 153 (66.2%) RD. Of 303 ER+Her- patients 26 (8.6%) achieved pCR while 277 (91.4%) RD. The cohort was divided into balanced populations with 476 patients used for training (80%) and test (20%) rounds of model development, while 118 patients were reserved as a validation set. Combining a “winnowing” process to remove genes with least predictive power, and hundreds of thousands of step-wise runs, followed by ranking genes based on conditional probabilities, we developed a 17-gene cassette (BA100) which was locked-down in the validation set with ROC (AUC) = 0.818. With a cut-off of 83% sensitivity and 68% specificity (PPV 0.4; NPV 0.94), BA100 achieved a 16% true positive rate (true pCR) and 55% true negative rate (true RD) identifying 76% of the patients who achieved pCR, and 69% of the patients with RD. In TNBC, BA100 classified 29% as true positives (TP), 36% as false positive (FP), 30% true negative (TN), and 4.8% false negative (FN). Kaplan Meier (KM) curves showed a significant difference in 5-year disease-free survival (5Y DFS) between TP and TN (p=0.00453) or FP (p=2.09E-06). However, FP had even worse outcomes than TN patients. To improve the TP rate, additional genes expressed in TNBC plus the original 325 genes were subjected to a second round of gene selection to discriminate between TP and FP, resulting in a 16-gene cassette (BA100.1). With a cut-off of 95% sensitivity and 73% specificity (PPV 0.7; NPV 0.95), applying BA100.1 reduced the FP rates from 24% to 9%, while correctly identifying 88% of RD in the validation set. KM curves showed no significant difference in 5Y DFS between 124 TNBC (53.7%) classified as TN versus 29 TNBC (12.6%) classified as FP, while a significant difference in survival rate was found between TNBC classified as TN vs TP (Cox Proportional Harzard p=8.42e-05). Taken together, we developed a predictive test consisting of two gene cassettes that accurately identified 71% (88/104) of pCR, and 88% (417/469) of RD patients. Gene cassettes include several transcriptional repressors, PI3K signal transduction, components of telomerase, DNA repair genes, fatty acid metabolism and estrogen-independent proliferation. The test stratified TNBC with differential response to chemotherapy and survival rates so that novel approaches can be used without delay. Further validation will confirm the test utility. Citation Format: Fournier MV, Chen J, Obenauer J, Goodwin EC, Tannenbaum SH, Brufsky AM. A predictive test for neoadjuvant chemotherapy in breast cancer identifies a subset of triple negative patients with resistant disease and the poorest prognosis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-10-08.

Abstract P3-03-06: Telomerase polymorphism and breast cancer

Abstract Introduction: Telomerase is an enzyme responsible for telomere maintenance in almost all human cancer cells, but generally not expressed in somatic ones. The minimum essential components of telomerase are the catalytic subunit, telomerase reverse transcriptase (TERT), and a non- coding RNA (TERC); TERT reverse transcribes telomere DNA using TERC as the template (1). Given the fundamental role of TERT in oncogenesis, polymorphisms of genes related to telomerase may influence the expression levels of this enzyme, influencing the host's susceptibility to tumor progression and metastasis [2,3]. Some studies have linked these polymorphisms with susceptibility and/or survival of patients with breast cancer [2,4,5,6]. In this work, we analyzed a region in the second intron of the gene TERT, located in chromosome 5p15.33, by sequencing, in order to find mutations associated with clinical aspects. The DNA of 65 patients with breast cancer were evaluated. Methodology: The population consisted of breast cancer patients who accepted to participate in the study, attended from March 2015 to September 2016 at a public hospital (HUB) and a private center for cancer treatment (Cettro®), both in Brasília city – Brazil. Standard DNA extraction from blood samples was performed by dehydration and precipitation with saturated NaCl solution. The concentration and purity of the DNA were determined by spectrophotometry using the NanoDrop One equipment® (Themo Scientific, Madison, USA). The region of interest was amplified by conventional PCR. The primer sequences were: F: 5'-ATG CGA CAG TTC GTG GCT CA-3 'and R: 5'-ATC CCC TGG CAC TGG ACG TA-3'. The sequencing was performed in the AB 3500 Genetic Analyzer® (Applied Biosystems). Data were analyzed by using software Chromas Lite ® (2.01, Technelysium Pty Ltd.) and the BLAST (NCBI - http://blast.ncbi.nlm.nih.gov/Blast.cgi). GraphPad Prism software version 7.02 was used for statistical analyzes. Results and Conclusion: A total of 65 patients were selected for DNA analysis. We identified a recurrent mutation (C>T) among them. The allelic frequencies were 0,79 for C and 0,21 for T. Only three patients had genotype TT, but 21 were heterozygous (CT). This polymorphism was significantly associated with tumor grade, the presence of T allele were implicated in more aggressive (grade 3) tumors (p=0.028, chi-square test). Those patients whith genotype CT were at greater risk them others whith CC genotype of having high grade (3) breast tumors (odds ratio = 6.13, 95% confidence interval 1.46, 25.74; relative risk 1.9, 95% confidence interval 1.02, 3.59). Citation Format: Alvares MM, Rodrigues KS, Matos JN, Oliveira DM. Telomerase polymorphism and breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-03-06.

The age of heterozygous telomerase mutant parents influences the adult phenotype of their offspring irrespective of genotype in zebrafish

Background: Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested. Methods: Here we use a zebrafish telomerase mutant which harbours a nonsense mutation in tert to investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths. Results: We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate that tert homozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents. Conclusions: These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation.

Pof8 is a La-related protein and a constitutive component of telomerase in fission yeast

Telomerase reverse transcriptase (TERT) and the non-coding telomerase RNA subunit (TR) constitute the core of telomerase. Here we now report that the putative F-box protein Pof8 is also a constitutive component of active telomerase in fission yeast. Pof8 functions in a hierarchical assembly pathway by promoting the binding of the Lsm2-8 complex to telomerase RNA, which in turn promotes binding of the catalytic subunit. Loss of Pof8 reduces TER1 stability, causes a severe assembly defect, and results in critically short telomeres. Structure profile searches identified similarities between Pof8 and telomerase subunits from ciliated protozoa, making Pof8 next to TERT the most widely conserved telomerase subunits identified to date.

ETS1, nucleolar and non-nucleolar TERT expression in nevus to melanoma progression.

TERT (telomerase reverse transcriptase) is the catalytic component of telomerase. TERT shows little expression in normal somatic cells but is commonly re-expressed in cancers, facilitating immortalization. Recently-discovered TERT promoter mutations create binding sites for ETS-family transcription factors to upregulate TERT. ETS1 is reported to be important for TERT upregulation in melanoma. However it is unclear when in melanoma progression TERT and ETS1 proteins are expressed. To elucidate this question, ETS1 and TERT immunohistochemistry were performed on a panel of benign (n=27) and dysplastic nevi (n=34), radial growth phase (n=29), vertical growth phase (n=25) and metastatic melanomas (n=27). Lesions were scored by percentage of positive cells. ETS1 was readily detectable in all lesions, but not in normal melanocytes. TERT was located in either the nucleolus, the nucleoplasm (non-nucleolar) or both. Non-nucleolar TERT increased in prevalence with progression, from 19% of benign nevi to 78% of metastases. It did not however correlate with cell proliferation (Ki-67 immunostaining), nor differ significantly in prevalence between primary melanomas with or without a TERT promoter mutation. These results demonstrate that ETS1 is expressed very early in melanoma progression, and interestingly only non-nucleolar TERT correlates clearly in prevalence with melanoma progression. It can be acquired at various stages and by mechanisms other than promoter mutations.

Acute telomerase components depletion triggers oxidative stress as an early event previous to telomeric shortening

Loss of function of dyskerin (DKC1), NOP10 and TIN2 are responsible for different inheritance patterns of Dyskeratosis congenita (DC; ORPHA1775). They are key components of telomerase (DKC1 and NOP10) and shelterin (TIN2), and play an important role in telomere homeostasis. They participate in several fundamental cellular processes by contributing to Dyskeratosis congenita through mechanisms that are not fully understood. Presence of oxidative stress was postulated to result from telomerase ablation. However, the resulting disturbed redox status can promote telomere attrition by generating a vicious circle, which promotes cellular senescence. This fact prompted us to study if acute loss of DKC1, NOP10 and TINF2 can promote redox disequilibrium as an early event when telomere shortening has not yet taken place. We generated siRNA-mediated (DKC1, NOP10 and TINF2) cell lines by RNA interference, which was confirmed by mRNA and protein expression analyses. No telomere shortening occurred in any silenced cell line. Depletion of H/ACA ribonucleoproteins DKC1 and NOP10 diminished telomerase activity via TERC down-regulation, and produced alterations in pseudouridylation and ribosomal biogenesis. An increase in the GSSG/GSH ratio, carbonylated proteins and oxidized peroxiredoxin-6 was observed, in addition to MnSOD and TRX1 overexpression in the siRNA DC cells. Likewise, high PARylation levels and high PARP1 protein expression were detected. In contrast, the silenced TINF2 cells did not alter any evaluated oxidative stress marker. Altogether these findings lead us to conclude that loss of DKC1 and NOP10 functions induces oxidative stress in a telomere shortening independent manner.

PARN Modulates Y RNA Stability and Its 3'-End Formation.

Loss-of-function mutations in 3'-to-5' exoribonucleases have been implicated in hereditary human diseases. For example, PARN mutations cause a severe form of dyskeratosis congenita (DC), wherein PARN deficiency leads to human telomerase RNA instability. Since the DC phenotype in PARN patients is even more severe than that of loss-of-function alleles in telomerase components, we hypothesized that PARN would also be required for the stability of other RNAs. Here, we show that PARN depletion reduces the levels of abundant human Y RNAs, which might contribute to the severe phenotype of DC observed in patients. Depletion of PAPD5 or the cytoplasmic exonuclease DIS3L rescues the effect of PARN depletion on Y RNA levels, suggesting that PARN stabilizes Y RNAs by removing oligoadenylated tails added by PAPD5, which would otherwise recruit DIS3L for Y RNA degradation. Through deep sequencing of 3' ends, we provide evidence that PARN can also deadenylate the U6 and RMRP RNAs without affecting their levels. Moreover, we observed widespread posttranscriptional oligoadenylation, uridylation, and guanylation of U6 and Y RNA 3' ends, suggesting that in mammalian cells, the formation of a 3' end for noncoding RNAs can be a complex process governed by the activities of various 3'-end polymerases and exonucleases.

New insights into melanoma development

Telomerase promoter mutations extend cellular life span and increase melanoma susceptibility

The age of heterozygous telomerase mutant parents influences the adult phenotype of their offspring irrespective of genotype in zebrafish

Background: Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested.Methods: Here we use a zebrafish telomerase mutant which harbours a nonsense mutation intertto investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths.Results: We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate thatterthomozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents.Conclusions: These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation.

Single-Molecule Studies of Telomeres and Telomerase.

Telomeres are specialized chromatin structures that protect chromosome ends from dangerous processing events. In most tissues, telomeres shorten with each round of cell division, placing a finite limit on cell growth. In rapidly dividing cells, including the majority of human cancers, cells bypass this growth limit through telomerase-catalyzed maintenance of telomere length. The dynamic properties of telomeres and telomerase render them difficult to study using ensemble biochemical and structural techniques. This review describes single-molecule approaches to studying how individual components of telomeres and telomerase contribute to function. Single-molecule methods provide a window into the complex nature of telomeres and telomerase by permitting researchers to directly visualize and manipulate the individual protein, DNA, and RNA molecules required for telomere function. The work reviewed in this article highlights how single-molecule techniques have been utilized to investigate the function of telomeres and telomerase.

Epigenetic perturbation driving asleep telomerase reverse transcriptase: Possible therapeutic avenues in carcinoma.

In the last decade, implications of human telomerase reverse transcriptase (hTERT), a component of ribonucleoprotein telomerase in aging, senescence, and stem cell are highly evident. Besides, the activation of hTERT is also being documented several cancer types including carcinoma. The awakening of telomerase during carcinoma initiation and development is being seen with different perspectives including genetic and epigenetic tools and events. In view of several tumor progenitors genes (also referred as epigenetic mediators), telomerase is placed as key enzyme to achieve the carcinoma phenotype and sustain during the progression. It is true that swaying of telomerase in carcinoma could be facilitated with dedicated set of epigenetic modulators and modifiers players. These epigenetic alterations are heritable, potentially reversible, and seen as the epigenetic signature of carcinoma. Several papers converge to suggest that DNA methylation, histone modification, and small non-coding RNAs are the widely appreciated epigenetic changes towards hTERT modulation. In this review, we summarize the contribution of epigenetic factors in the telomerase activation and discuss potential avenues to achieve therapeutic intervention in carcinoma.

Fundamental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells.

Aberrant activation of telomerase occurs in 85–90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments.

Transcriptional Regulation of Telomerase Reverse Transcriptase (TERT) by MYC.

Telomerase elongates telomeres and is crucial for maintaining genomic stability. While stem cells and cancer cells display high telomerase activity, normal somatic cells lack telomerase activity primarily due to transcriptional repression of telomerase reverse transcriptase (TERT), the catalytic component of telomerase. Transcription factor binding, chromatin status as well as epigenetic modifications at the TERT promoter regulates TERT transcription. Myc is an important transcriptional regulator of TERT that directly controls its expression by promoter binding and associating with other transcription factors. In this review, we discuss the current understanding of the molecular mechanisms behind regulation of TERT transcription by Myc. We also discuss future perspectives in investigating the regulation of Myc at TERT promoter during cancer development.

RETINAL VASCULAR TORTUOSITY AND EXUDATIVE RETINOPATHY IN A FAMILY WITH DYSKERATOSIS CONGENITA MASQUERADING AS FAMILIAL EXUDATIVE VITREORETINOPATHY

To report a novel presentation of dyskeratosis congenita masquerading as familial exudative vitreoretinopathy. Observational case series involving single family and literature review. A brother and sister were diagnosed with familial exudative vitreoretinopathy at ages 4 and 2, respectively. Both patients were managed with laser photocoagulation. Eight years after the initial presentation, both siblings developed pancytopenia secondary to bone marrow failure. Laboratory work-up revealed severely shortened telomere length in both patients, and genetic testing revealed a missense mutation in the gene that encodes the reverse transcriptase component of telomerase, confirming the diagnosis of dyskeratosis congenita. The father of both children was a carrier of the same mutation, who exhibited marked retinal vascular tortuosity of the second-order vessels. Dyskeratosis congenita is a severe multisystem disorder, which should be considered in cases of pediatric exudative retinopathies with concurrent signs and/or symptoms of bone marrow failure.

Visualization of Human Telomerase Localization by Fluorescence Microscopy Techniques.

Human telomerase is a ribonucleoprotein (RNP) that synthesizes DNA repeats at the ends of chromosomes and maintains telomere length and genome stability. The enzyme is comprised of telomerase RNA (hTR) (which provides the template for telomere addition) and several protein subunits including telomerase reverse transcriptase (hTERT) (the catalytic component). Intracellular trafficking of the enzyme has emerged as an important factor in the regulation of telomerase activity. Telomerase trafficking between nuclear Cajal bodies (proposed sites of telomerase biogenesis and regulation) and telomeres (sites of action) is regulated by the cell cycle in concordance with telomere synthesis during S phase. Here, we describe fluorescence microscopy approaches to visualize the subcellular localization of the essential RNA component of telomerase (hTR) relative to Cajal bodies and telomeres in cultured human cells. These methods include fluorescence in situ hybridization (to detect hTR and telomeric DNA) and immunofluorescence (to detect Cajal bodies and telomere binding proteins). Because telomerase localization to telomeres is normally restricted to S phase, we also describe methods to synchronize and analyze cells within this phase of the cell cycle.

Analysis of Yeast Telomerase by Primer Extension Assays.

Telomeres are specialized nucleoprotein structures located at eukaryotic chromosomal termini, which are required for chromosome stability and are maintained by a reverse transcriptase named telomerase. Budding yeast has served as an extremely useful model system for analyzing telomere maintenance because the organism offers a wide range of genetic and biochemical tools. Several milestones in telomerase research have been reached through investigation of the yeast system. For example, the consequence of telomerase loss was first characterized in the budding yeast Saccharomyces cerevisiae. The catalytic component of telomerase (telomerase reverse transcriptase; TERT) was likewise initially cloned from this organism. Moreover, much of the current understanding of the structure and function of the telomerase complex was derived from yeast studies. In this chapter, we discuss one of the most useful tools for investigating yeast telomerase mechanisms and regulation: the primer extension assay. This assay can be used to examine the overall activity as well as the processivity of telomerase, which represents a unique aspect of telomerase enzymology. It can also be employed to analyze the mechanisms of telomerase regulatory proteins.

Regulation of the Human Telomerase Gene TERT by Telomere Position Effect-Over Long Distances (TPE-OLD): Implications for Aging and Cancer.

Telomerase is expressed in early human development and then becomes silenced in most normal tissues. Because ~90% of primary human tumors express telomerase and generally maintain very short telomeres, telomerase is carefully regulated, particularly in large, long-lived mammals. In the current report, we provide substantial evidence for a new regulatory control mechanism of the rate limiting catalytic protein component of telomerase (hTERT) that is determined by the length of telomeres. We document that normal, young human cells with long telomeres have a repressed hTERT epigenetic status (chromatin and DNA methylation), but the epigenetic status is altered when telomeres become short. The change in epigenetic status correlates with altered expression of TERT and genes near to TERT, indicating a change in chromatin. Furthermore, we identified a chromosome 5p telomere loop to a region near TERT in human cells with long telomeres that is disengaged with increased cell divisions as telomeres progressively shorten. Finally, we provide support for a role of the TRF2 protein, and possibly TERRA, in the telomere looping maintenance mechanism through interactions with interstitial TTAGGG repeats. This provides new insights into how the changes in genome structure during replicative aging result in an increased susceptibility to age-related diseases and cancer prior to the initiation of a DNA damage signal.