Telomere structure and shortening in telomerase-deficient Trypanosoma brucei

Telomerase consists of a reverse transcriptase (TERT) and an RNA that contains a template for telomere-repeat extension. Telomerase is required to prevent telomere erosion and its activity or lack thereof is important for tumorigenesis and ageing. Telomerase has been identified in numerous organisms but it has not been studied in kinetoplastid protozoa. Trypanosoma brucei, the causative agent of African sleeping sickness, evades the host immune response by frequently changing its variant surface glycoprotein (VSG). The single expressed VSG is transcribed from one of ∼20 subtelomeric ‘Expression Sites’, but the role telomeres might play in regulating VSG transcription and switching is unknown. We identified and sequenced the T.brucei TERT gene. Deleting TERT resulted in progressive telomere shortening of 3–6 bp per generation. In other organisms, the rate of telomere shortening is proportional to the length of the terminal 3′ single-strand overhang. In T.brucei, G-overhangs were undetectable (<30 nt) by in-gel hybridization. The rate of telomere shortening therefore, agrees with the predicted shortening due to the end replication problem, and is consistent with our observation that G-overhangs are short. Trypanosomes whose telomere length can be manipulated provide a new tool to investigate the role of telomeres in antigenic variation

Unexpected X Chromosome Skewing during Culture and Reprogramming of Human Somatic Cells Can Be Alleviated by Exogenous Telomerase

SummarySomatic tissues in female eutherian mammals are mosaic due to random X inactivation. In contrast to mice, X chromosome reactivation does not occur during the reprogramming of human female somatic cells to induced pluripotent stem cells (iPSCs), although this view is contested. Using balanced populations of female Rett patient and control fibroblasts, we confirm that all cells in iPSC colonies contain an inactive X, and additionally find that all colonies made from the same donor fibroblasts contain the same inactive X chromosome. Notably, this extreme “skewing” toward a particular dominant, active X is also a general feature of primary female fibroblasts during proliferation, and the skewing seen in reprogramming and fibroblast culture can be alleviated by overexpression of telomerase. These results have important implications for in vitro modeling of X-linked diseases and the interpretation of long-term culture studies in cancer and senescence using primary female fibroblast cell lines

A novel cross-sector telemedical approach to detect arrhythmia in primary care patients with palpitations using a patient-activated event recorder

Background: Patient-activated event recorders (ER) can facilitate diagnosis in unclear palpi­tations, however impact of ER screening on further treatment in clinical routine is unknown. We investigated the feasibility and clinical value of a network-based telemetric monitoring using a patient activated ER. Methods: The network consisted of 12 general practitioners (GP) and a department of car­diology (DC). GP-patients sent electrocardiograms (ECGs) twice daily and in case of palpitations. ECGs were transferred by email to GP and DC and analyzed independently by both. The therapeutic strategy was discussed between GP and DC. The monitoring period ended after 4 weeks or in case of detected arrhythmia. Results: A group of 184 consecutive patients were retrospectively analyzed. Mean age was 57.5 ± 14.4 years (range 17–82), 104 (56.5%) were female. Significant arrhythmia occurred in 71 (38.5%) patients: Recurrence of known paroxysmal atrial fibrillation (AF; n = 27, 14.7%), de novo AF (n = 19, 10.3%), premature complexes/bigeminus (n = 13, 7.1%), sinus tachycar­dia (n = 7, 3.8%), atrioventricular nodal reentrant tachycardia (n = 3, 1.6%), and ventricular tachycardia (n = 2, 1.1%). A therapeutic consequence resulted in 63 (88.7%) patients with de­tected arrhythmia: new oral anticoagulation (n = 29, 40.8%), new antiarrhythmic medication (n = 27, 38.0%), behavioral intervention (n = 19, 26.8%), electrophysiology-study/catheter ablation (n = 4, 5.6%), cardioversion (n = 2, 2.8%), implantable cardioverter-defibrillator- -implantation (n = 1, 1.4%), and left atrial appendage occluder (n = 1, 1.4%). Conclusions: The investigated cross-sector telemetric network is a feasible approach to detect arrhythmia in patients with palpitations and may have high impact on further treatment, notably in those at risk for stroke due to AF

Telomere Structure and Shortening in Telomerase-Deficient Trypanosoma brucei: Implications for Anitgenic Variation

A variety of parasites persist in their host through sequential expression of variant surface antigens. Intriguingly, the genes encoding these antigens are frequently found adjacent to the telomeres. Telomeres are nucleoprotein complexes consisting of tandem DNA repeats and proteins that bind to them. Their function is to protect chromosome ends from the DNA repair machinery that would otherwise recognize them as double-stranded breaks. With the help of the ribonucleoprotein telomerase they compensate for the gradual sequence loss that would otherwise arise from the inability of conventional DNA polymerases to replicate chromosome ends. In Trypanosoma brucei, the causative agent of African trypanosomiasis, the surface coat consists of a dense layer of Variant Surface Glycoproteins (VSG). The actively transcribed VSG is found in one of ~20 telomeric Expression Sites (ES). Antigenic variation can occur by transcriptional switching, reciprocal translocations, or duplicative gene conversion events between ES. In recent African isolates, duplicative gene conversion occurs at a high frequency and predominates, but the switching frequency decreases dramatically upon laboratory-adaptation. Very little is known about the regulation of antigenic variation. To address whether telomeres are involved in antigenic switching we created telomerasedeficient T. brucei. Telomerase-deficient parasites exhibited progressive telomere shortening at a rate of 3–6 bp/PD, which correlates with the end replication problem and G-overhang length. Upon reaching a critical length, short silent ES telomeres stabilized. Telomere decline was accompanied by loss of minichromosomes and rearrangements at intermediate chromosomes. Essential megabase chromosomes remained stable. After extensive telomere attrition, the active ES telomere stabilized, but the transcribed VSG was gradually lost from the population and replaced by a new VSG through duplicative gene conversion. We present a model in which subtelomeric break-induced replication-mediated repair at a short ES telomere leads to duplicative gene conversion and expression of a new VSG. By restoring telomerase, we studied telomere elongation dynamics. At the active ES, the rate of telomere elongation is inversely proportional to the initial telomere length. At silent ES, the rate of elongation remains constant. We propose a model where transcription-dependent chromatin remodeling permits telomere elongation by telomerase. We show that telomere growth at a rate of 6–8 bp/PD appears to be a unique feature of T. brucei. Lastly we demonstrate that fast-switching African T. brucei isolates have dramatically shorter telomeres than laboratory strains. We present a speculative model in which telomere growth and breakage affect the rate of antigenic switching

Biomarkers of Cellular Senescence and Skin Aging

Cellular senescence is an irreversible growth arrest that occurs as a result of different damaging stimuli, including DNA damage, telomere shortening and dysfunction or oncogenic stress. Senescent cells exert a pleotropic effect on development, tissue aging and regeneration, inflammation, wound healing and tumor suppression. Strategies to remove senescent cells from aging tissues or preneoplastic lesions can delay tissue dysfunction and lead to increased healthspan. However, a significant hurdle in the aging field has been the identification of a universal biomarker that facilitates the unequivocal detection and quantification of senescent cell types in vitro and in vivo. Mammalian skin is the largest organ of the human body and consists of different cell types and compartments. Skin provides a physical barrier against harmful microbes, toxins, and protects us from ultraviolet radiation. Increasing evidence suggests that senescent cells accumulate in chronologically aged and photoaged skin; and may contribute to age-related skin changes and pathologies. Here, we highlight current biomarkers to detect senescent cells and review their utility in the context of skin aging. In particular, we discuss the efficacy of biomarkers to detect senescence within different skin compartments and cell types, and how they may contribute to myriad manifestations of skin aging and age-related skin pathologies.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

Telomerase-Independent Stabilization of Short Telomeres in Trypanosoma brucei

In cancer cells and germ cells, shortening of chromosome ends is prevented by telomerase. Telomerase-deficient cells have a replicative life span, after which they enter senescence. Senescent cells can give rise to survivors that maintain chromosome ends through recombination-based amplification of telomeric or subtelomeric repeats. We found that in Trypanosoma brucei, critically short telomeres are stable in the absence of telomerase. Telomere stabilization ensured genomic integrity and could have implications for telomere maintenance in human telomerase-deficient cells. Cloning and sequencing revealed 7 to 27 TTAGGG repeats on stabilized telomeres and no changes in the subtelomeric region. Clones with short telomeres were used to study telomere elongation dynamics, which differed dramatically at transcriptionally active and silent telomeres, after restoration of telomerase. We propose that transcription makes the termini of short telomeres accessible for rapid elongation by telomerase and that telomere elongation in T. brucei is not regulated by a protein-counting mechanism. Many minichromosomes were lost after long-term culture in the absence of telomerase, which may reflect their different mitotic segregation properties

Telomere Structure and Function in Trypanosomes: A Proposal

Telomeres are specialized DNA-protein complexes that stabilize chromosome ends, protecting them from nucleolytic degradation and illegitimate recombination. Telomeres form a heterochromatic structure that can suppress the transcription of adjacent genes. These structures might have additional roles in Trypanosoma brucei, as the major surface antigens of this parasite are expressed during its infectious stages from subtelomeric loci. We propose that the telomere protein complexes of trypanosomes and vertebrates are conserved and offer the hypothesis that growth and breakage of telomeric repeats has an important role in regulating parasite antigenic variation in trypanosomes