Télomérase | EPFL Graph Search

La télomérase est une ADN polymérase ARN dépendante qui, lors de la réplication de l'ADN chez les eucaryotes, permet de conserver la longueur du chromosome en ajoutant une structure spécifique à chaque extrémité : le télomère (du grec τέλος, extrémité ou fin). Bien que composé de désoxyribonucléotides comme le reste du chromosome, le télomère est synthétisé suivant un mode différent de la réplication classique de l'ADN. Les télomérases sont des ribonucléoprotéines (assemblage d'ARN et de protéines) qui catalysent l'addition d'une séquence répétée spécifique à l'extrémité des chromosomes. Cette séquence riche en nucléotides T et G est (TTAGGG)n chez les chordés (donc chez l'espèce humaine), avec un nombre de répétition n de l'ordre de quelques centaines à quelques milliers. Le composant ARN de la télomérase sert de matrice pour la synthèse de l'ADN. Historique L'enzyme a été découverte par Elizabeth Blackburn, Carol Greider et Jack Szostak en 1985 qui ont reçu le Prix N

Identification and characterization of proteins that protect human telomeres from fragility

Anna Christina Näger

Difficulties to replicate telomeres - the ends of our chromosomes - can cause telomere shortening andgenome instability. These difficulties are due to the repetitive DNA sequence and distinct structures at telomeresthat challenge the semi-conservative DNA replication machinery. Among the proteins that help toovercome the obstacles to telomere replication are factors known to be mutated in genetic diseases (forinstance WRN, BLM, RTEL1, CTC1), but we suspect that there are still factors and pathways unknown.DNA replication defects at telomeres manifest as smeary or multiple telomeric fluorescence in situ hybridization(FISH) signals on metaphase chromosomes - commonly called telomere fragility. To identify proteinsthat protect human telomeres from fragility, we depleted 71 proteins (including 8 controls) from HeLa cellsusing siRNA pools and analysed their metaphase chromosomes for fragile telomeres. The majority of theseproteins had been identified by QTIP-iPOND - a proteomic analysis of chromatin near telomere replicationforks. Among the 71 proteins that were depleted in the siRNA screen, we identified 29 novel proteins thatprevent telomere fragility and are therefore crucial to maintain genome stability. The telomere fragilityscreen validated QTIP-iPOND as a technique to identify telomere replication proteins. The hits identified inour telomere fragility screen may for example have functions in regulating telomeric chromatin compositionand compaction, regulating transcription of the telomeric long non-coding RNA TERRA and its associationwith telomeric chromatin, dealing with replication stress and stalled replication forks, modulating DNAdamage signalling and repair, modulating sister chromatid cohesion, and preventing oxidative stress.One of the 29 novel proteins identified in the telomere fragility screen is chromobox 8 (CBX8) whose functionat telomeres was investigated in further detail in this study. CBX8 is a component of the canonical polycombrepressive complex 1 (PRC1), an epigenetic regulator of gene expression. However, CBX8 also hasfunctions that are independent of the PRC1 complex. We show that human CBX8 prevents telomere fragilitycaused by RNA-DNA hybrids which could stall replication forks and induce homologous recombination.CBX8 depletion or knockout did however not affect telomeric RNA-DNA hybrid levels. We hypothesize thatCBX8 may be involved in repair activities at stalled replication forks that arise when the replisome encounterspersistent RNA-DNA hybrids. Moreover, we found that CBX8 localizes to telomeric repeat-binding factor2 (TRF2)-deficient telomeres in a manner that is dependent on RNA-DNA hybrids and ATM signalling.We demonstrate that H3K27me3 (trimethylated histone 3 lysine 27) marks, which have previously beensuggested to help recruitment of CBX proteins to chromatin via the CBX chromodomain, are dispensable forCBX8 recruitment to TRF2-deficient telomeres. Furthermore, we found that CBX8 promotes non-homologousend joining (NHEJ)-mediated telomere fusions of dysfunctional telomeres. We therefore hypothesizethat CBX8 is involved in repair activities that influence the DNA repair pathway choice at TRF2-deficient telomeres.

EPFL2022

Insight into the Regulation of Telomerase Access to Telomeres by Shelterin Proteins

Elena Aritonovska

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase. Impaired telomerase function can lead to premature organismal aging, development of cancer and multisystem disorders such as dyskeratosis congenita. Telomerase access to telomeres may be regulated by a telomere-binding complex termed shelterin, which is composed of TRF1, TRF2, RAP1, TIN2, TPP1 and POT1 proteins. However the molecular basis for human telomerase recruitment to telomeres is not known. Here, we have directly investigated the process of telomerase recruitment via chromatin immunoprecipitation (ChIP) and fluorescence in situ hybridization (FISH). We find that depletion of two components of the shelterin complex – TPP1 and the protein that tethers TPP1 to the complex, TIN2 – results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Furthermore, we find that the double-stranded telomere binding protein, TRF2 is dispensable for telomerase association with telomeres. Deletion of the oligonucleotide/oligosaccharide-binding fold (OB-fold) of TPP1 further disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that the recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere. We propose that the loss of TRF2 can be compensated by the second double-stranded telomere binding protein, TRF1 that anchors TIN2/TPP1-recruited telomerase onto the telomeres. Dyskeratosis congenita (DC) is a multisystem disorder characterized with bone marrow failure, cancer predisposition and defective telomere maintenance. The TINF2 gene that encodes for the TIN2 shelterin protein is one of seven mutated genes identified in DC patients. Here, we have examined the effects of TIN2 DC mutants on telomere protection and shelterin protein stability. We find that exogenous expression of TIN2 DC mutants can rescue the TIN2-depleted phenotype, restoring TPP1 protein levels and telomere protection. Our findings indicate that TIN2 DC mutants preserve the intact telomere structure and protection. We propose that defective telomere elongation may be the underlying cause of impaired telomere maintenance in TIN2 DC patients.

EPFL2011