The human telomeric proteome during telomere replication

Chih-Yi Lin, Joachim Lingner, Gérald Joseph Paul Lossaint, Thomas Frédéric Lunardi, Aleksandra Vancevska
OXFORD UNIV PRESS, 2021
Article

Résumé

Telomere shortening can cause detrimental diseases and contribute to aging. It occurs due to the end replication problem in cells lacking telomerase. Furthermore, recent studies revealed that telomere shortening can be attributed to difficulties of the semi-conservative DNA replication machinery to replicate the bulk of telomeric DNA repeats. To investigate telomere replication in a comprehensive manner, we develop QTIP-iPOND - Quantitative Telomeric chromatin Isolation Protocol followed by isolation of Proteins On Nascent DNA - which enables purification of proteins that associate with telomeres specifically during replication. In addition to the core replisome, we identify a large number of proteins that specifically associate with telomere replication forks. Depletion of several of these proteins induces telomere fragility validating their importance for telomere replication. We also find that at telomere replication forks the single strand telomere binding protein POT1 is depleted, whereas histone H1 is enriched. Our work reveals the dynamic changes of the telomeric proteome during replication, providing a valuable resource of telomere replication proteins. To our knowledge, this is the first study that examines the replisome at a specific region of the genome.

Official source

https://infoscience.epfl.ch/record/291313?ln=fr

À propos de ce résultat

Cette page est générée automatiquement et peut contenir des informations qui ne sont pas correctes, complètes, à jour ou pertinentes par rapport à votre recherche. Il en va de même pour toutes les autres pages de ce site. Veillez à vérifier les informations auprès des sources officielles de l'EPFL.

Concepts associés

Chargement

Publications associées

Chargement

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

Chargement

Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.

1997

Chargement

Telomeric states in cancer development

Jana Majerská

Telomere dysfunction can unleash genome instability or permanently arrest cell proliferation, and thereby contribute to the processes of cancer and aging. Proper telomere function relies on many proteins that form part of its structure. Some telomeric proteins mediate telomere replication whereas others suppress the DNA damage response and DNA repair activities avoiding chromosome end-to-end fusions. However, our knowledge of the telomeric proteome and its pathological perturbations remains incomplete. Progress in telomere research has been partially hampered by a lack of systematic approaches and sensitive tools to analyze telomere composition and function. This thesis provides methodological improvements and applications that have helped to expand the repertoire of proteins involved in protection of telomere integrity. Firstly, this work offers a detailed implementation guide for the Quantitative Telomeric Chromatin Isolation Protocol (QTIP), a mass-spectrometry based technique, which enables identification of telomeric proteins and their quantitative changes between different cell states. Secondly, QTIP is used to characterize the alterations that occur in the telomeric proteome during cellular transformation. I have discovered that proteins involved in telomere DNA replication and DNA damage repair, as well as the telo-meric RNA TERRA are upregulated during cellular transformation. Thirdly, I demonstrate that several of the upregulated proteins counteract telomere fragility, which can be induced by oncogenic stress. SAMHD1 is one of the proteins induced upon cellular transformation. It is frequently mutated in cancer and Aicardi-Goutières syndrome. SAMHD1 association with telomeres is con-firmed and its function explored. I show that SAMHD1 deficiency leads to telomere breakage events when telomere replication is compromised by TRF1 depletion. These results suggest that telomeres break during replication when deprived of TRF1 and that SAMHD1 is required for telomeric DNA damage repair. Finally, the molecular basis of the telomere syndrome caused by CTC1 mutations is examined. Overall, SAMHD1 and CTC1 functions are characterized and added to the conundrum of proteins that facilitate faithful telomere replication.

EPFL2018

Chargement

Telomeric states in cancer development

Jana Majerská

EPFL2018

EPFL2022