Thèses sur le sujet « Telomere protection »

An SGA approach to discover cdc13-1ts supressors. Telomeres, the DNA-protein complexes at the end of eukaryotic chromosomes, are essential for chromosomal stability. In yeast, the telomeric single-strand binding protein Cdc13p has multiple important roles related to telomere maintenance: (1) telomeric"capping"--protection of telomeres by forming complexes with yKu70/80 and with Stn1p/Ten1p; (2) positive regulation of telomere replication via interaction with Est1p, which is a part of telomerase; (3) negative regulation of telomerase by the recruitment of telomere elongation suppressors Stn1p and Ten1p. In an attempt to identify genes that are involved in the deleterious outcome of an absence of Cdc13p, we screened the yeast gene knock-out library for genes that could suppress the growth defect of cdc13-1 cells at 33ê C. For this purpose, we performed an SGA array experiment. We scored for the ability of double mutant haploids to grow at 33ê C. Eventually, we hoped to find the elusive genes involved in telomere 5'-end processing (exonucleases). Based on the comparative analysis of growth properties of the strains (23ê C vs 33ê C), the initial screen identified up to 111 genes that displayed an apparent growth at 33ê C. In order to verify these results, diploids were regenerated, sporulated, microdissected, and haploid double mutants cdc13-1 yfg[deletion] were isolated from 38 potential cdc13-1 suppressors. Unfortunately, this verification failed to reproduce a suppression of the growth defect by any of the selected genes at any temperature. While disappointing, the results reemphasize that careful re-examination of large scale SGA approaches are indispensable before going on to more involved experimentation. Similarities and differences between adaptation to DNA double-strand break and to telomere uncapping in yeast Saccharomyces cerevisiae. It was previously shown that a certain proportion of telomerase negative survivor cells (both type I and type II cells) is able to survive in the absence of the telomere capping protein Cdc13p. These strains (named [deletion]13s) were characterized in great detail and one of their discovered features was a striking ability to continuously inactivate DNA-damage checkpoints. Based on structural similarities between DNA double strand breaks (DSB) and unprotected telomeres, we attempted to verify if the molecular mechanisms regulating adaptation to a single irreparable DSB also regulate adaptation to a loss of Cdc13p. For this purpose we created three tlc1[deletion] cdc13[deletion] strains also harboring DSB adaptation related mutations tid1[deletion], ptc2[deletion] and rfa1-t11. After deprotection of their telomeres, mutant survivor cells showed similar cell cycle progression patterns as compared to the cells where a single irreparable DSB was introduced. Adaptation defective mutants tid1[deletion] and ptc2[deletion] demonstrated an inability to adapt to telomere uncapping and to resume cell cycle. Interestingly, cells harboring the rfa1-t11 allele, which was reported to suppress adaptation defects of other mutations, did not show any distinguishable phenotype in terms of initial adaptation to telomere deprotection; i.e. rfa1-t11 mutant survivors do escape the G2/M arrest and re-enter the cell cycle. However, all three mutant survivor strains failed to produce viable [deletion]13 capping independent cells, which is consistent with the hypothesis that adaptation to loss of Cdc13p depends on the same pathway as the previously reported adaptation phenomenon. Finally, we report the surprising finding that if cells had once experienced an adapted [deletion]13 state, they will re-produce capping negative survivors much more readily. Thus, while a culture of type II survivor cells generates [deletion]13s at a rate of about 1×10 -5 events per division, cells that had been [deletion]13s and re-transformed with a Cdc13p carrying plasmid will produce capping independent cells at about 1×10-2 events per division. We are currently examining why these cells re-generate [deletion]13 cell lines more readily and suspect structural differences in telomere terminal sequence arrangements.

Telomerase is a ribonucleoprotein complex with a well-established role in telomere maintenance. There is growing evidence of non-canonical functions of telomerase that promote tumorigenesis. Here we have demonstrated that hTERT can mediate telomere protection independent of its canonical function in telomere maintenance, potentially enhancing positive feedback pathways that may facilitate oncogenesis. hTERT mediated telomere protection was found to be independent of catalytic activity and telomere recruitment, required nuclear export of hTERT, and required an intact N-terminal portion of hTERT. We have further shown that hTERT appears to regulate two seemingly independent pathways, with mutual requirement of both NFκB and Hsp70 to exert telomere protection. hTERT over-expression resulted in an increased localization of Hsp70 at the telomeric protein TRF2. This increase was dependent on the presence of the shelterin accessory protein Apollo, suggesting that Apollo acts as the bridge between Hsp70 and TRF2, facilitating the stabilization of TRF2 to form a fully capped telomere. Conversely, depletion of hTERT down-regulated NFκB signaling, with a concomitant increase in telomeric DNA damage and consequential G1 arrest. These two pathways may intersect physiologically through Hsp70 regulation of the NFκB pathway, further enforcing the feed-forward mechanism between hTERT and its transcriptional targets to promote oncogenesis.

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. In this work, we investigated the role of key telomeric proteins in protecting Saccharomyces cerevisiae telomeres from degradation. We show that the shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also Yku prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres. In particular, while Yku plays the major role in preventing initiation, Rif2 and Rap1 act primarily by limiting extensive resection. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases. Since Rif1 plays a very minor role in protecting wild type telomeres from degradation, we further investigated whether Rif1 participates in telomere protection in combination with other capping activities, like those exerted by the CST complex (Cdc13-Stn1-Ten1). We found that, unlike RIF2 deletion, the lack of RIF1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, lethality in cdc13 rif1Δ cells is partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. Like CDC13, RIF1 also genetically interacts with the Polα-primase complex, which is involved in the fill-in of the telomeric complementary strand. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex.

Telomeres are specialized nucleoprotein structures at the ends of linear chromosomes to maintain the integrity of chromosome ends. In human cells, telomeres are protected by a complex of six proteins bound to telomeric DNA, named shelterin. As a crucial component of shelterin, the human telomeric capping protein TRF2 is required for telomere stabilization and function. Recently, TRF2 was shown to form a complex with Apollo, a 5' -exonuclease of the metallo-beta-lactamase family. In this thesis, biochjemical, genetic, and cellular approaches were combined to understand the mechanism of 5' -exonuclease of Apollo in the telomere protection. First, in vitro evidence shows that TRF2 plays a dual role in controlling Apollo nuclease activity. TRF2 stimulates the nuclease activity of Apollo on TRF2-free DNA ends and inhibits it when it is bound proximately to the 5' end. This stimulatory effect of Apollo nuclease depends on the basic domain of TRF2. Second, the specific coupling between the Apollo nuclease activity and the dynamics of TRF2 DNA binding shows in vivo that TRF2 and Apollo are not only involved in the protection of the very-terminal structure of telomeres but also of the inner part of telomere repeat tracts, which is required for telomere integrity during S-phase. Moreover TRF2 and Apollo protect from DNA damage telomere repeat tracts inserted far away from chromosome ends, furthze indicating that TRF2 couples the exonuclease activity of Apollo to protect telomere during fork progression. Third, exonuclease activity of Apollo, which is required for the telomere protection, is directly regulated by TRF2. These data are consistent with a new model of telomere protection in with TRF2 couples the exonuclease activity of Apollo to replication in order to eliminate clastogenic and recombinogenic structures. The work in my thesis suggest that replicative senescence in human cells may not only be caused by loss of the terminal cap integrity but also by replication-linked DNA breakages and recombinations within the inner part of telomeres.

Several aspects of Drosophila telomere biology indicate that telomere protection can be regulated by an epigenetic mechanism. First, terminally deleted chromosomes can be stably inherited and do not induce damage responses such as apoptosis or cell cycle arrest. Second, the telomere protection proteins HP1 and HOAP localize normally to these chromosomes and protect them from fusions. Third, unprotected telomeres still contain HeT-A sequences at sites of fusions. Taken together these observations support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophilatelomeres from fusion. Work presented in this thesis demonstrates that the Drosophila proteins ATM and Nbs are required for the regulation of DNA damage responses similar to their yeast and mammalian counterparts. This work also establishes a role for the ATM and ATR DNA damage response pathways in the protection of both normal and terminally deleted chromosomes. Mutations that disrupt both pathways result in a severe telomere fusion phenotype, similar to HP1 and HOAP mutants. Consistent with this phenotype, HOAP localization at atm,atr double mutant telomeres is completely eliminated. Furthermore, telomeric sequences are still present, even at the sites of fusions. These results support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophila telomeres from fusion.

TTAGGG repeat factor 2 (TRF2) is a protein that plays an important role in capping telomere ends from DNA damage responses. Telomere DNA consists of double strand repeats of the TTAGGG sequence ending with a 3'single-stranded overhang of the guanine strand (the G-strand overhang). TRF2 protects telomeres from being recognized as double-stranded breaks. It is thought that this protection is performed through the formation of T-loop structures and recruitment of proteins into a complex called shelterin. The exact mechanism of T-loop formation is unknown. I show with in vitro biochemical studies that TRF2 specifically interacts with telomeric ss/ds DNA junctions and binding is sensitive to the sequence of the G-strand overhang and double-stranded DNA sequence at the junction. Binding assays with TRF2 truncation mutants suggest that TRF2 interacts with both the double-stranded DNA through the C-terminal DNA binding domain and the G-strand overhang through the N-terminus. Mobility shifts and atomic force microscopy with truncation mutants bound to telomeric DNA also show that a previously uncharacterized "linker" region within TRF2 is involved in DNA-specific TRF2 oligomerization. From these observations, I suggest that TRF2 forms protective loops by oligomerizing through both a previously characterized dimerization domain and the linker region. I propose that loop formation involving the telomere ends is accomplished through direct interactions between TRF2 and the G-strand overhang. In addition to DNA protection, a new role has emerged for TRF2 in sensing DNA damage. TRF2 can be phosphorylated within its dimerization domain by ATM and recruited to DNA damage foci in cells. The inhibition of TRF2 function alone has been shown to induce senescence and apoptosis in vascular endothelial cells. Since the common stimuli for a senescence phenotype is activation of a DNA damage response, I studied the relationship between DNA damage and TRF2 phosphorylation. Ex-vivo characterization of DNA damage-induced changes in vascular smooth muscle cells (VSMC) was undertaken. VSMC treated with H202 induced an increase in reactive oxygen species (ROS), and 8-oxo-guanine accumulation resulting in cell cycle arrest, chromatin condensation and a senescent phenotype. Interestingly phosphorylated TRF2 and ATM were also up regulated. Balloon injury was used to test the connection between phosphorylated TRF2 and senescence during vascular remodeling in rat arteries. Vascular remodeling as judged by neointima formation was associated with accumulation of 8-oxo-guanine, DNA damage signaling, including phosphorylated TRF2, an increase in cell cycle inhibitors and senescence. These events were exaggerated in aged animals and are consistent with a role in telomere dysfunction, and age related diseases.

Upon fertilization, the early embryo sustains most of the cellular processes using the maternally deposited reserves in the egg itself until the zygotic gene expression takes charge. Among the plethora of essential components provided by the mother are small non-coding RNAs called PIWI-interacting RNAs (piRNAs), which provide immunity to the zygote against transposon challenge. In this thesis, I have presented three different functions of piRNAs in Drosophila melanogaster- in maintenance of genomic integrity, telomere protection and their role as an adaptive immune system against genomic parasites. In Chapter 2, I have described the phenotypic effects of the loss of piRNA function in early embryos. The mutations affecting the piRNA pathway are known to cause embryonic lethality. To describe this lethality in detail, I have shown that all the characterized piRNA mutants show compromised zygotic genomic integrity during early embryogenesis. In addition, two piRNA pathway components, Aubergine (Aub) and Armitage (Armi) are also required for telomere resolution during early embryogenesis. Aub and Armi recruit telomeric protection complex proteins, HOAP and HP1, to the telomeric ends and thus avoid activation of the Non-homologous end joining (NHEJ) DNA repair pathway at the telomeres. There are about 120 transposon families in Drosophila melanogaster and piRNA pathway mutations cause activation of many of the resident transposons in the genome. In Chapter 3, I have described the effects of infection by a single transposon, P-element, in naïve strains by introduction through the zygote. Activation of the P-element leads to desilencing of unrelated transposons, causing accumulation of germline DNA damage which is linked to severely reduced fertility in the hybrid females. However, there is partial restoration of fertility as the hybrid progeny age, which correlates with P-element piRNA production and thus P-element silencing. Additionally, a number of transposons mobilize into piRNA generating heterochromatic clusters in the genome, and these insertions are stably inherited in the progeny. Collectively our data shows that piRNA production can be triggered in the adults in an absence of maternal contribution and that piRNAs serve as an adaptive immune system which helps resolve an internal genetic conflict between the host and the parasite. In an effort to understand the phenotypic effects of piRNA dysfunction in Drosophila, we have uncovered new exciting roles for piRNAs in development and presented evidence how transposons can act as architects in restructuring the host genome.

Protection of telomeres (POT1) binds chromosome ends, recognizing single-strand telomeric DNA via two oligonucleotide/oligosaccharide binding folds (OB-folds). The Arabidopsis thaliana POT1a and POT1b paralogs are atypical: they do not exhibit telomeric DNA binding, and they have opposing roles in regulating telomerase activity. AtPOT1a stimulates repeat addition processivity of the canonical telomerase enzyme, while AtPOT1b interacts with a regulatory lncRNA that represses telomerase activity. Here, we show that OB1 of POT1a, but not POT1b, has an intrinsic affinity for telomeric DNA. DNA binding was dependent upon a highly conserved Phe residue (F65) that in human POT1 directly contacts telomeric DNA. F65Amutation of POT1aOB1 abolished DNA binding and diminished telomerase repeat addition processivity. Conversely, AtPOT1b and other POT1b homologs from Brassicaceae and its sister family, Cleomaceae, naturally bear a non-aromatic amino acid at this position. By swapping Val (V63) with Phe, AtPOT1bOB1 gained the capacity to bind telomeric DNA and to stimulate telomerase repeat addition processivity. We conclude that, in the context of DNA binding, variation at a single amino acid position promotes divergence of the AtPOT1b paralog from the ancestral POT1 protein.

Le cellule eucariotiche prevengono l’instabilità genomica attivando un complesso sistema biochimico chiamato Risposta ai Danni al DNA (DDR). Le proteine chinasi Mec1 e Tel1 di S. cerevisiae, ortologhe di ATR ed ATM umane, giocano un ruolo centrale nella DDR. Queste proteine attivano il checkpoint da danni al DNA il quale coordina la riparazione dei danni al DNA con la progressione del ciclo cellulare. Il ruolo della proteina Tel1 è particolarmente evidente in presenza di rotture a doppia elica del DNA (DSB). I DSB possono essere riparati tramite ricombinazione omologa la quale richiede la degradazione dell’estremità 5’ della rottura (resection). Tel1 contribuisce alla riparazione dei DSB promuovendo l’inizio della resection. Nonostante le funzioni di Tel1 nella DDR, l’assenza di Tel1 conferisce alle cellule di lievito solo una moderata sensibilità alla camptotecina (CPT), un inibitore delle topoisomerasi di tipo I. Dato che derivati della CPT sono attualmente usati in chemioterapia, comprendere il ruolo di Tel1 in risposta alla CPT è rilevante per lo sviluppo di nuove terapie anticancro. Oltre a ciò, Tel1 è importante per il mantenimento dei telomeri, i quali vengono replicati grazie ad una trascrittasi inversa chiamata telomerasi. In particolare, Tel1 promuove il reclutamento della telomerasi e quindi l’omeostasi dei telomeri. La telomerasi è inattivata nella maggior parte dei tessuti umani che di conseguenza sono soggetti ad un progressivo accorciamento dei telomeri. Quando i telomeri divengono criticamente corti, si ha il blocco della divisione cellulare in un processo noto come senescenza replicativa che limita la proliferazione cellulare agendo da meccanismo oncosoppressore. La senescenza è innescata dall’attivazione del checkpoint da danni al DNA governato da Mec1/ATR e Tel1/ATM. In particolare, il meccanismo attraverso il quale Tel1/ATM induce la senescenza è ancora ignoto. Durante il mio dottorato ho quindi seguito due distinti progetti allo scopo di far luce sui meccanismi molecolari che coinvolgono Tel1 in risposta alla CPT e nell’induzione della senescenza. Riguardo al primo progetto, sia in lievito che in mammifero la CPT induce la reversione delle forche replicative. Cellule tel1∆ sono caratterizzate da un ridotto livello di forche regresse indotte dalla CPT rispetto a cellule selvatiche. In risposta alla CPT, l’assenza dell’attività nucleasica di Mre11 ripristina livelli normali di forche regresse in cellule tel1∆. Inoltre, l’inattivazione della proteina Mrc1 mitiga la sensibilità a CPT di cellule tel1∆ e previene la reversione delle forche in cellule selvatiche, in cellule tel1∆ e in cellule senza l’attività nucleasica di Mre11. Nel loro insieme questi risultati indicano che Tel1 stabilizza le forche regresse generate da Mrc1 in presenza della CPT inibendo l’attività nucleasica di Mre11 a livello di queste strutture di DNA. Riguardo al secondo progetto, per studiare il ruolo di Tel1 nell’induzione della senescenza ho sfruttato cellule di lievito senza telomerasi e l’allele mutante TEL1-hy184 identificato precedentemente come soppressore dei difetti di checkpoint di cellule mec1∆. Subito dopo l’inattivazione della telomerasi la variante Tel1-hy184 accelera la senescenza rispetto a cellule che esprimono la forma selvatica di Tel1. L’aumentata senescenza indotta da Tel1-hy184 è causata dall’attivazione di un checkpoint completamente dipendente dalla proteina Rad9 e solo in parte dipendente da Mec1. Inoltre, Tel1-hy184 non sembra aumentare il livello di ssDNA alle estremità di DNA. Ciò suggerisce che Tel1 induce la senescenza replicativa contribuendo direttamente all’attivazione del checkpoint in presenza di telomeri disfunzionali. Nel complesso, i risultati che ho ottenuto durante il mio dottorato permettono di comprendere meglio le funzioni di Tel1/ATM nel mantenimento della stabilità genomica.
Eukaryotic cells prevent genomic instability by activating a complex network of safeguard pathways called DNA Damage Response (DDR). S. cerevisiae Mec1 and Tel1 protein kinases, orthologs of human ATR and ATM, play a central role in the DDR. These proteins activate a checkpoint cascade which coordinates DNA damage repair with cell cycle progression. The role of Tel1 is particularly evident in the presence of DNA Double-Strand Breaks (DSBs), one of the most cytotoxic forms of DNA lesions. DSBs can be repaired by Homologous Recombination (HR), which requires the degradation of 5’-ended strands of the break (resection). Tel1 contributes to DSB repair by promoting resection initiation. Despite Tel1 functions in DDR, the absence of Tel1 confers a moderate sensitivity to camptothecin (CPT), an inhibitor of type I DNA topoisomerases. Since CPT derivatives are currently used in chemotherapy, understanding the molecular basis of tel1Δ mutant sensitivity to CPT is relevant for the development of anti-cancer therapies based on combined treatments with CPT derivatives and ATM inhibitors. In addition, Tel1 is important for the maintenance of telomeres, which are replicated by a reverse transcriptase called telomerase. In particular, Tel1 promotes the recruitment of telomerase and therefore telomere homeostasis. Telomerase is inactivated in most human tissues, which undergo progressive telomere shortening. When telomeres become critically short, a block of cell division, known as replicative senescence, limits cell proliferation, thus acting as a cancer-suppressor mechanism. Senescence is triggered by the activation of a checkpoint response governed by Mec1/ATR and Tel1/ATM. While Mec1/ATR is known to block cell division in the presence of extended ssDNA, the molecular mechanism by which Tel1/ATM triggers senescence is still unclear. During my PhD I have managed two different projects with the aim to shed light into the molecular mechanisms that involve Tel1 in response to CPT and in the induction of replicative senescence. Regarding the first project, in both yeast and mammals, CPT induces replication fork reversal, which has been proposed to stabilize stalled replication forks, thus providing time for the repair of CPT-induced lesions and supporting replication restart. tel1∆ cells have a reduced amount of CPT-induced reversed forks compared to wild type cells. The lack of Mre11 nuclease activity restores wild-type levels of reversed forks in CPT-treated tel1Δ cells, without affecting fork reversal in wild-type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild-type, tel1Δ, and mre11 nuclease-deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT. Altogether, these data indicate that Tel1 stabilizes Mrc1-dependent reversed forks generated in the presence of CPT by counteracting Mre11 nucleolytic activity at these structures. Regarding the second project, to studying the role of Tel1/ATM in the induction of senescence, I took advantage of telomerase-deficient yeast cells, which are considered a reliable model of replicative senescence, and the TEL1-hy184 allele, previously identified because it was able to suppress the checkpoint defects of Mec1-deficient cells. Upon telomerase inactivation, Tel1-hy184 accelerates senescence compared to wild type Tel1, while the lack of Tel1 was found to delay senescence. The enhanced senescence in telomerase-negative TEL1-hy184 cells depends on the activation of a checkpoint that is completely Rad9-dependent and only partially dependent on Mec1. Furthermore, Tel1-hy184 does not appear to increase ssDNA at DNA ends, suggesting that Tel1 induces replicative senescence by directly contributing to checkpoint signaling at dysfunctional telomeres. Taken together, the results that I have obtained during my PhD allow to better understand the functions of Tel1/ATM in the maintenance of genome stability.

The purpose of the present study was to examine if childhood sexual abuse (CSA) was associated with decreases in mean telomere length (TL), and if social support and/or optimism moderated this association. The study included 99 Caucasian female monozygotic twins, ranging in age from 19-48 (Mage = 30.5, SD = 7.8) at Time 1. Linear mixed effects models were employed to test study hypotheses. Analyses with all participants did not detect an effect of CSA exposure or severity on mean TL, nor were there effects with optimism. However, in analyses that only included women exposed to abuse, increases in social support were associated with increases in mean TL. Further, for women who experienced non-genital abuse, social support was positively associated with mean TL. Findings from the current study clarify the role of CSA in telomere attrition, and factors that may protect against the negative biological effects of CSA.

Les télomères sont des séquences d’ADN, généralement répétées en tandem, localisées à l’extrémité des chromosomes linéaires. Une des fonctions principales des télomères est de différencier l’extrémité des chromosomes des cassures double-brin, et ainsi de prévenir l’activation des voies de réparation de l’ADN. Chez les mammifères, cette fonction est plus spécifiquement assurée par le complexe shelterin. Il s’agit d’un complexe hétérogène composé de six protéines distinctes : TRF1, TRF2, POT1, RAP1, TPP1 et TIN2, qui interagit spécifiquement avec l’ADN télomérique. Au sein de ce complexe, les protéines RAP1 et TRF2 coopèrent afin d’empêcher l’extrémité des chromosomes d’être perçue comme un dommage de l’ADN, ce qui autrement aboutirait à des fusions inter-chromosomiques suite au processus de réparation. La protéine TRF2 se lie directement à la molécule d’ADN dans laquelle elle s’enroule de façon spécifique. Cette propriété est primordiale pour générer une structure d’ADN en forme de boucle, appelée t-loop, et dont le bon fonctionnement des télomères dépend. Les travaux effectués au cours de cette thèse ont mis en évidence deux scenarii indépendants dans lesquels la protéine RAP1 assure un rôle critique dans la stabilité des télomères. Premièrement, RAP1 peut prévenir les fusions inter-chromosomiques dans des cellules exprimant une forme altérée de TRF2 incapable de former des t-loops. Deuxièmement, l’inhibition de RAP1 dans des cellules en sénescence réplicative conduit à l’activation des voies de réparation de l’ADN et à la formation de fusions inter-chromosomiques. Ces observations font écho à des résultats précédents obtenus dans des cellules HeLa traitées avec l’inhibiteur de la télomérase BIBR1532, et dont l’expression de la protéine RAP1 était abolie par shRNA. De plus, j’ai montré que les fusions interchromosomiques engendrées par la perte de RAP1 sont dépendantes de la ligase IV, qui est un acteur principal de la voie de réparation de l’ADN par recombinaison non-homologue (NHEJ). Dans l’ensemble, ces travaux démontrent l’importance de la protéine RAP1 dans la stabilité des télomères lorsque la protéine TRF2 est non fonctionnelle, mais aussi dans des situations physiologiques telles que la sénescence réplicative
In mammals, the shelterin complex is the guardian of telomere stability. It operates through a set of six proteins (TRF1, TRF2, POT1, RAP1, TPP1 and TIN2) that binds telomeric DNA and protects it from being recognized as DNA double-strand breaks and therefore control DNA repair and DNA damage response pathways. Among them, RAP1 and TRF2 cooperate and together protect chromosome extremities from end-to-end fusions. TRF2 is seen as a major factor to control telomere DNA topology by wrapping DNA around itself in a right handed manner. This property of TRF2 is required to promote the formation of t-loops, special DNA structures at telomeres that are considered as protective barriers to DNA damage response and fusion. Here we demonstrate two independent situations where RAP1 dysfunction is critical for telomere protection. First, in cells expressing a wrapping-deficient TRF2 allele that cannot form t-loops, RAP1 appears as a backup anti-fusion mechanism. Second, RAP1 downregulation in replicative senescent cells leads to telomere fusions and DNA damage response activation. This is consistent with similar observations in HeLa cells treated with the telomerase inhibitor BIBR1532, and in which RAP1 expression was abolished by an inducible shRNA system. In addition, we show that fusions triggered by RAP1 loss are dependent upon ligase IV, which is a key player of the classical non-homologous end-joining (c-NHEJ) repair pathway. Altogether, these results indicate that RAP1 takes over telomere protection when TRF2 cannot properly function or in the normal physiological situation, such as replicative senescence

Sous conditions de stress métabolique ou environnemental, l’activation instantanée de voies moléculaires puissantes permet aux cellules de prévenir la formation et l’accumulation d’agrégats protéiques toxiques. HSF1 (Heat Shock Factor 1) est le facteur de transcription majeur capable d’orchestrer cette réponse cellulaire au stress et cela via l’activation de protéines au rôle protecteur nommées chaperonnes. Cependant, il est aujourd’hui évident que les fonctions initialement attribuées au facteur HSF1 s’étendent bien au-delà de l’activation de la transcription de chaperonnes. En effet, il a été démontré que HSF1 joue un rôle essentiel dans l’activation et le remodelage de régions répétées appartenant à l’hétérochromatine péricentromérique sous stress thermique et plus récemment qu’HSF1 contribuerait significativement au processus de tumorigenèse dans différents types de cancers. Dans cette étude, nous avons identifié pour la première fois les régions subtélomériques comme étant une nouvelle cible génomique d’HSF1 sous conditions de stress thermique. Nous avons démontré, que la liaison directe et spécifique d’HSF1 avec plusieurs de ces régions sous stress thermique est à l’origine d’une surexpression de longs ARNs non codants issus des télomères, aussi connus sous le nom de TERRA. De façon intéressante nous avons trouvé que cette transcription était corrélée à un enrichissement de la marque épigénétique répressive H3K9me3 au niveau télomérique. De plus, nos données ont permis de démontrer que l’intégrité de la chromatine télomérique était significativement atteinte sous conditions de stress thermique. Nous observons à la fois, une dissociation partielle de la protéine TRF2 (Telomeric repeat-binding factor 2) et une accumulation de dommages à l’ADN détectés grâce au marqueur moléculaire H2AX-P, au niveau des télomères. Finalement, nos résultats ont également permis de souligner un rôle d’HSF1 dans le maintien de cette intégrité télomérique. L’ensemble de ce travail établit un premier lien entre la voie cellulaire puissante de réponse au stress, son acteur majeur HSF1 et les régions de l’hétérochromatine télomérique, dans des lignées de cellules humaines cancéreuses. Ces données fournissent des indications précieuses sur une voie de maintien de télomères sous stress et nous permettant de proposer un modèle dans lequel cette nouvelle fonction d’HSF1 aux télomères pourrait être étroitement liée à l’expression des ARNs non codants télomériques. Sur la base de nos données ainsi que sur les multiples publications démontrant l’implication d’HSF1 dans la tumorigenèse, la définition exacte du rôle d’HSF1 au niveau de l’intégrité des télomères dans un contexte pathologique comme le cancer apparait aujourd’hui comme un défi prometteur
In response to metabolic or environmental stress, cells rapidly activate powerful defense mechanisms to prevent the formation and accumulation of toxic protein aggregates. The main orchestrator of this cellular response is HSF1 (Heat Shock Factor 1), a transcription factor involved in the up-regulation of protein-coding genes with protective roles. However, it is now becoming clear, that HSF1 function extends beyond what was previously predicted and that HSF1 can contribute to pericentromeric heterochromatin remodeling and activation as well as to efficiently support malignancy. In this study, we identify subtelomeric DNA as a new genomic target of HSF1 upon heat shock (HS). We show that HSF1 binding to subtelomeric regions plays an essential role in the upregulation of TERRA lncRNAs transcription and in the accumulation of repressive H3K9me3 histone mark at telomeres upon HS. Additionally, we demonstrate that HS significantly affects telomere capping and telomere integrity. We bring evidence of a partial TRF2 telomeric-binding factor dissociation and we reveal an accumulation of DNA damage at telomeres using the DNA damage marker H2A.X-P. In line with this, we bring solid evidences that under heat shock, HSF1 contributes to preserve telomere integrity by significantly limiting telomeric DNA damage accumulation. Altogether, our findings therefore reveal a new direct and essential function of HSF1 in transcription activation of TERRA and in telomere protection upon stress in human cancer cell lines. This work provides new insights into how telomeres are preserved under stressful heat shock conditions and allow us to propose a model where HSF1 may exert its protective function at telomeres via the expression of TERRA ncRNAs. Based on our results and given the important role of HSF1 in tumor development, defining the role of HSF1 with regard to telomere stability in tumor development already emerges as a promising challenge

Les télomères sont les extrémités des chromosomes et sont protégés par le complexe shelterin pour ne pas être reconnus comme des cassures accidentelles d'ADN double brin. Un enjeu majeur dans la recherche sur les télomères est de comprendre comment l'intégrité des extrémités des chromosomes au cours de l'oncogenèse est modifiée. La perte de répétitions télomériques à chaque division cellulaire peut être compensée par l’activité de l’enzyme télomérase qui ajoute "de novo" des motifs télomériques. Bien que la télomérase soit surexprimée dans la majorité des cellules cancéreuses, l’impact de modifications des protéines shelterin qui coiffent les télomères dans le processus oncogénique n'est pas clair. Dans la première étude, nous avons analysé l'état des télomères de patients atteints de la Leucémie Lymphoïde Chronique (LLC) uniquement au stade précoce de la LLC, stade A de Binet. La LLC est une leucémie commune aux pays occidentaux et se développe chez les personnes âgées. Nous avons montré que la majorité des patients atteints de la maladie présente des télomères dysfonctionnels. Les dommages télomériques ne corrèlent pas avec la longueur des télomères ou le statut mutationnel des patients, mais corrèlent avec la faible expression des protéines télomériques TIN2 et TPP1. Dans la seconde étude, nous avons montré que l'expression de l'oncogène H-RasV12 confère une résistance accrue vis-à-vis des télomères dysfonctionnels et aucun défaut de croissance n’est observable lorsque TRF2, une protéine télomérique est inhibée. Cet effet dépend de l'expression de l'interleukine 6 (IL-6), révélant un rôle inattendu de cette cytokine dans la fonction des télomères. De plus, la co-inhibition de TRF2 et IL-6 dans les cellules RasV12 conduit à blocage G2 / M associé à une séparation des chromatides sœurs. Ces résultats suggèrent que l'induction de la tumorigénicité par l’oncogène Ras protège les télomères contre les dommages et que la ségrégation des chromosomes est dépendante de TRF2 et IL-6
Telomeres protect the chromosome extremities from being repaired and recognized as accidental DNA breaks double-strand. A major issue in telomere research is to understand how systems monitoring the integrity of chromosomes ends change during oncogenesis. Although telomerase overexpression occurs in the majority of cancer cells, whether other types of telomere changes play roles in the oncogenic process is unclear. In the first study, we analyzed the status of telomeres in patients with early stage Chronic Lymphocytic Leukemia (B-CLL), which is a common leukemia in Western countries that develops in the elderly. We showed that the majority of CLL patients exhibit telomeric dysfunction. It does not correlate with telomere length or mutation status of patients, but correlates with low expression of telomeric proteins TIN2 and TPP1. In the second study, we showed that expression of the H-RasV12 oncogene confers increased resistance to telomere uncapping and protects against to growth defects when TRF2, a telomeric protein, is depleted. This effect depends upon the expression of interleukin 6 (IL-6), revealing an unexpected role for this cytokine in telomere function. Notably, the co-inhibition of TRF2 and IL-6 in RasV12 cells led to G2/M block associated with an increased incidence of premature sister chromatid separation. These findings suggest that the induction of a tumorigenic state by oncogenic Ras protects against telomere damage and chromosome segregation defects in TRF2-compromised cells through an increase of IL-6 expression

Telomeres are the physical ends of linear chromosomes in eukaryotes. Telomeres not only protect chromosome ends from being recognized as double-strand breaks but also maintain the chromosome terminal sequences. These processes involve a number of telomere-related proteins. A major challenge in the field is to elucidate the full constitution of telomere-associated proteins and to understand how different protein complexes are regulated at chromosome termini. Here, I report the identification and characterization of STN1 (Suppressor of cdc thirteen, 1), CTC1 (Conserved Telomere maintenance Component 1) and TEN1 (Telomeric pathways in association with Stn1, 1) in Arabidopsis. CTC1/STN1/TEN1 (CST) forms a trimeric complex that specifically associates with telomeres. Loss of any component of the CST induces catastrophic telomere loss, disrupted telomere end architecture, and massive chromosome end-to-end fusions. Thus, CST plays an essential role in chromosome end protection. I also show that CST function at telomeres is independent of a previously characterized capping complex KU70/KU80, and that ATR is responsible for a checkpoint response in plants lacking CTC1/STN1. Additionally, I present data showing that Arabidopsis POT1a (Protection Of Telomere 1, a) has evolved as a telomerase recruitment factor. Unlike POT1 in other eukaryotes which binds and protects ss telomeric DNA, AtPOT1a interacts with telomerase RNA (TER). Based on an evolutionary analysis, we found that the POT1a lineage is under positive selection in the Brassicaceae family in which Arabidopsis belongs. Mutations of two positive selection sites significantly reduce POT1a?s activity in vivo. These data suggest POT1a is under pressure to evolve from a telomeric DNA binding protein to a TER binding protein. I also discovered that POT1a interacts with the novel telomere capping protein CTC1 in vitro and in vivo. Thus, I hypothesize that POT1a acts as a telomerase recruitment factor linking this enzyme to the chromosome termini via interacting with TER and CTC1. Finally, I dissected the functional domains of POT1a and demonstrated that both the N-terminus and the C-terminus of POT1a are required for its function in vivo. In summary, my work has uncovered several new and essential telomereassociated proteins that provide new insight into mechanisms of chromosome end protection and maintenance.

博士
國立臺灣大學
微生物學研究所
105
Telomere homeostasis is regulated by both telomerase and end protection mechanisms. Short telomere can activate DNA damage sensing kinases ATM/ATR for telomerase recruitment. However, it is still not clear whether telomere shortening also regulates end protection. Here I reveal a feedback end protection mechanism which regulated by yeast ATM/ATR under telomere stress. Rap1 is phosphorylated by Tel1 and Mec1 kinases at serine 731, and this phosphorylation is strengthened by DNA damage and telomere shortening. Loss of Rap1 phosphorylation decreases the interaction between Rap1 and its interacting partner Rif1, which further weakens the strength of end protection. Reduction of Rap1-Rif1 association impairs telomere length regulation and increases the change of telomere-telomere recombination. However, impaired Rap1 phosphorylation neither influences the telomere-telomere fusion nor the telomeric silencing. These results indicate that ATM/ATR DNA damage checkpoint signal controls the telomere protection by strengthening the Rap1-Rif1 interaction at short telomere, and the checkpoint kinase regulates both telomerase recruitment and end capping pathways to maintain telomere homeostasis.

Telomeres are DNA-protein structures that protect eukaryotic chromosome ends from illegitimate recombination and degradation. Telomeres become shortened with each cell division unless telomerase, a reverse transcriptase, is activated. In addition to playing a protective role of chromosome ends, telomeres and telomere binding proteins are also essential for regulating telomere length and telomerase access. The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage and negatively regulating telomere length. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein-interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded DNA (dsDNA) binding protein TRF2. I sought to determine which of these functions--ssDNA, TPP1, or TRF2 binding--was required for POT1-mediated telomere localization, protection, and length regulation. Using separation-of-function POT1 mutants deficient in one of POT1's three functions, I found that binding to TRF2 fosters robust loading of POT1 onto telomeric chromatin and regulates telomere length, but is dispensable in the protection of telomeres. Although it remains unclear what role TPP1 binding plays in telomere length regulation, I found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1-mediated protection of telomeres, possibly by excluding RPA from telomeres.

Dissertation