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<p><h2> </h2></p> <p>Telomeres are protein-DNA complexes found at the ends of human chromosomes. The function of telomeres is to protect chromosome ends from being recognized as damaged DNA. This protection is essential in preventing the erosion of telomeres, which has been shown to lead to genomic instability, a hallmark of cancer and aged cells. Precise regulation of telomere length and function is crucial to cell survival, and defects in this regulation are related to tumorigenesis and aging related disorders. The proteins that bind telomere DNA play an indispensable role in telomere maintenance. TRF1, <em>t</em>elomere <em>r</em>epeat binding <em>f</em>actor 1, is a protein that directly binds to mammalian telomeric DNA and participates in regulating telomere length. Post-translational modifications, such as phosphorylation, have been shown to modulate TRF1 function. The results presented here demonstrate that two phosphorylation sites on TRF1, S367 and T371, are involved in regulating the function and localization of TRF1. TRF1 S367 is phosphorylated by ATM, and this phosphorylation removes TRF1 from telomere DNA and directs TRF1 to sites of proteasome degradation. On the other hand, the phosphorylation of TRF1 at T371 prevents the association of TRF1 with telomere DNA but also protects TRF1 from degradation. We have demonstrated that the phosphorylation of T371 by CDK1 is important for the resolution of sister chromatids in mitosis. In interphase cells, in response to the induction of DNA damage, TRF1 phosphorylated at T371 is recruited to sites of damage and is involved in promoting efficient homologous recombination and in conferring checkpoint activation and cell survival. The work presented within this thesis sheds light on the regulation of TRF1 function by phosphorylation events and reveals novel functions of TRF1.</p> / Doctor of Philosophy (PhD)

<p>TRF1 is an essential shelterin protein that binds to double stranded telomeric DNA. TRF1 is best known for its role as a negative regulator of telomere length. Post-translational modifications, like phosphorylation, have been shown to regulate TRF1 function in cells. Mass spectrometric analysis revealed three potential TRF1 phosphorylation sites, threonine 271, serine 279 and threonine 305 <em>in vivo</em>. To analyze the function of these three potential phosphorylation sites, phosphomimic (aspartic acid, D) and non-phosphorylatable (alanine, A) mutations were made to be analyzed <em>in vitro</em>. Through <em>in vitro</em> gel shift assays, the phosphomimic mutation at threonine 271 mutant exhibits a DNA binding defect, whereas serine 279 and threonine 305 mutants have no binding defects. However, <em>in vivo</em> analysis needs to be conducted in order to determine whether this binding defect is authentic.</p> <p>PIN2 is a TRF1 isoform, which is identical in its protein sequence with the exception of 20 amino acids (residue 296-316 of TRF1). The second study looks to characterize any potential functional differences between PIN2 and TRF1. <em>In vitro</em>, CDK1 kinase assay was conducted on TRF1 and PIN2 mutants to assess whether there was a difference in phosphorylation. The kinase assay revealed that both PIN2 threonine 351 and TRF1 threonine 371 are both phosphorylated by CDK1. However, the net phosphorylation level of PIN2 by CDK1 is far lower than the net phosphorylation level of TRF1. An <em>in vitro</em> gel shift assay was also conducted to analyze the binding difference between TRF1 threonine 371 and PIN2 threonine 351 mutants. The DNA binding assay revealed that TRF1 threonine 371D mutant has a binding defect, whereas PIN2 threonine 351D mutant has no binding defect. However, this data needs to be verified to determine whether a PIN2 threonine 351D mutant behaves like a phosphomimic.</p> / Master of Science (MSc)

TRF2 is an essential telomeric protein involved in preventing the telomere ends from being recognized as DNA breaks. I have shown that TRF2 does not appear to play a major role in the UV -induced DNA damage response in IMR90, Cockayne syndrome or XPC deficient cells. TRF2 binds telomeric DNA via its Myb domain and also contains an N-terminal basic domain. Expression of TRF2MMM causes telomere fusions, whereas TRF2^(ΔB) causes rapid deletion of telomeric DNA, as both phenotypes result in senescence. These phenotypes are dependant upon recombination events. Thus, the basic domain of TRF2 may be essential to suppress recombination events at telomeres. However, it is not fully understood what amino acid residues in the basic domain of TRF2 are indispensable to maintain its function. By creating mutations in the arginine residues in the basic domain of TRF2, I have shown that the positive charge of the basic domain alone is not sufficient to maintain its protective function. By expressing these TRF2 mutants in the presence or absence of the Myb domain in HT1080 and BJ/hTERT cells, I have been able to recapitulate the TRF2^(ΔB) and TRF2^(ΔBΔM) decreased proliferation and senescence phenotypes. Furthermore, by analyzing anaphase and metaphase chromosomes and performing Southern blotting, I have shed light on the molecular mechanisms responsible for the deleterious phenotypes observed in the TRF2 mutants. Amino acid changes from arginines to lysines introduced into the basic domain of TRF2 results in a significant increase in telomere doublets. However, when these TRF2 mutants are expressed in the absence of the Myb domain, a significant increase in telomere fusions events occur. Collectively, my results indicate that more than one arginine residue in the basic domain is essential to maintain the protective function of TRF2, as these arginine residues may act as substrates for protein arginine methyltransferases. / Thesis / Master of Science (MSc)

Les télomères sont des structures nucléoprotéiques nécessaires à la protection des extrémités des chromosomes contre les dégradations ou fusions induites par les processus de réparation de lADN. Ils sont constitués de complexes protéiques associés à des répétitions en tandem dun motif 5-(TTAGGG)-3 sous forme double brin de plusieurs kilobases, et finalisés par une extrémité 3simple brin de la même séquence de quelques centaines de bases. On observe in vitro un raccourcissement des télomères à chaque division cellulaire, ce même fait est corrélé in vivo avec le vieillissement. Lorsque les télomères se raccourcissent et atteignent une taille critique, les cellules entrent en sénescence réplicative qui se définit par un arrêt de croissance définitif et viable des cellules. La télomérase est une ADN polymerase ARN-dépendante qui allonge le télomère en lui ajoutant des séquences répétitives TTAGGG. Elle comprend une composante ARN (hTR) qui sert de matrice et une composante catalytique à activité transcriptase inverse (hTERT). Lexpression seule dhTERT suffit à immortaliser différents types cellulaires. La télomérase est fortement exprimée dans la majorité des cellules tumorales alors que son activité est difficilement détectable dans la plupart des cellules somatiques. Ces observations font de la télomérase une cible dintérêt pour des nouvelles thérapies anticancéreuses. Une de ces nouvelles stratégies consiste en lutilisation de molécules capables de stabiliser les structures en G-quadruplexe de lADN. La stabilisation des G-quadruplexes télomériques rend les télomères inaccessibles pour la télomérase et inhibe son activité par la séquestration de son substrat. Lobjectif de cette thèse est dévaluer la réponse cellulaire induite par le traitement cellulaire de deux ligands des G-quadruplexes au niveau du protéome des cellules WI38 transfectées pour exprimer hTERT. Les deux ligands, TMPyP4 et la télomestatine, inhibent la télomérase mais ont une spécificité différente pour les diverses structures G-quadruplexes. En premier lieu, nous avons étudié leffet de la transfection dhTERT sur des cellules fibroblastiques humaines (WI38). Cette première étude a été conduite afin de caractériser ladaptation cellulaire résultante de limmortalisation des cellules WI38. Par la suite, celle-ci permettra de comparer ces résultats avec ceux obtenus lors de létude protéomique de leffet des ligands des G-quadruplexes. Nous avons montré que hTERT induit une augmentation de la capacité fonctionnelle du réticulum endoplasmique ainsi quune modulation des signaux cellulaire Ca2+-dépendant. Nous proposons que cette adaptation cellulaire est responsable dune résistance accrue vis-à-vis de différents stress environnementaux. Dautres protéines impliquées dans des mécanismes doncogenèse ont été identifiées et sont différentiellement exprimées entre les cellules parentales et les cellules transfectées. Lanalyse protéomique des traitements cellulaires indique que TMPyP4 induit une altération du protéome beaucoup plus prononcée que celle induite par la télomestatine. Ceci est probablement dû au manque de spécificité de TMPyP4 pour les G-quadruplexes télomériques. TMPyP4 induit, entre autres, une sous-expression massive des hnRNPs, une modulation de la voie protéasomale, une diminution probable de la traduction et une surexpression de plusieurs chaperonnes moléculaires. La télomestatine induit notamment une surexpression de la protéine BCL2A1 qui est impliquée dans les processus de résistance aux agents anticancéreux et une probable augmentation de la traduction. Les deux ligands ont des effets communs sur la variation dexpression des chaperons CCT (sou-expression), de lHSP90 alpha (surexpression) et de lhnRNP D (sous-expression). LHSP90 est également surexprimée dans les cellules hTERT-WI38 par rapport aux cellules parentales. Cette protéine fait actuellement lobjet de nombreuses recherches visant à inhiber son activité du fait de son implication en oncogenèse ainsi que dans la modulation de lactivité de la télomérase. Enfin, nous avons montré lintérêt de ce type détude protéomique dans lévaluation dagents à vocation thérapeutique préalablement aux études cliniques.

Telomeres form protective caps at the ends of linear chromosomes. They consist of repetitive DNA nucleotides and associated proteins of the shelterin complex. In vitro telomeres become shorter during cell division and when a critical shortness is reached they trigger a DNA damage response that leads to replicative senescence or apoptosis. Telomere shortening is a recognised hallmark of cellular ageing and seems to be also associated with organismal ageing. Telomere length (TL) and the rate of shortening vary across individuals and several studies have found that short telomeres and fast telomere depletion are associated with poor survival and early onset of age related diseases. However, longitudinal studies are needed to better understand the relationship of TL and TL dynamics with longevity measures. Relevant studies on livestock species are largely missing from the literature. In the dairy industry, farmers are forced to cull a considerable percentage of their heifers and cows at a young age due to fertility problems or diseases. As a consequence many replacement heifers have to be reared to maintain a specific herd size. This results in increased costs, consumption of resources, and damage to the environment. Breeding for an improved productive lifespan is difficult because longevity measures are recorded at the end of life and are known to have a low heritability. Therefore, the expected genetic improvement is generally slow, but could be considerably accelerated if an early life heritable biomarker was identified that is predictive of productive lifespan and could be used for animal selection. The question is if TL could be used as such a biomarker. The objectives of this thesis were to 1) develop robust methods to measure average relative leukocyte TL (RLTL) in cattle, 2) examine RLTL dynamics with age at a population as well as at an individual level, 3) estimate genetic parameters and 4) assess the association of RLTL and RLTL dynamics with productive lifespan. A quantitative polymerase chain reaction (qPCR) based assay developed for human studies was adapted to cattle and delivered robust results (repeatability > 80%, coefficient of variation=0.05). Different DNA extraction methods were tested for their effect on RLTL measurements and it was demonstrated that fast silica based DNA extraction methods are suitable for telomere projects which can improve the sample throughput and enable large-scale projects. Subsequently, RLTL in 1328 whole blood samples of 308 Holstein Friesian dairy cows and additionally in 284 whole blood samples of 38 female calves was measured. Repeatability and random regression models were used for the statistical analysis of telomere data. RLTL decreased considerably within the first year of life, but remained relatively stable afterwards at population level. Animals varied significantly in their amount and direction of telomere change. The genetic correlation between consecutive measurements in the same individual weakened with increasing sample interval from r=1 to r=0.69 which indicates that TL in the beginning of life might be under a different genetic control than TL later in life. For the first time in a livestock species we calculated heritability estimates for RLTL which were high (0.32-0.38) and remained constant over life. Long telomeres at birth were not predictive of better productive lifespan. However, animals with long RLTL at the ages of one and five years had a survival advantage. Also, animals that showed less average RLTL attrition over their lives remained in production for longer. TL dynamics differed among individuals and a considerable subset of individuals demonstrated telomere lengthening between consecutive measurements. On average, telomeres tend to shorten early in life and then remain relatively constant. While TL is a heritable trait throughout lifetime, telomere change is not heritable. Short TL at specific ages and telomere attrition over life were associated with poorer productive lifespan.

CD4 T-cell depletion is a hallmark of HIV/AIDS, but the underlying mechanism is still unclear. We have recently shown that ataxia-telangiectasia-mutated (ATM) deficiency in CD4 T cells accelerates DNA damage, telomere erosion, and cell apoptosis in HIV-infected individuals on antiretroviral therapy (ART). Whether these alterations in ART-treated HIV subjects occur in vitro in HIV-infected CD4 T cells remains unknown. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the telomeric DNA damage response (DDR) and cellular apoptosis in highly permissive SupT1 cells, followed by the validation of our observations in primary CD4 T cells with active or drug-suppressed HIV infection. Specifically, we established an in vitro HIV T-cell culture system with viral replication and raltegravir (RAL; an integrase inhibitor) suppression, mimicking active and ART-controlled HIV infection in vivo. We demonstrated that HIV-induced, telomeric DDR plays a pivotal role in triggering telomere erosion, premature T-cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This in vitro model provides a new tool to investigate HIV pathogenesis, and our results shed new light on the molecular mechanisms of telomeric DDR and CD4 T-cell homeostasis during HIV infection. IMPORTANCE The hallmark of HIV infection is a gradual depletion of CD4 T cells, with a progressive decline of host immunity. How CD4 T cells are depleted in individuals with active and virus-suppressed HIV infection remains unclear. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the chromosome end (telomere) DNA damage response (DDR) and cellular apoptosis in a T-cell line (highly permissive SupT1 cells), as well as in primary CD4 T cells with active or drug-suppressed HIV infection. We demonstrated that HIV-induced telomeric DDR plays a critical role in inducing telomere loss, premature cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This study sheds new light on the molecular mechanisms of telomeric DDR and its role in CD4 T-cell homeostasis during HIV infection.

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