Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice.

Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice.

Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Telomeres are the ends of linear chromosomes which are protected by a multi-protein complex called shelterin. The proper maintenance of telomeres involves strict control over the length of the TTAGGG telomeric repeat sequences. In part, this is achieved through the action of the shelterin complex component TRF1. TRF1 binds to duplex telomere DNA and acts as a suppressor of telomerase-dependent telomere elongation, however the exact mechanism by which it achieves this is currently unknown. Recent observations with a phospho-specific TRF1 antibody indicate that phosphorylated (pT371)TRF1 localizes at Cajal bodies. Cajal bodies are subnuclear organelles with myriad functions, one of which is to recruit the subunits of the telomerase holoenzyme for assembly and the subsequent targeting of the enzyme to telomeres for elongation. The results presented here demonstrate that this association of phosphorylated (pT371)TRF1 to Cajal bodies is highly specific, requiring its DNA binding capability, and occurring only in Cajal bodies which are not actively involved with telomere extension. While the function of this association has not been elucidated, the data are suggestive of a telomerase-related role being played by phosphorylated (pT371)TRF1 at Cajal bodies, possibly related to its function in suppressing telomere elongation. CSB is a multifunction protein which is implicated in transcription-coupled repair (TCR), base excision repair (BER), and control of transcription. Certain mutations and truncations of CSB are known to cause Cockayne syndrome (CS) in humans, an autosomal recessive progeria with devastating consequences. Unlike other progeria, CS patients do not display increased cancer incidence. Despite this fact, CSB is upregulated and in many cancers. In these cells, removal of CSB leads to apoptosis and increased sensitivity to chemotherapeutic drugs which suggests a dependency on CSB. The CSB of some non-CS cancer patients has also been found to be mutated at several recurring SNPs through the CSB gene. Preliminary examination of some of these SNPs suggests that they may invoke a change in the efficiency of TCR repair of UV-induced DNA damage. The results presented here demonstrate that, for the SNPs examined, there is no significant change in the repair of UV damage as assessed by colony survival assays post UV-treatment. While this may rule out an effect on TCR by these cancer-associated SNPs, it is possible that they may have an effect on CSB’s involvement in other vital cellular processes. / Thesis / Master of Science (MSc)

TRF1, telomeric-repeat binding factor 1, is a component of the six-subunit protein complex, referred to as shelterin, which is essential for not only regulating telomere length maintenance but also protecting mammalian telomeres from being recognized as damaged DNA. TRF1 acts as a negative mediator of telomerase-dependent telomere elongation in telomerase-expressing cells, whereas it promotes alternative lengthening of telomeres (ALT) activity by regulating ALT features including the production of extrachromosomal telomere-repeat (ECTR) DNA such as C-circles, and ALT-associated promyelocytic leukemia bodies, or APBs. The activity of TRF1 is tightly regulated by post-translational modification such as phosphorylation. This thesis sets out to investigate the function of TRF1 phosphorylation on threonine-271 (T271) and threonine-371 in telomere maintenance. The results presented in this thesis demonstrate that TRF1 phosphorylation on T271 positively regulates the association of TRF1 to telomeric DNA in telomerase expressing cells. In ALT cells, TRF1 phosphorylation on both T271 and T371 is shown to be important for the formation of APBs. Furthermore, the work presented here suggests that transcription-associated DNA damage mediates the association of phosphorylated (pT371)TRF1 with APBs. / Thesis / Master of Science (MSc) / TRF1, telomeric-repeat binding factor 1, is a component of the shelterin complex, which is essential for regulating telomere length maintenance and protecting mammalian telomeres from being recognized as damaged DNA. TRF1 acts as a negative mediator of telomerase-dependent telomere elongation in telomerase-expressing cells, whereas it promotes alternative lengthening of telomeres. The activity of TRF1 is tightly regulated by phosphorylation. This thesis sets out to investigate the function of TRF1 phosphorylation on threonine-271 and threonine-371 in telomere maintenance. Understanding how post-translational modifications on TRF1 may be linked to telomere homeostasis will be crucial for our understanding in cancer cell biology.

<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)

TRF1 est une protéine télomérique essentielle pour la stabilité et la régulation de la longueur des télomères. Son expression est altérée dans de nombreux cancers humains, et son inhibition, dans un contexte p53 déficient, favorise le développement de tumeurs chez la souris. Nous montrons ici que l'inhibition de TRF1 dans les fibroblastes primaires humains conduit à une accumulation télomérique de γ-H2AX et à une activation de la voie de réponse aux dommages de l'ADN dépendante des kinases ATR/Chk1, menant rapidement les cellules vers la sénescence. En revanche, lorsque les voies p53 et pRb sont défaillantes, les cellules échappent à la sénescence. L'érosion accrue des télomères engendre alors une fragilité télomérique et une instabilité chromosomique, caractérisées par la présence de fusions entres chromatides soeurs et de signaux multi-télomériques (MTS). Un niveau élevé de MTS, associés à la présence de télomères courts, est également retrouvé après la surexpression de TRF1. Cette fragilité télomérique conduit à une extension de la capacité proliférative des cellules, due à une stabilisation de la longueur des télomères par réactivation de la télomérase. Nous proposons que la fragilité des télomères, induit par l'altération de la charge télomérique de TRF1, conduit à une instabilité chromosomique qui facilite la réactivation de la télomérase et à des anomalies chromosomiques comparables à celles retrouvées dans les tumeurs. La dérégulation de l'expression de TRF1 joueraient un rôle dans la progression tumorale des cellules p53 et pRb déficientes. / TRF1 is a telomere-binding protein which is essential for both telomere stability and telomere length regulation. TRF1 depletion in the context of p53 deficiency promotes tumor development in the mouse, and TRF1 expression is altered in some human cancers. We report here that inhibition of TRF1 in human primary fibroblast results in rapid induction of senescence, which is concomitant with telomeric accumulation of γ-H2AX and phosphorylation of the ATR downstream checkpoint kinase Chk1. Abrogation of p53 and pRb pathways bypasses senescence but leads to accelerated telomere shortening and early onset of chromosomal instability, including sister chromatid fusions and the occurrence of multi-telomeric signals (MTS) related to telomere fragility. MTS are also elevated in TRF1-overexpressing cells and are coincident with the presence of short telomeres. Elevated telomere fragility was associated with greater immortalization potential and resultant cells maintained their telomeres via telomerase reactivation. We propose that changes in TRF1 occupancy at telomeres lead to telomere-fragility driven chromosome instability, which facilitates the reactivation of telomerase and engenders cancer-relevant chromosomal aberrations. These events would occur at early stages of the tumor progression process in the context of an impaired p53 and pRb response.

<p>TRF1, a shelterin protein, is a negative mediator of telomere length maintenance. Phosphorylation has been shown to play an important role in modulating TRF1 function. T137 and S249 of TRF1 have been indentified to be candidate phosphorylation sites in vivo, and one of my thesis objectives was to examine their role in regulating TRF1 function. Both T137 and S249 have each been changed to either alanine (nonphosphorylatable) or phosphomimic mutation. The TRF1 mutants were introduced into a TRF1-depleted cell line. Southern analysis revealed that neither T137 nor S249 of TRF1 is involved in telomere length maintenance. Immunoprecipitation studies showed that T137 and S249 are not required for TRF1 interaction with TIN2. In vitro gel-shift assays indicated that T137 and S249 are not important for TRF1 binding to telomeric DNA. Taken together, these results suggest that T137 and S249 may not be required for TRF1 function in telomere length maintenance. Human Est1A has been suggested to play a role in telomere length maintenance. To identify the domain of hEst1A involved in telomere length maintenance, a number of deletion constructs were generated and retrovirally introduced into HT1080 cells. Southern analysis revealed that the RID domain may positively regulate telomere length maintenance whereas the first 220 amino acids at the N-terminus may be a negative mediator of telomere length maintenance. In S. cerevisiae, Est1 recruits telomerase to telomeres in a Tel1- (homolog of ATM) and MRX-dependent manner. To assess whether atm-1 and smg-6 may function in the same genetic pathway of regulation of telomere length in C.elegans, the single mutant strain atm-1(gk186) was crossed with three of smg-6 mutant strains (tm1308, ok1794 and r896) to generate double mutants. Southern analysis revealed that deletion of ATM-1 or SMG-6 (tm1308) results in telomere shortening, suggesting that atm-1 and smg-6 may function in the same genetic pathway to regulate telomere length maintenance.</p> / Master of Science (MSc)

Nos indivíduos com obesidade, o excesso de tecido adiposo, exerce um papel fundamental induzindo um estado inflamatório crônico e sistêmico. A obesidade mimetiza processos celulares semelhantes aos do envelhecimento tais como a deterioração de tecidos e órgãos e diminuição na capacidade de reparo dos danos induzidos ao DNA. Nesse contexto, as citocinas pró-inflamatórias induzem atritos ao DNA que impactam, principalmente nas regiões mais susceptíveis dos cromossomos, os telômeros. Os telômeros, presentes nas extremidades dos cromossomos, estão associados a um complexo proteico denominado complexo shelterin. O complexo shelterin é formado por 6 proteínas (TRF1, TRF2, TIN2, POT1, TPP1 e RAP1), que junto com proteínas acessórias como a disquerina (DKC1), participam da regulação do comprimento dos telômeros e protegem os cromossomos dede atividades indesejadas de erosão enzimática, recombinação não-homóloga e fusão das terminações cromossômicas. Nos últimos anos, foram estabelecidas relações positivas entre condições patológicas clinicamente diferentes, como as moduladas por inflamação, e o comprimento dos telômeros. Recentemente, nosso grupo demonstrou telômeros encurtados em indivíduos portadores de obesidade mórbida. Assim o objetivo do presente trabalho foi explorar fatores adicionais associados ao metabolismo telomérico, como a expressão gênica das proteínas do complexo shelterin e citocinas pró-inflamatórias, as quais podem contribuir para o encurtamento acelerado de telômeros. Utilizamos amostras de células mononucleares de sangue periférico (PBMC) de indivíduos adultos saudáveis (n = 27) e indivíduos adultos portadores de obesidade (n = 39). Quantificamos a expressão gênica por transcrição reversa e PCR quantitativa (RT-qPCR) de todos os genes do complexo shelterin, DKC1, IL-1β e TNF-α. Nossos resultados demonstram um perfil de expressão gênica alterado quando comparada a expressão gênica das proteínas analisadas nos dois grupos estudados, controles e portadores de obesidade. Os indivíduos portadores de obesidade mostraram um perfil significativamente elevado dos genes TRF1, POT1, RAP1 e DKC1 (P < 0,05). Não observamos correlação de expressão gênica entre os diferentes genes e o comprimento dos telômeros nos grupos estudados, mas sim com a DKC1 na obesidade. Entretanto, quando analisamos as associações entre os genes de complexo shelterin observamos mudanças significativas nas associações intra-grupo dependentes da condição de obesidade. Nossos resultados salientam a complexa e intrincada rede de fatores associados e desregulados durante o processo fisiopatológico da obesidade. Estudos adicionais serão necessários acrescentando novos fatores para tentar dissecar a regulação coordenada do comprimento dos telômeros na homeostase e no processo patológico da obesidade. / In individuals with obesity, the excess of adipose tissue plays a key role in inducing a chronic and systemic inflammatory state. Like aging, obesity mimics cellular processes such as deterioration of tissues and organs and decreased ability to repair age-dependent DNA damages. In this context, the proinflammatory cytokines induce DNA damage that impact, especially in the most susceptible regions of the chromosomes, the telomeres. The telomeres, present at the ends of the chromosomes, are associated with a protein complex called the shelterin complex. The shelterin complex consists of 6 proteins (TRF1, TRF2, TIN2, POT1, TPP1 and RAP1), which together with accessory proteins such as dyskerin (DKC1), participate in telomere’s length regulation and protect chromosomes from undesired erosion, enzymatic activities, non-homologous recombination and fusion of chromosomal terminations. In recent years, positive relationships have been established between clinically different pathological conditions, such as those modulated by inflammation, and telomeres’ length. Recently, our group demonstrated shortened telomeres in individuals with morbid obesity. Thus, the aim of the present study was to explore additional factors associated with telomeres’ metabolism, such as gene expression of the shelterin complex components and proinflammatory cytokines, which may contribute to the accelerated shortening of the telomeres. We used peripheral blood mononuclear cells (PBMC) samples from healthy adults (n = 27) and adults with obesity (n = 39). We quantified gene expression by reverse transcription and quantitative PCR (RT-qPCR) of all shelterin complex genes, DKC1, IL-1β and TNF-α. Our results demonstrate an altered gene expression profile when compared to the gene expression of the proteins analyzed in the two studied groups, controls and individuals with obesity. Individuals with obesity showed a significantly elevated profile of TRF1, POT1, RAP1 and DKC1 (P < 0.05) genes. We did not observe correlation of gene expression between the different shelterin genes and the length of telomeres in the studied groups, but with DKC1 in obesity. However, when we analyzed the associations between the shelterin complex genes we observed significant changes in the intra-group associations dependent on the obesity condition. Our results highlight the complex and intricate network of associated and deregulated factors during the pathophysiological process of obesity. Further studies are needed together with the inclusion of additional factors to try to dissect the coordinated regulation of telomeres’ length in homeostasis and in the pathological process of obesity.