Hematopoietic cell populations exhibiting detectable telomerase activity and elongated telomere lengths display strong engraftment survivability in humans during transplants. We investigated telomerase activity and telomere length in umbilical cord blood hematopoietic cell populations obtained from ViaCell Inc. at various intervals of a two-week ex vivo stem cell amplification process. Telomerase activity is increased with time in ViaCell's amplification process, perhaps in response to the removal of differentiated cells and expansion of primitive hematopoietic stem cell populations in tissue culture media containing a mixture of growth factors. Two of ViaCell's cell culture fractions were analyzed for telomere length using a TLA. Our results showed relatively long telomere lengths for day-0 and day-14 cord populations, and that despite an upregulation of telomerase activity in Day-14 samples, a loss of about 2 kb of telomeric DNA occurs. Our data are consistent with a model in which the increase in telomerase activity in day-14 ex vivo amplified cord blood hematopoietic cells relative to fresh cord is sufficient to reduce, but not prevent, telomere shortening caused by cell proliferation. Lastly, we investigated various culture conditions that could potentially upregulate telomerase activity in the Day-14 amplified cells. However none of the treatments tested altered telomerase activity. Our detection of increased telomerase activity and relatively long telomere lengths in ViaCell's Day-14 ex vivo amplified cord blood stem cell fraction provides support for ViaCell's Selective Clonogenic AmplificationTM indicating a high engraftment potential for these cells.

Dans la plupart des eucaryotes, il n’existe qu’une seule région centromérique par chromosome et celle-ci est capable d’être liée au fuseau mitotique via le complexe du kinétochore. Dans ce contexte, la présence de deux centromères est un défi pour une séparation normale. Au cours de la mitose, la capture des deux centromères de la même chromatides vers les pôles opposés génère un pont d’anaphase résultant en une rupture entre les centromères. Les extrémités libérées peuvent être fusionnées bout à bout recréant ainsi un dicentrique. Le chromosome entre alors dans un cycle de Rupture Cassure Pont, capable quelques cycles d’entrainer des modifications profondes du nombre de copies de gène qui peuvent contribuer à l'oncogenèse et résistance à la chimiothérapie. Malgré son importance, le mécanisme de rupture reste pour une grande partie inexploré. Ce projet permet l’analyse de la rupture des chromosomes dicentriques en utilisant le modèle de la levure bourgeonnante, Saccharomyces cerevisiae. Nous utilisons des souches dicentriques conditionnelles dans lequelles un chromosome, portant un centromère conditionnel sous le contrôle de deux promoteurs inductibles au galactose, est fusionné à un autre chromosome natif par recombinaison homologue. Nous avons observé que les chromosomes dicentriques ont tendance à casser dans le voisinage des deux centromères. La région de la rupture se répand sur ~ 30 kb vers l'autre centromère. Une insertion d’un fragment d’ADN 1-kb possédant un centromère ectopique dans un chromosome avec un centromère conditionnelle établit un point chaud d’environs 30 kb indiscernables des points chauds à centromères natifs. En outre, la taille de zone de rupture n’est pas corrélée à la distance intercentromerique (des intervalles de 30-600 kb ont été testés). Cela indique que la plus forte propension à rompre est une conséquence de la structure ou de la fonction des centromères et est sans rapport avec les séquences environnantes des chromosomes. Il est encore difficile de savoir si la rupture aux centromères a une fonction physiologique, mais nous pouvons supposer que ce point chaud peut favoriser les réarrangements d'ADN dans ces régions permettant ainsi l’inactivation du centromère et donc le retour à un caryotype stable. Globalement dans la S.cerevisiae, les dicentriques cassent dans les régions péricentromériques ou dans les fusions de télomères quand ils sont présents. Fait intéressant, les séquences télomériques internes, à savoir les répétitions TG₁₋₃, établissent plusieurs points chauds de rupture à une fréquence similaire. En perspective, il serait intéressant d'aborder les questions suivantes : 1) Quelles sont les caractéristiques qui rendent une région plus sujette à la casse ? 2) Quelles sont les positions de rupture au niveau des nucléotides ? 3) Existe-t-il un contrôle de la cassure des chromatides exercé dans la cellule ? 4) Quelle peut être la fonction biologique des points chauds de cassures ? / In most eukaryotes, there is one defined centromeric region per chromosome that links it to the spindle apparatus via the kinetochore complex. In this context, the presence of two centromeres is a challenge for an accurate segregation. During mitosis, the capture of the two centromeres of the same chromatid to opposite poles generates anaphase bridges that results in breakage between the centromeres. The released ends can be fused end-to-end thus recreating dicentric. It enters breakage-fusion-bridge cycles that, in multiple rounds, can result in large gene copy number alterations that can contribute to oncogenesis and chemotherapy resistance. Despite of its significance, the mechanism of breakage remains for a large part unexplored. This project adresses the dicentric breakage using a budding yeast, Saccharomyces cerevisiae. We use conditional dicentric strains, where a chromosome, bearing a conditional centromere under the control of two galactose-inducible promoters, is fused to another native chromosome by homologous recombination. We observed that dicentric chromosomes tend to break in the vicinity of the two centromeres. The breakage region spreads over ~30 kb towards the other centromere. An insertion of a 1-kb ectopic centromere in a chromosome with a conditional centromere establishes a ~30 kb hot spot indistinguishable from the hot spots at native centromeres. Furthermore, the size of breakage region is unrelated to an intercentromeric distance (30-600 kb intervals were tested). This indicates that the higher propensity to break is a consequence of centromere structure or function and is unrelated to the native surrounding sequences. It is yet unclear whether breakage at centromeres has a physiological function but we can speculate that this hot spot may favour local DNA rearrangements that result in centromere inactivation and thus the return to a stable karyotype. Overall in budding yeast, dicentrics break at pericentromeric regions or at the telomere fusions when they are present. Interestingly, internal telomeric sequences, i.e. TG₁₋₃ repeats, establish several breakage hot spots with a similar frequency. In perspective, it would be interesting to address the following questions: 1) What are features that make a region more prone to breakage? 2) What are the positions of breakage at nucleotide level? 3) Is there a coordination of dicentric chromatid breakage? 4) What can be the biological function of dicentric breakage hot spots?

O estresse é muito presente na vida das pessoas, e grande parte deste é de origem social. O estresse contínuo induz a uma série de efeitos nocivos no organismo. Estes efeitos também podem ser observados no cérebro, principalmente no hipocampo, onde promove alterações na sua morfologia e função. Recentemente, demonstrou-se uma redução do tamanho dos telômeros em leucócitos de mulheres com alto grau de estresse. Os telômeros são seqüências repetitivas dos nucleotídeos TTAGGG, responsáveis por proteger as extremidades dos cromossomos, e sua manutenção e alongamento é mediado, principalmente, pela enzima telomerase. Telômeros e telomerase têm sido alvos de muitos estudos envolvendo o envelhecimento e morte celular. Assim, o presente trabalho estudou os efeitos do estresse psicossocial prolongado em um modelo animal de subjugação/submissão sobre a biologia telomérica do hipocampo. O modelo de subjugação consistiu em um pareamento dos animais experimentais com um camundongo agressivo por 30 minutos. Camundongos machos jovens adultos da linhagem C57BL/6J passaram por este modelo de estresse diariamente por 21 dias. Observou-se um aumento na temperatura corpórea basal em repouso no 21º dia, bem como diminuição na hipertermia reativa a um estresse agudo de manipulação, demonstrando a efetividade do modelo em indução de estresse. Os tamanhos dos telômeros, a expressão gênica da enzima telomerase e sua atividade protéica foram investigados no hipocampo dos camundongos que sofreram o estresse crônico e comparados com animais do grupo controle. Observou-se, após o período de indução do estresse, uma diminuição em aproximadamente 58% no tamanho relativo médio dos telômeros das células hipocampais nos camundongos submetidos ao estresse psicossocial, quando comparados ao grupo controle. A quantidade de RNAm relativo ao gene da telomerase, bem como sua atividade enzimática, mostraram-se similares entre os grupos controle e experimental. Estes resultados sugerem que o estresse psicossocial prolongado reduz o tamanho dos telômeros do hipocampo de camundongos e que esta redução não parece ser devido a alterações gênicas ou enzimáticas da telomerase. Com este estudo procuramos entender melhor os mecanismos celulares e genômicos cerebrais modulados pelo estresse psicológico prolongado, de natureza social. Uma vez que muitos distúrbios psiquiátricos e neurodegenerativos são provenientes do estresse crônico, nossos resultados fornecem mais subsídios para se evidenciar a importância do ambiente social na saúde mental dos indivíduos. / Stressor stimuli are part of the daily life and the major part is derived from the social context. Long term stress can be very harmful, and induce several noxious events in the organism. These effects can also be perceived in the central nervous system, particularly in the hippocampus, in which morphological and functional damages may be observed. Recently a correlation of perceived stress and telomere length was demonstrated in leukocytes of stressed women. Telomeres, which are in tanden repeats of the nucleotides TTAGGG, are responsible to protect the extremities of the chromosomes, and it´s maintenance and elongation is mainly mediated by the enzyme telomerase. Telomeres and telomerase have been the targets of many studies in the last years, concerning aging and cell death. In this regard, the present study has investigated the effects of prolonged psychosocial defeat stress model in the telomeric biology of the hippocampus. Male C57Bl/6J young adult mice were submitted to a 21 days of psychosocial stress. We observed a raise in body temperature, as well as a decrease in reactive hyperthermia to the handling stress, demonstrating the effectiveness of this stress model. The telomere length, the transcript levels of telomerase mRNA, and the activity of the enzyme telomerase were also investigated in the hippocampus of psychosocially stressed mouse. A decrease of 58% in average telomere length was observed in the hippocampal cells of stressed mice when compared to control group. Levels of telomerase mRNA and telomerase activity were similar between control and defeat groups. In this study, we intend to better understand the cellular and genomic mechanisms promoted by long term psychological stress, with social nature, in brain. Since many psychiatric and neurodegenerative disorders have chronic stress underlying them, our results reinforce the importance of the social environment on individual mental health.

O estresse é muito presente na vida das pessoas, e grande parte deste é de origem social. O estresse contínuo induz a uma série de efeitos nocivos no organismo. Estes efeitos também podem ser observados no cérebro, principalmente no hipocampo, onde promove alterações na sua morfologia e função. Recentemente, demonstrou-se uma redução do tamanho dos telômeros em leucócitos de mulheres com alto grau de estresse. Os telômeros são seqüências repetitivas dos nucleotídeos TTAGGG, responsáveis por proteger as extremidades dos cromossomos, e sua manutenção e alongamento é mediado, principalmente, pela enzima telomerase. Telômeros e telomerase têm sido alvos de muitos estudos envolvendo o envelhecimento e morte celular. Assim, o presente trabalho estudou os efeitos do estresse psicossocial prolongado em um modelo animal de subjugação/submissão sobre a biologia telomérica do hipocampo. O modelo de subjugação consistiu em um pareamento dos animais experimentais com um camundongo agressivo por 30 minutos. Camundongos machos jovens adultos da linhagem C57BL/6J passaram por este modelo de estresse diariamente por 21 dias. Observou-se um aumento na temperatura corpórea basal em repouso no 21º dia, bem como diminuição na hipertermia reativa a um estresse agudo de manipulação, demonstrando a efetividade do modelo em indução de estresse. Os tamanhos dos telômeros, a expressão gênica da enzima telomerase e sua atividade protéica foram investigados no hipocampo dos camundongos que sofreram o estresse crônico e comparados com animais do grupo controle. Observou-se, após o período de indução do estresse, uma diminuição em aproximadamente 58% no tamanho relativo médio dos telômeros das células hipocampais nos camundongos submetidos ao estresse psicossocial, quando comparados ao grupo controle. A quantidade de RNAm relativo ao gene da telomerase, bem como sua atividade enzimática, mostraram-se similares entre os grupos controle e experimental. Estes resultados sugerem que o estresse psicossocial prolongado reduz o tamanho dos telômeros do hipocampo de camundongos e que esta redução não parece ser devido a alterações gênicas ou enzimáticas da telomerase. Com este estudo procuramos entender melhor os mecanismos celulares e genômicos cerebrais modulados pelo estresse psicológico prolongado, de natureza social. Uma vez que muitos distúrbios psiquiátricos e neurodegenerativos são provenientes do estresse crônico, nossos resultados fornecem mais subsídios para se evidenciar a importância do ambiente social na saúde mental dos indivíduos. / Stressor stimuli are part of the daily life and the major part is derived from the social context. Long term stress can be very harmful, and induce several noxious events in the organism. These effects can also be perceived in the central nervous system, particularly in the hippocampus, in which morphological and functional damages may be observed. Recently a correlation of perceived stress and telomere length was demonstrated in leukocytes of stressed women. Telomeres, which are in tanden repeats of the nucleotides TTAGGG, are responsible to protect the extremities of the chromosomes, and it´s maintenance and elongation is mainly mediated by the enzyme telomerase. Telomeres and telomerase have been the targets of many studies in the last years, concerning aging and cell death. In this regard, the present study has investigated the effects of prolonged psychosocial defeat stress model in the telomeric biology of the hippocampus. Male C57Bl/6J young adult mice were submitted to a 21 days of psychosocial stress. We observed a raise in body temperature, as well as a decrease in reactive hyperthermia to the handling stress, demonstrating the effectiveness of this stress model. The telomere length, the transcript levels of telomerase mRNA, and the activity of the enzyme telomerase were also investigated in the hippocampus of psychosocially stressed mouse. A decrease of 58% in average telomere length was observed in the hippocampal cells of stressed mice when compared to control group. Levels of telomerase mRNA and telomerase activity were similar between control and defeat groups. In this study, we intend to better understand the cellular and genomic mechanisms promoted by long term psychological stress, with social nature, in brain. Since many psychiatric and neurodegenerative disorders have chronic stress underlying them, our results reinforce the importance of the social environment on individual mental health.

Break Induced Replication (BIR), a mechanism by which cells heal one-ended double-strand breaks, involves the invasion of a broken strand of DNA into a homologous template, and the copying of tens to hundreds of kilobases from the site of invasion to the telomere using a migrating D-loop. Here we show that if BIR encounters an interstitial telomere sequence (ITS) placed in its path, BIR terminates at the ITS 12% of the time, with the formation of a new telomere at this location. We find that the ITS can be converted to a functional telomere by either direct addition of telomeric repeats by telomerase, or by homology-directed repair using natural telomeres. This termination and creation of a new telomere is promoted by Mph1 helicase, which is known to disassemble D-loops. We also show that other sequences that have the potential to form new telomeres, but lack the unique features of a perfect telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can cause chromosome truncations if BIR is made less processive by loss of Pol32 or Pif1. These findings together indicate that features of the ITS itself, such as secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by Mph1, promoting telomere formation at the site.

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.

Fanconi anaemia (FA) is an inherited disorder characterized by bone marrow failure, cancer predisposition and congenital abnormalities. The 12 known FA genes have been implicated in homologous recombination (HR), a process involved in telomere maintenance. A complex of at least 7 FA proteins promotes FANCD2 monoubiquitination and nuclear foci formation. FANCD2 colocalizes and interacts with HR proteins, however the role of FANCD2 in HR is unclear. Telomeres in dividing human somatic cells shorten until they reach a critical length, triggering most cells to undergo senescence or apoptosis. Rare immortal cells escape this crisis by expressing telomerase, or activating the Alternative Lengthening of Telomeres (ALT) pathway, which involves HR. FA core complex proteins and FANCD2 colocalize with telomeric foci in ALT, but not telomerase positive cells. Localization of FANCD2 to ALT telomeric foci requires monoubiquitination by the FA core complex, but is independent of ATM and ATR. FANCD2 primarily colocalizes with ALT telomeric DNA within ALT-associated PML bodies (APBs). Electron spectroscopic imaging and FISH experiments show that APBs contain extra-chromosomal telomeric repeat (ECTR) DNA that is non-nucleosomal. Depletion of FANCD2 causes marked increases in ECTR in ALT, but not telomerase positive cells. Overexpression of BLM, the helicase mutated in Bloom syndrome, also causes an ALT-specific increase in ECTR DNA. FANCD2 coimmunoprecipitates with BLM in ALT cells, and FANCD2 localization to ALT telomeric foci requires BLM expression. FANCD2-depleted ALT cells have reduced viability, signs of mitotic catastrophe, and multiple types of telomeric abnormalities, including increases in telomeric recombination, entanglements, colocalization with DNA repair proteins, and expression of fragile site characteristics. SiRNA depletion of FANCD2 does not cause overexpression of BLM, however codepletion of BLM with FANCD2 suppresses the telomere phenotypes caused by FANCD2 knockdown. Together this suggests that FANCD2 regulates BLM-dependent recombination and amplification of telomeric DNA within ALT cells.

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. In this work, I reported the completion of a transposon mutagenesis screen in budding yeast and the identification of five novel genes (RRD1, CIK1, CTF18, RTS1, and IRC6) critical for telomere healing. The characterization of Rrd1 led to the surprising finding that Rrd1 facilitates telomere healing at DSBs with little or no TG-rich sequences but not at DSBs with long tracts of telomeric sequences. Pph3, a PP4 phosphatase, acts in conjunction with Rrd1 to promote telomere healing. Conversely, Mec1, the ATR ortholog, phosphorylates Cdc13 on its S306 residue to suppress its accumulation at DSBs. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DSBs. Next, I found that Cik1 and its kinesin partner Kar3 are both important for telomere healing. Importantly, Kar3 contributes to telomere healing through its motor function. In contrast to Rrd1, Kar3 contributes to telomere healing regardless of telomeric sequence lengths adjacent to the break. Finally, Cik1 and Kar3 have a general role in DNA repair and physically associate with DSBs, which is dependent on the process of anchoring DSBs to nuclear periphery. In conclusion, I identified a mechanism by which the ATR family of kinases enforces genome integrity, a phosphoregulatory loop that underscores the contribution of Cdc13 to the fate of DNA ends, and a kinesin complex critical for the spatial organization of DNA repair.

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. In this work, I reported the completion of a transposon mutagenesis screen in budding yeast and the identification of five novel genes (RRD1, CIK1, CTF18, RTS1, and IRC6) critical for telomere healing. The characterization of Rrd1 led to the surprising finding that Rrd1 facilitates telomere healing at DSBs with little or no TG-rich sequences but not at DSBs with long tracts of telomeric sequences. Pph3, a PP4 phosphatase, acts in conjunction with Rrd1 to promote telomere healing. Conversely, Mec1, the ATR ortholog, phosphorylates Cdc13 on its S306 residue to suppress its accumulation at DSBs. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DSBs. Next, I found that Cik1 and its kinesin partner Kar3 are both important for telomere healing. Importantly, Kar3 contributes to telomere healing through its motor function. In contrast to Rrd1, Kar3 contributes to telomere healing regardless of telomeric sequence lengths adjacent to the break. Finally, Cik1 and Kar3 have a general role in DNA repair and physically associate with DSBs, which is dependent on the process of anchoring DSBs to nuclear periphery. In conclusion, I identified a mechanism by which the ATR family of kinases enforces genome integrity, a phosphoregulatory loop that underscores the contribution of Cdc13 to the fate of DNA ends, and a kinesin complex critical for the spatial organization of DNA repair.

Fanconi anaemia (FA) is an inherited disorder characterized by bone marrow failure, cancer predisposition and congenital abnormalities. The 12 known FA genes have been implicated in homologous recombination (HR), a process involved in telomere maintenance. A complex of at least 7 FA proteins promotes FANCD2 monoubiquitination and nuclear foci formation. FANCD2 colocalizes and interacts with HR proteins, however the role of FANCD2 in HR is unclear. Telomeres in dividing human somatic cells shorten until they reach a critical length, triggering most cells to undergo senescence or apoptosis. Rare immortal cells escape this crisis by expressing telomerase, or activating the Alternative Lengthening of Telomeres (ALT) pathway, which involves HR. FA core complex proteins and FANCD2 colocalize with telomeric foci in ALT, but not telomerase positive cells. Localization of FANCD2 to ALT telomeric foci requires monoubiquitination by the FA core complex, but is independent of ATM and ATR. FANCD2 primarily colocalizes with ALT telomeric DNA within ALT-associated PML bodies (APBs). Electron spectroscopic imaging and FISH experiments show that APBs contain extra-chromosomal telomeric repeat (ECTR) DNA that is non-nucleosomal. Depletion of FANCD2 causes marked increases in ECTR in ALT, but not telomerase positive cells. Overexpression of BLM, the helicase mutated in Bloom syndrome, also causes an ALT-specific increase in ECTR DNA. FANCD2 coimmunoprecipitates with BLM in ALT cells, and FANCD2 localization to ALT telomeric foci requires BLM expression. FANCD2-depleted ALT cells have reduced viability, signs of mitotic catastrophe, and multiple types of telomeric abnormalities, including increases in telomeric recombination, entanglements, colocalization with DNA repair proteins, and expression of fragile site characteristics. SiRNA depletion of FANCD2 does not cause overexpression of BLM, however codepletion of BLM with FANCD2 suppresses the telomere phenotypes caused by FANCD2 knockdown. Together this suggests that FANCD2 regulates BLM-dependent recombination and amplification of telomeric DNA within ALT cells.